1st International Immunonutrition Workshop Abstract (2008) 100p

1st International

Immunonutritio

n Workshop,

Valencia, 3–5

October 2007,

Valencia, Spain

Polyphenols and immunity

F. Sánchez de Medina and A. Zarzuelo

Immunomodulation and antioxidant capacity of

hydroxytyrosol present at oleic acid-rich sunflower oil

L. E. Díaz, S. Gómez, E. Nova, J. Romeo and A. Marcos

Arginine and the immune system

L. Tomás-Cobos, R. Miñambres, A. Rodrigo, M.

Navarro and D. Tomás

Effects of synbiotics on intestinal and immune function

E. Nova, B. Viadel, M. Blasco and A. Marcos

Exercise and the immune system

E. Ortega

Nutrition and immunity in the elderly

M. de la Fuente

The effects of dietary fish oil on chemokine secretion by

murine peritoneal and spleen cells

H. H. Arnardottir, D. H. Petursdottir and I.

Hardardottir

Immunomodulatory and anti-inflammatory potential of a

malleable protein matrix composed of concentrated

fermented whey proteins

J. Beaulieu, E. Trottier, R. Dubuc, C. Dupont and P.

Lemieux

Nutrition and inflammatory processes

P. C. Calder, R. Albers, J.-M. Antoine, S. Blum, R.

Bourdet-Sicard, G. A. Ferns, G. Folkerts, P. S.

Friedmann, G. S. Frost, F. Guarner, M. Løvik, S.

Macfarlane, P. D. Meyer, L. M'Rabet, M. Serafini, W.

van Eden, J. van Loo, W. vas Dias, S. Vidry, B. M.

Winklhofer-Roob and J. Zhao

Malnutrition, stress and immunodepression: their

interrelationship with fungal infections

I. Cesaroni, F. Negro and M. Guerrini

Impaired immune function in an animal model for cancer

cachexia

J. Faber, D. Kegler, P. Vos, A. van Helvoort and J.

Garssen

Impact of chia ( Salvia hispanica L.( on the immune

system: preliminary study

I. Fernandez, S. M. Vidueiros, R. Ayerza, W. Coates

and A. Pallaro

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In vitro and in vivo immunomodulating effects of

traditionally-prepared extract and purified compounds

from Cetraria islandica

J. Freysdottir, S. Omarsdottir, K. Ingolfsdottir, A.

Vikingsson and E. S. Olafsdottir

Oral lactoferrin and glycine display in vivo synergistic

anti-inflammatory activity

A. Hartog and J. Garssen

Interactions between dendritic cells, probiotic bacteria and

the adipokine leptin

S. C. Knight, N. R. English and A. I. F. Blakemore

A new n-3 PUFA-enriched formula for enteral nutrition

modulates oxidative LDL and inflammatory cytokines

J. Olza, R. Moreno, C. M. Aguilera, A. Pérez de la Cruz,

A. Gil and M. D. Mesa

Conjugated linoleic acid feeding during rat suckling period

enhances intestinal IgA production

F. J. Pérez-Cano, M. Molero, C. Ramírez-Santana, M.

Castell, C. Castellote and A. Franch

Detection of biological activities promoted by proteins from

supernatant fractions of Lactobacillus plantarum in a

promyelocytic cell line

E. Puertollano, M. A. Puertollano, L. Cruz-Chamorro,

G. Alvarez de Cienfuegos and M. A. de Pablo

Oral health and nutritional biochemical variables in adult

patients with AIDS

N. Slobodianik, P. Perris, A. Squassi, G. Sánchez,

M. S. Feliu and N. Bordoni

Gut microbes and obesity in adolescents

M. Sotos, I. Nadal, A. Marti, A. Martínez, M.

Martin-Matillas, C. Campoy, M. A. Puertollano, J.

Wärnberg, A. Marcos and Y. Sanz

PUFA in the pathogenesis and treatment of patients with

multiple sclerosis

L. S. Harbige, E. Pinto, M. Xiang, M. Leach and M. K.

Sharief

Potentiation of systemic humoral immune response in

suckling rats by conjugated linoleic acid (CLA)

C. Ramírez-Santana, F. J. Pérez-Cano, M. Castell, C.

Castellote and A. Franch

Effects of a diet with polyphenol-rich cereal

supplementation on the function and redox state of

peritoneal leucocytes from mice: differences between a

short (5 weeks) and long (20 weeks) period of

supplementation

P. Álvarez, C. Alvarado, L. Jimenez and M. de la

Fuente

Structure and antigenicity changes in 7S soyabean allergen

by enzymic deglycosylation

M. Amigo-Benavent, M. Villamiel and M. D. del

Castillo

Effects of IL-6 on amylase secretion and calcium signalling

in pancreatic AR42J cells: modulation by membrane fatty

acid composition

N. Audi, M. A. Martínez, M. D. Mesa, E.

Martínez-Victoria, M. Mañas and M. D. Yago

Effect of oil (sunflower oil) consumption with added

hydroxytyrosol (natural antioxidant) on antioxidant

variables in leucocytes from healthy adults

I. Baeza, N. M. de Castro, L. E. Díaz, A. Marcos and M.

de la Fuente

Expression of Toll-like receptors on peritoneal

macrophages and dendritic cells from old mice treated with

soyabean isoflavones and green tea

I. Baeza, N. M. de Castro, L. Arranz and M. de la

Fuente

The effects of organic v. conventional diets on immune

variables in rats

A. M. Baranska, E. Rembialkowska, E. Hallmann, L.

Lueck, J. M. Cooper and C. Leifert

Inflammatory mediators in overweight adolescents:

association with insulin sensitivity, body composition and

metabolic syndrome

R. Burrows, L. Leiva, A. M. Tong, J. Aldunate and E.

Díaz

Immunological response in coeliac disease is age related

M. Calzado, E. Donat, B. Polo, B. Baena and C. Ribes

Hormonal and immune changes with age: effect of energy

restriction

P. Cano, G. Segovia, M. P. Fernandez-Mateos, A. del

Arco, V. Jimenez, F. Mora and A. I. Esquifino

Use of inducible class II and MHC class I-related chain A/B

expression on T lymphocytes as an in vitro screen for the

immunomodulatory properties of dietary products

C. Carter, K. Varley and B. Clark

Dietary intake of nutrients of great interest in

immunonutrition to prevent muscle damage in soccer

players

C. Conejos, A. Giner, J. Mañes and J. M. Soriano

Total antioxidant capacity of refrigerated orange juice

treated with pulsed electric fields

C. Cortés, F. Barba, M. J. Esteve, R. González and A.

Frígola

Examination of host immune resistance against Listeria

monocytogenes infection in cyclophosphamide

(CPA)-treated mice after dietary lipid administration

L. Cruz-Chamorro, M. A. Puertollano, E. Puertollano,

C. Carazo, G. Alvarez de Cienfuegos and M. A. de

Pablo

Effect of dietary oils on oxidative stress and cytokine

production by murine macrophages

R. de la Puerta Vázquez, M. A. Fernández Arche, A.

Márquez Martín and V. Ruiz-Gutierrez

Seasonal changes in the neuroimmunoendocrine system

activity in young adult male rats administered with a

balanced controlled diet from weaning

A. I. Esquifino, M. P. Fernandez-Mateos, V. Jimenez,

J. Rios and P. Cano

Effect of long-chain fatty alcohols from orujo olive oil on

nitric oxide and eicosanoid generation

M. A. Fernández Arche, R. de la Puerta Vázquez, A.

Márquez Martín and V. Ruiz-Gutierrez

Fermented soyabean products as hypoallergenic food

J. Frias, Y. S. Song, C. Martínez-Villaluenga, E.

Gonzalez de Mejia and C. Vidal-Valverde

Immunomodulatory properties of Lactobacillus salivarius

are not limited to the intestine

J. Galvez, B. Arribas, M. E. Rodríguez-Cabezas, E.

Bailon, M. Comalada, D. Camuesco, J. Xaus and A.

Zarzuelo

Vitamin E as an IgE inhibitor: stability during cold storage

of human milk

G. García-Llatas, R. Lacomba, F. Ortega, A. Alegría, R.

Barberá and D. Silvestre

Melatonin supplementation modifies experimental chronic

colitis in mice

M. Golab, M. Dudziak and K. Skwarlo-Sonta

Effect of dietary supplementation on lymphocytes subsets

and liver-related variables in hospitalised patients with

anorexia nervosa

S. Gómez, L. E. Díaz, E. Nova, G. Morandé and A.

Marcos

Borage ( Borago officinalis) oil supplementation in relation

to monocyte chemoattractant protein 1 expression in

healthy subjects

M. Xiang, E. Pinto, M. A. Rahman, M. Leach and L. S.

Harbige

Vitamin D deficiency among older adults in England

remains a cause for concern!

V. Hirani, A. Ali and K. Tull

Evaluation of diet, anthropometry and immunocompetence

of young male athletes

D. Lacerda, V. Cotta-de-Almeida, A. Montero, W.

Savino and A. Marcos

Effect of inulin and probiotic bacteria on iron availability in

beans ( Phaseolus vulgaris L.)

J. M. Laparra, R. P. Glahn and D. Miller

Effect of different doses of creatine supplementation on

endogenous creatine synthesis

B. Li, M. Xiang, L. S. Harbige, X. Li and H. Ai

Effect of cessation of creatine supplementation on

endogenous creatine synthesis

X. Li, M. Xiang, L. S. Harbige and H. Ai

Activation of innate immunity by a probiotic bacterium and

a fermented milk containing this micro-organism

C. Maldonado Galdeano, A. de Moreno de Leblanc, C.

Dogi, S. Chaves, E. Carmuega, R. Weill and G.

Perdigón

Milk-derived supplement inhibits in vitro lymphocyte

proliferation and IL-2 production

S. Marín-Gallén, F. J. Pérez-Cano, M. Castell, C.

Castellote and A. Franch

Influence of fruit intake and antioxidants on the prevention

of opacities of the lens

M. I. Martínez, J. Martínez-Raga, J. Raga, A. Alegre,

M. C. Gimeno, E. Llusar and I. Alfonso-Sanchez

Alcohol consumption in secondary-school students: effects

on plasma total antioxidant capacity

M. I. Martínez, J. Martínez-Raga, A. Alegre, J. A.

Dominguez and I. Alfonso-Sanchez

Synthesis of galactooligosaccharides with prebiotic

potential during hydrolysis of lactose by Lactozym 3000 L

HP G

C. Martínez-Villaluenga, A. Cardelle-Cobas, N. Corzo,

A. Olano and M. Villamiel

Raffinose family oligosaccharides of lupin ( Lupinus albus L.

cv multolupa) as a potential prebiotic

C. Martínez-Villaluenga, J. Chicholoska, A. Kliber and

K. Gulewicz

Antioxidant and anti-inflammatory potency of different

wheat varieties and fractions

N. Mateo Anson, R. v. d. Berg, R. Havenaar, G.

Haenen and A. Bast

Elicitation of an allergic reaction in mice orally sensitized to

whey or casein proteins

A. J. Nauta, B. Schouten, B. C. A. M. van Esch, S. van

Doorn, G. A. Hofman, L. W. J. van den Elsen, L. E. M.

Willemsen, L. M. J. Knippels and J. Garssen

Effect of enteral nutrition on plasma soluble adhesion

molecules in an elderly population

J. Olza, M. D. Mesa, R. Moreno, A. Pérez de la Cruz, A.

Gil and C. M. Aguilera

Serum C3c and C4c concentrations and adenosine

deaminase activity of children with cystic fibrosis:

preliminary study

P. Perris, M. S. Feliu, S. Barbeito, I. Strasnoy, M.

Ferraro and N. Slobodianik

Introduction of complementary foods to the infant diet

within the first year of life: evaluation of general

recommendations using Achievable Benchmarks of Care®

N. Pastor, B. Soler, C. Lifschitz and and The

Generación Study Group

Plasma protein supplements modulate the activation of

gut-associated immune system induced by Staphylococcus

aureus enterotoxin B in rats

A. Pérez-Bosque, L. L. Miró, J. Polo, L. Russell, J.

Campbell, E. Weaver, J. Crenshaw and M. Moretó

Dietary fish oil increases IL-4 secretion by murine

splenocytes by an effect on accessory cells

D. H. Petursdottir and I. Hardardottir

Effects of borage ( Borago officinalis) oil supplementation

on the expression of monocyte and T-cell adhesion

molecules in healthy volunteers

E. Pinto and L. S. Harbige

Effect on lymphoproliferation and in vitro Ig production of

splenocytes from suckling rats when supplemented with

conjugated linoleic acid

C. Ramírez-Santana, F. J. Pérez-Cano, M. Castell, A.

Franch and C. Castellote

Anti-inflammatory effects of cocoa in rat

carrageenin-induced paw oedema

S. Ramos-Romero, E. Ramiro-Puig, F. J. Pérez-Cano,

C. Castellote, A. Franch and M. Castell

Lactobacillus fermentum exerts a beneficial effect in an

experimental model of rheumatoid arthritis in mice

M. E. Rodríguez-Cabezas, F. Fisac, E. Bailon, M.

Comalada, D. Camuesco, J. Xaus, A. Concha, P.

Talavera, A. Nieto, A. Zarzuelo and J. Galvez

Immunological changes after dehydration resulting from

physical effort in a hot environment

J. Romeo, D. Jimenez-Pavón, M. Cervantes-Borunda,

J. Wärnberg, L. E. Díaz and A. Marcos

Intake of some immunonutrients and ecoimmunonutrients

by young high-level basketball players

G. Santos, M. A. Buil, J. A. Ferrero, J. J. Delgado, J.

Silva and J. M. Soriano

Moderate ingestion of beer reduces inflammatory and

oxidative brain events induced by aluminium in mice

A. Schultz, R. Oliver, M. Bautista, M. J.

Gonzalez-Muñoz, I. Meseguer, A. Peña, M. I.

Sanchez-Reus, J. Benedí and F. J. Sánchez-Muniz

Impact of glycation on duodenal digestibility of

Bowman-Birk inhibitors

J. M. Silván and M. D. del Castillo

Lymphocyte subpopulations in Venezuelan preschool

children of high socio-economic status

L. Solano and D. Llovera

Timing of gluten introduction and quantity and nature of

gluten-containing foods consumed by infants in Valencia,

Spain

B. Soto, A. López and C. Ribes

Selenium deficiency in adults infected with HIV in the era of

highly-active antiretroviral therapy

M. Stambullian, M. S. Feliu, C. M. López, A. E. Piñeiro,

I. Cassetti and N. Slobodianik

Can dietary supplements reduce cell-surface expression of

molecules involved in organ transplantation?

K. Varley, B. Clark and C. Carter

Psychological stress and immune function: establishing

both acute and chronic psychological stress models in

balb/c mice

E. Vázquez, A. Barranco, M. Manzano, M. L. Jiménez

and R. Rueda

Effects of a synbiotic on intestinal and immune functions of

healthy adults

B. Viadel, E. Nova, J. E. Carreres and A. Marcos

Changes in nutritional status and their effects on serum

and thymus zinc levels and serum copper:zinc

S. M. Vidueiros, I. Fernandez, A. E. Piñeiro, N.

Slobodianik and A. Pallaro

Impact of a cereal-based diet on intestinal villi of growing

rats

S. M. Vidueiros, I. Fernandez, N. Slobodianik, M. E.

Roux and A. Pallaro

Breast-milk levels of long-chain PUFA in Kazakhstan and

Sweden

M. Xiang, L. S. Harbige and R. Zetterström

Time-course study of high-dose creatine supplementation

for endogenous creatine synthesis

M. Xiang, L. S. Harbige, X. Li, B. Li and H. Ai

The effect of moderate dose of EPA+DHA on lipid profile

and inflammatory markers in middle-aged men

H. M. Yusof and P. C. Calder

A specific mixture of short-chain galacto-oligosaccharides

and long-chain fructo-oligosaccharides induced an

anti-allergic Ig profile in infants at risk for allergy

A. J. Nauta, S. Arsnalognu, G. Boehm, G. Moro, J.

Faber, E. Knol, B. Ruiter, E. van Hoffen, L. M'Rabet

and J. Garssen

Immune-enhancing role of vitamin C and zinc and effect on

clinical conditions

S. Beveridge, E. S. Wintergerst, S. Maggini and D.

Hornig

Contribution of selected vitamins and trace elements to

immune function

S. Maggini, E. S. Wintergerst, S. Beveridge and D.

Hornig

Differential effect of Bifidobacterium species characteristic

of the gut microbiota of breast-fed and formula-fed infants

on in vitro cytokine production

E. Puertollano, T. Pozo, I. Nadal, A. Marcos, Y. Sanz

and E. Nova

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Polyphenols and immunity

F. Sanchez de Medina and A. ZarzueloDepartment of Pharmacology, School of Pharmacy, University of Granada, Spain

Flavonoids are biologically-active polyphenolic compounds with antioxidative, antineoplasic, cardiovascular protective and anti-inflammatory properties. Pharmacological therapy is essential in inflammatory bowel disease but has many adverse effects and doesnot cure the disease. Flavonoids are excellent candidates because of their anti-inflammatory properties and their low toxicity. Severalflavonoids have been shown to exert intestinal anti-inflammatory activity in vivo, including (mg/kg) quercitrin 1–5(1), rutin 10–25(2), morin25(3,4), hesperidin and diosmin 10–25(5). However, the mechanism of action is unclear. Since inflammation is associated by significantoxidative stress, this mechanism may be relevant. Indeed, flavonoid treatment counters colitis-induced glutathione depletion. On the otherhand, quercitrin treatment reduces macrophage infiltration in the dextran sulfate sodium colitis model(6). The effects of flavonoids onprimary macrophages have been studied and their structure–activity relationship characterized(7). A number of flavonoids inhibit macro-phage proliferation (but not cell viability) and some additionally reduce TNF and inducible NO synthase (iNOS) expression, probablyinterfering with the NF-kB pathway. The structural determinants of activity include the C-2——C-3 double bond, the catechol group in theB ring and the 2-position of the B ring.

Most of these flavonoids are glycosides, which are known to be hydrolysed by bacterial enzymes in the gut. Since luteolin and quercetinare not active in vivo and aglycone flavonoids are absorbed in the small intestine it is likely that glycosides act as prodrugs, releasing thebiologically-active aglycone in the lumen and preventing their premature absorption, which has been proven in the case of quercitrin(8). Inparticular, a faecal homogenate was shown to mediate quercetin release from quercitrin in vitro, and the resulting aglycone retained TNF,iNOS and IL-1b inhibitory activity in murine bone marrow-derived macrophages. This principle probably applies to the other heterosideswith known intestinal anti-inflammatory activity.

In conclusion, flavonoids have intestinal anti-inflammatory activity that is associated with macrophage inhibition and antioxidativeeffects. Further investigation of the mechanistic aspects of flavonoid pharmacological action is underway.

1. Sanchez de Medina F, Galvez J, Romero JA & Zarzuelo A (1996) J Pharmacol Exp Ther 278, 771–779.2. Galvez J, Cruz T, Crespo E et al. (1997) Planta Med 63, 409–414.3. Galvez J, Coelho G, Crespo ME et al. (2001) Aliment Pharmacol Ther 15, 2027–2039.4. Ocete MA, Galvez J, Crespo ME et al. (1998) Pharmacology 57, 261–270.5. Crespo ME, Galvez J, Cruz T, Ocete MA & Zarzuelo A (1999) Planta Med 65, 651–653.6. Camuesco D, Comalada M, Rodriguez-Cabezas ME et al. (2004) Br J Pharmacol 143, 908–918.7. Comalada M, Ballester I, Bailon E et al. (2006) Biochem Pharmacol 72, 1010–1021.8. Comalada M, Camuesco D, Sierra S et al. (2005) Eur J Immunol 35, 584–592.

Proceedings of the Nutrition Society (2008), 67 (OCE), E1 doi:10.1017/S0029665108006101


Immunomodulation and antioxidant capacity of hydroxytyrosolpresent at oleic acid-rich sunflower oil

L. E. Dıaz, S. Gomez, E. Nova, J. Romeo and A. MarcosInstituto del Frıo, Spanish Council for Scientific Research, Madrid, Spain

Typical components of the Mediterranean diet such as olive oil and red wine contain high concentrations of phenols that may haveimportant antioxidant and immunomodulation roles. The main phenols identified in extra-virgin olive oil belong to different classes:simple phenols such as 3,4-dihydroxyphenylethanol and p-hydroxyphenylethanol; secoiridoids e.g. oleuropein, the aglycone of ligstrosideand their respective decarboxylated dialdehyde derivatives(1,2). These phenols have been suggested to prevent oxidative damage andbeneficially modify immune and inflammatory responses(3). The aim of the present study was to evaluate the effect of oil (sunflower oil)containing added hydroxytyrosol (HT; ‘Oleoactive from Koipesol’; Sos Cuetara SA, Madrid, Spain) consumed at 45–50 mg/d, on theimmune cells and oxidation variables in healthy adults. Thus, twenty-two healthy subjects of both genders (20–45 years) were recruitedfor a cross-over design study. The subjects were divided into two groups of eleven and assigned to one of two treatments for a period of8 weeks: group A, 3 weeks of oil with added HT, 2 weeks of wash-out and 3 weeks of sunflower oil without HT; group B, 3 weeks of oilwithout HT, 2 weeks of wash-out and 3 weeks of oil with added HT. Leucocytes were analysed using an automatic blood-cell counter.T (CD3, CD4, CD8) and B (CD19) lymphocyte subsets and natural killer cells (CD56 + 16) were studied by flow cytometry usingperipheral blood marked with monoclonal antibodies. The oxidative and phagocytic capacities of polymorphonuclear leucocytes werequantified in vitro after incubating lymphocytes with opsonised Escherichia coli. Finally, serum Ig levels were measured by nephelometry.All variables were analysed at the beginning of the study and at 3, 5 and 8 weeks. No significant changes in leucocytes, differential cellcounts and lymphocyte subsets were observed in the two groups during the study. Nevertheless, the oxidative capacity showed a tendencyto increase in both groups after consuming oil with added HT. On the other hand, in group A the percentages of lymphocytes, monocytes,eosinophils and basophils and the Ig levels showed a tendency to increase after the consumption of oil with added HT. However, in groupB leucocyte counts, the percentage of neutrophils and Ig levels showed a tendency to increase (Figure). In conclusion, HT could improvethe immune response, but further studies with increasing levels of intake or periods of consumption of HT are required to establishwhether the effects are significant.

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This study has been supported by Sos Cuetara SA.

1. Owen RW, Giacosa A, Hull WE, Haubner R, Wurtele GB Spiegelhalder & Bartsch H (2000) Lancet Oncol 1, 107–112.2. Bonoli M, Montanucci M, Gallina, Toschi T & Lercker G (2003) J Chromatogr 1011A, 163–172.3. Carliccio MA, Siculella L, Ancora MA et al. (2003) Arteroscl Tromb Vasc Biol 23, 622–629.



Arginine and the immune system

L. Tomas-Cobos, R. Minambres, A. Rodrigo, M. Navarro and D. TomasAinia Technological Centre, Parque Tecnologico de Valencia, c Benjamın Franklin 5-11, E 46980 Paterna, Valencia, Spain

L-Arginine (arg) has been classified as a semi-essential amino acid. In addition to participating in protein synthesis, L-arg has been shownto be a powerful mediator of multiple biological processes, including the release of several hormones, collagen synthesis during woundhealing, antitumour activity and immune cell responses. L-Arg is metabolized in macrophages, endothelial cells, hepatocytes, kidneys cellsand certain tumour cells by three enzymic pathways: inducible NO synthase (iNOS); arginase I; arginase II. In macrophages L-arg ismetabolized by iNOS to produce citrulline and niticoxide, which is one of the principal cytotoxic mechanisms in these cells(1). Theavailability of L-arg to modulate the immune system has lead to the this amino acid being considered to be an immunonutrient(2).

The almond (Prunus amygdalus) is a nut with a high energy and nutritional value. The main components are vitamin E, unsaturatedfatty acids, fibre and a high proportion of arg-rich protein(3). The aim of the present study was to investigate the effects of almond, as anarg-rich food, on the immune system. Thus, NO production and genes encoding pro-inflammatory mediators such as cyclooxygenase-2(COX-2), iNOS and TNFa were evaluated in a macrophage cell line of Raw 264.7 cells to test the effect of almond foods in the form ofalmonds or a commercial almond cream (an almond-based product containing (g/kg): fat 177, protein 70, carbohydrate 360, water 370).

Based on the bioavailability of almonds in vivo, the almond foods were subjected to two pre-treatment procedures: (1) enzymictreatment (trypsin and proteinase K); (2) simulated digestion (gastric digestion with pepsin and intestinal digestion with a pancreatin–biliary extract). Raw 264.7 cells were treated with the pre-treated or non-pre-treated almond foods. As a positive control cells werestimulated with lipopolysaccharide (LPS) and L-arg. After 6 h of treatment iNOS, TNFa and COX-2 gene expression were analysed byRT–PCR (b-actin and 18S genes were used as an internal control) and after 24 h of treatment NO levels were determined using the Griessreaction(4).

The data indicated that NO production was increased in cells treated with almond foods when compared with untreated cells. NOproduction by cells stimulated with almonds was decreased in presence of iNOS inhibitor (1400W)(5). The production of NO requires ahigher concentration of digested samples than enzymic pre-treated samples. iNOS, TNFa and COX-2 gene expression was induced by thealmond product, although higher levels were found in LPS-induced cells (positive control).

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In conclusion, these preliminary results suggest that almonds can stimulate the activity of the macrophages in Raw 264.7 cells, and sostimulate the immune system. Thus, almonds could be considered to be an ‘immunomodulator’(6). However, further investigation isneeded to establish the concentration at which almonds are immunomodulatory in vivo.

1. Rodriguez PC, Zea AH, Desalvo J et al. (2003) J Immunol 17, 1232–1239.2. Grimble RF (2001) Proc Nutr Soc 60, 389–397.3. Mataix J, Manas M, Llopis J & Martinez de Victoria E (1998) Tabla de Composicion de Alimentos Espanoles (Spanish Food Composition Tables),

4th ed. Granada, Spain: Instituto de Nutricion y Tecnologıa de Alimentos.4. Kolb JP, Paul-Eugene N, Damais C, Yamaoka K, Drapier JC & Dugas B, (1994) J Biol Chem 269, 9811–9816.5. Garvey EP, Oplinger JA, Furfine ES et al. (1997) J Biol Chem 272, 4959–4963.6. Efron DT & Barbul A (2000) Nutrition 16, 73–74.



Effects of synbiotics on intestinal and immune function

E. Nova1, B. Viadel2, M. Blasco2 and A. Marcos1

1Immunonutrition Group, Metabolism and Nutrition Department, Instituto del Frıo (CSIC), Madrid, Spain and2Department of New Products, AINIA,Technological Center, Valencia, Spain

A synbiotic results from the combination of a probiotic and a prebiotic in a single product that is used as a healthy dietary supplement inthe restoration and maintenance of colonic flora. The prebiotics most commonly used in the EU are fructo-oligosaccharides (FOS), inulinand galacto-oligosaccharides (GOS). The prebiotic component (non-digestible carbohydrates) selectively increases the survival rate of aparticular probiotic or several probiotic strains during intestinal transit and thus their effect on the gastrointestinal tract. In this sense, FOShas been reported to increase bile resistance in bifidobacteria. However, except for the prebiotic effect, the evidence to support thepurported effects of synbiotics on health is still scarce in man. There is also the need to establish what differences there are in the observedeffects of the synbiotic product v. the prebiotic alone. New prebiotics, other than FOS, GOS and inulin, are being assayed for novelapplications. b-Glucans, for example, have shown a lactobacillogenic effect(1) and biotechnology is being employed for the production ofhuman-milk oligosaccharides that might facilitate the development of a healthy microbiota in non-breast-fed infants.

The effects of synbiotic therapies on intestinal function of different types of critically-ill patients have been investigated in a fewstudies(2). Different synbiotic preparations have also proved useful in preliminary studies of the management of patients with short bowelsyndrome(3), ulcerative colitis(4,5), acute pancreatitis(6) and allergy(7). A study has been carried out to evaluate the effects of the con-sumption for 6 weeks of a synbiotic product containing Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillusparacasei ssp. paracasei, Streptococcus thermophilus, Bifidobacterium sp. (24 · 108 colony-forming units/d) and FOS on the intestinalmicrobiota and self-reported intestinal function, as well as on immune function of generally healthy adults. Although no differences inself-reported improvement were found with treatment of mild gastrointestinal symptoms present at baseline such as constipation, flatu-lence, postprandial bloating or dyspepsia, there was a significant improvement overall in symptoms and in motility in the synbiotic groupcompared with the placebo group. Intestinal microbiota did not change as a result of synbiotic consumption, and the only significant effectof treatment (ANOVA; P=0.05) on immune variables was on the concentration of serum soluble L-selectin, which showed a decrease inthe synbiotic group. This outcome might lead to a more beneficial profile of the inflammatory markers in relation to the prevention ofatherosclerosis and CVD. A trend towards a decrease in CD3 - (CD56 + 16)+ cells (natural killer cells) was found in the women in thesynbiotic group. This effect was not observed in men, probably because their values at baseline were at the lower limit of the normalrange. No effect of treatment was found in other lymphocyte subsets or other immune variables such as the phagocytic activity ofmonocytes and granulocytes and inflammatory proteins such as C-reactive protein and caeruloplasmin.

1. Snart J, Bibiloni R, Grayson T et al. (2006) Appl Environ Microbiol 72, 1925–1931.2. Jain PK, McNaught CE, Anderson AD, MacFie J & Mitchell CJ (2004) Clin Nutr 23, 467–475.3. Kanamori Y, Sugiyama M, Hashizume K, Yuki N, Morotomi M & Tanaka R (2004) J Pediatr Surg 39, 1686–1692.4. Haskey N & Dahl WJ (2006) Nutr Rev 64, 132–138.5. Furrie E, Macfarlane S, Kennedy A, Cummings JH, Walsh SV, O’Neil D & Macfarlane GT (2005) Gut 54, 242–249.6. Olah A, Belagyi T, Poto L, Romics L Jr & Bengmark S (2007) Hepatogastroenterology 54, 590–594.7. Ogawa T, Hashikawa S, Asai Y, Sakamoto H, Yasuda K & Makimura Y (2006) FEMS Immunol Med Microbiol 46, 400–409.



Exercise and the immune system

E. OrtegaDepartment of Physiology, Faculty of Sciences, University of Extremadura, 06071-Badajoz, Spain

The immune system is a system for self-recognition and maintaining homeostasis. It is an extremely complex network that extendsthroughout the body, and it can recognize and defend the organism against a theoretical infinity of challenges. The participants in innateimmune mechanisms are macrophages and neutrophils, along with natural killer (NK) cells, complement and defensins, and they con-stitute the first line of defence. All its constituents need a basic capacity to distinguish between self and foreign, and danger or non-dangersignals with the involvement of Toll-like receptors (TLR). By engulfing, processing and presenting antigens, macrophages form thecritical link to the specific branch of the immune system that mainly comprises the various subpopulations of lymphocytes and theirproducts. While regular moderate exercise is very likely to be associated with decreased susceptibility to infection, excessive exhaustiveexercise has been associated with symptoms of transient immunosuppression, leading to increased susceptibility to infection. Thisoutcome is particularly the case for athletes in a competitive setting, who are frequently subjected to physical, psychological andenvironmental stress as well as to inadequate nutrition that may cause immunosuppression.

However, recently there has been excessive generalization of the notion that moderate exercise is beneficial and intense exercise isharmful for the immune system. The latest studies, however, have revealed that this general finding cannot be extended to phagocytosis.Some stages of the phagocytic process, in particular chemotaxis and phagocytosis, are stimulated by both moderate and intense exercise,which may counter some of the pronounced suppressive effects of intense exercise on lymphocytes and NK responses.

Exercise-induced changes in the immune system are mediated by the stress hormones, mainly glucocorticoids and catecholamines.Exercise-induced stress also results in the release of the 72 kDa heat-shock protein (Hsp72), which also has marked effects on immunity.Stress hormones and proteins may also be considered as ‘stress mediators’ in the exercise-induced stimulation of phagocytes and as‘danger signals’ for the immune system during intense exercise. Stimulation of chemotaxis and phagocytosis of neutrophils and macro-phages by glucocorticoids and noradrenaline at physiological exercise-induced concentrations has been reported(1). Post-exercise Hsp72concentrations also stimulate neutrophil phagocytosis and chemotaxis through TLR-2 together with its cofactor CD14.

Chemotaxis and phagocytosis of neutrophils and macrophages are two important functions in the inflammatory response. However,while inflammation plays an important role in host defence, uncontrolled inflammatory reactions are responsible for the initiation andprogression of autoimmune and inflammatory diseases. In this context, it has also been found that intense exercise also alters the pro-inflammatory–anti-inflammatory cytokine balance, which is critical for inflammatory and autoimmune diseases; mainly in women, whoare more susceptible to these types of pathologies. It has been reported that glucocorticoids, noradrenaline and Hsp72 modulate the releaseof pro-inflammatory cytokines by phagocytes and other immune cells(2,3). In addition pro-inflammatory cytokines, such as IL-1, IL-6, IL-8, interferon-g and TNFa can inhibit food intake. IL-1, together with catecholamines and glucocorticoids, may regulate glucose home-ostasis during an immune response; probably, as recently reported, serving to divert glucose to inflamed tissues to satisfy the high energycost of the immune response(4). During exercise muscle-derived IL-6 has been also shown to affect metabolic responses, such as increaseglucose metabolism, lipid oxidation and lipolysis(5).

Thus, it is necessary to bear in mind that the immune system is a homeostatic regulatory system that operates in situations of highenergy costs such as exercise-induced stress, and, moreover, it is involved in disease situations that also need high energy contributions.

This work has been partially supported by 2PR04A076 and DEP2006-56187-C04-03/PREV.

1. Ortega E (2003) Exerc Immunol Rev 9, 70–93.2. Elenkov I & Chrousos GP (2002) Ann NY Acad Sci 996, 290–303.3. Asea A, Kabingu E, Stevenson MA & Calderwood SK (2000) Nat Med 6, 435–442.4. Besedovsk HO & del Rey A (2007) Brain Behav Immun. 21, 34–44.5. Steenberg A (2003) Exer Immunol Rev 9, 41–47.



Nutrition and immunity in the elderly

M. de la FuenteDepartment of Physiology, Faculty of Biology, Complutense University of Madrid, Madrid, Spain

Ageing is a universal process that may be defined as a progressive, endogenous and irreversible accumulation of adverse changes thatincrease vulnerability to disease and finally to death. The aging process is very heterogeneous, showing different rates of physiologicalchanges in the various systems of the organism and in the diverse members of a population of the same chronological age. This diversityjustifies the introduction of the concept of ‘biological age’, which is very useful in assessing the level of aging experienced by eachindividual and therefore his life expectancy. Currently, it is known that immune functions change with age. Since the functional capacityof the immune system is a marker of health and longevity, several immune functions (Table) have been proposed as markers of biologicalage and predictors of longevity. These immune functions have been standardized at different ages in mice and human subjects and showsimilar changes with aging in leucocytes from both species. Moreover, a model of premature aging in mice as well as very high longevityin mice and human populations (very old mice and centenarians) has been used. In the prematurely-aging mice the functional variablesinvestigated have been shown to have values characteristic of chronologically-older animals, and these mice also show a significantlydecreased lifespan. In very old mice and centenarians these immune functions have values similar to those for adults (6 months and 30years respectively). The cause of immunosenescence has also been investigated, and since the oxidation theory is now the most widelyaccepted explanation of the ageing process, the age-related imbalance between free radical production and antioxidant defences has beenanalysed, with a higher production of the former, denominated oxidative stress, in the immune cells from subjects throughout ageing(Table). Since free radicals are needed for many physiological processes including immune function, prevention of an imbalance isrequired to maintain good health. Oxidative stress is responsible for a large number of pathologies, many of which occur more frequentlywith ageing. The immune cells from prematurely-ageing mice show oxidative stress whereas leucocytes from very old mice and cen-tenarians show values of oxidant and antioxidant compounds similar to those in cells from adults. One of the mechanisms involved is theactivation of NF-kB. A diet with adequate amounts of antioxidants neutralizes the oxidative stress of immune cells in ageing and thereforethe function of these cells improves, both in mice and human subjects, showing values close to those in adults (Table)(1–3). Miceconsuming a diet with antioxidants for only 5 weeks increase their lifespan. Thus, good nutrition with compounds rich in antioxidants canprevent the age-related deterioration of the immune system and help to achieve a healthy longevity.

Aging Antioxidant supplementation

FunctionsChemotaxis, phagocytosis Decrease Increase (=adult)Lymphoproliferation in response to mitogens Decrease Increase (=adult)IL-2 release, NK activity Decrease Increase (=adult)Adherence, TNFa release Increase Decrease (=adult)Oxidant compoundsExtracellular superoxide anion, PGE2 Increase Decrease (=adult)Oxidized gluthatione Increase Decrease (=adult)

Antioxidant defencesReduced gluthatione Decrease Increase (=adult)Superoxide dismutase and catalase activities Decrease Increase (=adult)Gluthatione peroxidase and reductase activities Decrease Increase (=adult)

This work has been supported by the MEC (BFU 2005–06777) and RETICEF (RD06/0013/0003) (ISCIII) of Spain.

1. De la Fuente M, Hernanz A & Vallejo MC (2005) Antioxid Redox Signal 7, 1356–1366.2. Alvarado C, Alvarez P, Puerto M, Gausseres N, Jimenez L & De la Fuente M (2006) Nutrition 22, 767–777.3. Alvarez P, Alvarado C, Mathieu F, Jimenez L & De la Fuente M (2006) Eur J Nutr 45, 428–438.



The effects of dietary fish oil on chemokine secretion bymurine peritoneal and spleen cells

H. H. Arnardottir, D. H. Petursdottir and I. HardardottirBiochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland

Dietary fish oil, rich in n-3 PUFA, affects immune function partly by its effects on cytokine secretion by peritoneal and splenic macro-phages and T-cells(1,2,3). The effects of dietary fish oil on secretion of chemokines by these cells have been less studied. Macrophageinflammatory protein 1a (MIP-1a) and monocyte chemotactic protein 1 (MCP-1) are chemokines that act on and are secreted bymonocytes, macrophages and T-cells(4). MIP-1a may promote a T-helper (Th) 1-type immune response(5), whereas MCP-1 is thought topromote a Th2-type immune response(6). The aim of the present study was to determine the effects of dietary fish oil on MIP-1a andMCP-1 secretion by murine peritoneal macrophages and splenic macrophages and T-cells ex vivo.

Mice were fed diets supplemented with (g/kg) 180 fish oil+ 20 maize oil or 200 maize oil for 6 weeks (n 10). Resident peritonealmacrophages were stimulated with lipopolysaccharide (LPS) with or without antibodies against TNFa or IL-10. Total spleen cells werestimulated with LPS or antibodies against CD3 and CD28. Concentration of the chemokines MIP-1a and MCP-1 in the medium wasmeasured by ELISA.

Dietary fish oil did not affect LPS-induced MIP-1a secretion but decreased LPS-induced MCP-1 secretion by resident peritonealmacrophages (329 v. 174 pg/ml respectively, P=0.01). The effect of dietary fish oil on MCP-1 secretion was not mediated by an effect onTNFa or IL-10 production as it was not affected by antibodies against TNFa or IL-10. On the other hand, dietary fish oil increased LPS-induced MIP-1a secretion by splenic macrophages (see Figure) but had little effect on MCP-1 secretion. Dietary fish oil decreased anti-CD3/anti-CD28-induced MIP-1a secretion by splenic T-cells (see Figure) but had no effect on MCP-1 secretion.

These results demonstrate that dietary fish oil affects chemokine secretion by peritoneal and splenic macrophages as well as T-cells andthat its effects depend on the cell type and location. These effects of dietary fish oil on chemokine secretion may help explain its effects onimmune function in which chemokines play an important role.

MIP-1α secretion by total splenocytesstimulated with LPS or anti-CD3/CD28

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1. Calder PC (1997) Ann Nutr Metab 41, 203–234.2. Petursdottir DH, Olafsdottir I & Hardardottir I (2002) J Nutr 132, 3740–3743.3. Petursdottir DH & Hardardottir I (2007) J Nutr 137, 665–670.4. Rollins BJ (1997) Blood 90, 909–928.5. Schrum S, Probst P, Fleischer B & Zipfel PF (1996) J Immunol 157, 3598–3604.6. Gu L, Tseng S, Horner RM, Tam C, Loda M & Rollins BJ (2000) Nature 404, 407–411.



Immunomodulatory and anti-inflammatory potential of a malleableprotein matrix composed of concentrated fermented whey proteins

J. Beaulieu1, E. Trottier2, R. Dubuc1, C. Dupont1 and P. Lemieux1,2

1Institut National de la Recherche Scientifique-Institut Armand-Frappier (INRS-IAF) 531 Blvd Des Prairies, Building 18,

Laval, Quebec H7V 1B7, Canada and 2Technologie Biolactis 500 Cartier West Suite 218, Laval, Quebec H7V 5B7, Canada

A novel nutraceutical ingredient, the malleable protein matrix (MPM), is obtained from the fermentation of pasteurized whey with aunique probiotic strain isolated from kefir grains. It is also composed of capsular exopolysaccharides, vitamins, minerals (such as Ca) andpeptides generated during the fermentation process. MPM is a complex biological mixture in which synergistic effects between thecomponents are expected. Immunocompetent-animal studies have shown that MPM increases the polymorphonuclear cell counts(1), butalso modulates the overall level of glutathione in circulating cells(1)and cytokine production (Fig. 1). These results suggest that MPMexerts a definitive stimulation of the innate immunity and that consumption of MPM could be either beneficial or detrimental on theimmune system.

In order to assess the resulting effect of MPM, an oxazolone-induced atopic contact dermatitis model (ACD) was used to inducesystemic inflammation. The results showed that MPM did not promote any detrimental side effects. On the contrary, MPM exhibited apositive and significant anti-inflammatory effect comparable with that of hydrocortisone but without the side effects(2). Using this model amarked reduction in ear inflammation was demonstrated in MPM-treated mice, and this effect was correlated with inhibition of neutrophilextravasation in the tissue(2). The in vivo ‘air pouch’ model, which represents the pathological characteristics of arthritis, also demon-strated a 50% inhibition of neutrophil infiltration in MPM-treated mice, which was correlated with an important reduction in cytokine andchemokine production following stimulation (J Beaulieu, D Girard, C Dupont and P Lemieux, unpublished results).

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Fig. 1. Systemic cytokine production in healthy mice treated with water orMPM for 2 weeks. IFNg, interferon-g; TNFa, TNFa; GM-CSF, granulocyte-macrophage colony-stimulating factor.

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Fig. 2. Systemic cytokine production in the ACD model in water- or MPM-treated mice.IFNg, interferon-g; TNFa, TNFa; GM-CSF, granulocyte-macrophage colony-stimulatingfactor.

The evaluation of cytokine production in healthy animals indicates that the stimulation of immunity is not related to activation ofclassical T-helper 1 or T-helper 2 pathways (Fig. 1). Furthermore, in the ACD model the cytokine production also indicates that thesepathways are not implicated in the anti-inflammatory effect of the MPM, and IL-2 appears to have a role in the mechanism of action(Fig. 2). The modulation of cytokine production in animal models suggests that MPM regulates a newly-discovered subpopulation oflymphocytes that acts specifically on neutrophils, which could explain the dual effects obtained with MPM, i.e. the capacity to stimulateimmunity as well as exhibiting important anti-inflammatory effects. This mechanism of action has not previously been associated with anutraceutical product, suggesting that MPM may become an alternative and a functional food of choice for those individuals sufferingfrom autoimmune diseases, as well as being able to stimulate infectious defence.

1. Beaulieu J, Dubuc R, Beaudet N, Dupont C & Lemieux P. (2007) J Med Food 10, 67–72.2. Beaulieu J, Dupont C & Lemieux P. (2007) J Inflamm (Lond) 4, 6.



Nutrition and inflammatory processes

P. C. Calder1, R. Albers2, J.-M. Antoine3, S. Blum4, R. Bourdet-Sicard3, G. A. Ferns5, G. Folkerts6,P. S. Friedmann1, G. S. Frost5, F. Guarner7, M. Løvik8, S. Macfarlane9, P. D. Meyer10,L. M’Rabet11, M. Serafini12, W. van Eden13, J. van Loo14, W. vas Dias15, S. Vidry16,

B. M. Winklhofer-Roob17 and J. Zhao18

1School of Medicine, University of Southampton, Southampton SO16 7PX, UK, 2Unilever Health Institute, 3130 AC,

Vlaardingen, The Netherlands, 3Danone Vitapole, Palaiseau 91767, France, 4Nestle Research Centre, Lausanne 26,

Switzerland, 5School of Biomedical & Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK, 6Department of

Pharmacology & Pathophysiology, University of Utrecht, 3508 TB, Utrecht, The Netherlands, 7Digestive System Research

Unit, Hospital General Vall d’Hebron, 08035 Barcelona, Spain, 8Division of Environmental Medicine, Norwegian Institute

of Public Health, Nydalen, 0403 Oslo, Norway, 9Division of Pathology and Neuroscience, Dundee University, Dundee DD1

9SY, UK, 10Sensus, 4804 RA, Roosendall, The Netherlands, 11Numico-Research, 6700 CA, Wageningen, The Netherlands,12Unit of Human Nutrition, National Institute for Nutrition, 00178 Roma, Italy, 13Faculty of Veterinary Medicine, University

of Utrecht, 5384 CL, Utrecht, The Netherlands, 14Raffinerie Tirlemontoise - Orafti, 3300 Tienen, Belgium, 15Seven Seas Ltd,

Marfleet, Hull HU9 5NJ, UK, 16ILSI Europe, 1200 Brussels, Belgium, 17Institute of Molecular Biosciences, University of

Graz, 8010 Graz, Austria and 18Yakult Europe, 1332 EN, Almere, The Netherlands

The ILSI Europe Task Force on ‘Nutrition and Immunity in Man’ aims to better understand the effects of diet or nutrients on variousaspects of immune function in essentially healthy individuals. In 2005 the Task Force commissioned an activity focusing on ‘the impact ofnutrition on inflammation’. The aim of this activity was to review current knowledge focusing on common mechanisms and markers ofinflammation, the role of inflammation in various diseases and conditions, and the potential for modulation of inflammation by nutrition.The aim was addressed by establishing an Expert Group, drafting a document and holding a Workshop to discuss the draft document andto finalise the conclusions. The finalised document will be published.

The Workshop was held in 2006 and gathered together clinicians, immunologists, pharmacologists and nutritionists in order to consider:(a) the role of inflammation in a range of distinct pathological conditions (inflammatory bowel diseases, coeliac disease, asthma, chronicobstructive pulmonary disorder, atopic dermatitis, psoriasis, rheumatoid arthritis, atherosclerosis, obesity) including the identification ofcommon and unique molecular and cellular responses and signalling pathway; (b) the mechanism of action of common anti-inflammatorydrugs; (c) the potential pro- and anti-inflammatory roles of specific dietary components (PUFA, vitamins C and E, carotenoids, flavonoids,prebiotics, probiotics).

A number of conclusions were reached. Inflammation is a normal part of the host immune response to infection and to other insults; itinitiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. Normally, thehost is tolerant to microbes and other environmental components that do not pose a threat. This tolerance involves only a limited hostresponse or an active response that is tightly controlled. Where an inflammatory response does occur it is normally well regulated in orderthat it does not cause excessive damage to the host, is self-limiting and resolves rapidly. Pathological inflammation involves a loss oftolerance and/or of regulatory processes, although the reasons for this loss are not clear. Whatever the site of inflammation or the nature ofthe trigger, common mediators of inflammation include certain cytokines (TNFa, IL-1b, IL-6, interferon-g), chemokines (IL-8, monocytechemoattractant peptide-1), eicosanoids (PGE2, 4-series leukotrienes), matrix metalloproteases and reactive oxygen species, and signallingpathways often involve the activation of NF-kB. Several nutritional strategies, including n-3 PUFA, antioxidants vitamins, plant flavo-noids, prebiotics and probiotics may be able to amelioriate chronic inflammatory processes. However, nutritional studies rely heavily oncell culture and animal models, and more studies in human subjects are needed. Although nutritional studies have focused on therapy ofinflammatory conditions, appropriate nutrition may lower the risk of such conditions occurring, but strong evidence of this effect iscurrently lacking.



Malnutrition, stress and immunodepression: their interrelationshipwith fungal infections

I. Cesaroni, F. Negro and M. GuerriniLABBCE, La Plata, Argentina

Prolonged stress, malnutrition and inappropriate nutrition represent 90% of the causes of immunodepression in subjects without basalpathologies, and are key to the entry into epithelia and endothelia of fungal species coexisting in the environment, as they provide byimmunological deficit an optimal and permissive host. Both persistent emotional and nutritional disorders converge in metabolic stress,with hyperproduction of free radicals and an increase in cAMP. On the one hand, excessive production of free radicals, not neutralizedbecause of the antioxidant deficit, impacts on the skin, thus generating tissue microlesions. On the other hand, cAMP accumulationproduced by energy consumption associated with stress acts as a negative modulator of cellular immunity that decreases the activity ofT-cells and macrophages. Consequently, there is a decrease in levels of IL, interferon-g , TNFa and Ig, providing optimal conditions forthe entry of opportunistic pathogens. These outcomes are manifest in numerous cases as severe surface mycoses, and in some cases asprofound and systemic mycoses.

When patients with mycosis in different zones (n>2400) were analysed 60% showed onychomycosis (infection of finger and toenails)and 40% a dermal mycosis, some with genetic immunological restrictions similar to psoriasis(1) and vitiligo (20% of those studied). In theremainder of cases mycosis was coincident with immunodepression in patients such as badly-nourished and/or malnourished children andelderly adults, breast-feeding mothers, postnatal mothers, alcohol- and drug-dependent patients, patients undergoing prolonged treatmentwith quimiotherapeutics, radiotherapeutics and corticosteroids, with emotional stress and other less-frequent cases.

Analysis of observed causes and outcomes with an unexpected finding has led to the postulation of a treatment model based on 350patients with psoriasis participating in a randomised double-blind study that included a natural probiotic solution based on four species ofLactobacillus associated with Saccharomyces cerevisiae in oat solution. This product is a leading probiotic solution because of its topicaland oral use. Periodic consumption of the probiotic(2) allows recovery of the immunomodulating action of the intestinal mucosa, whichcontains 70% of the body’s immune cells. Furthermore, its topical application facilitates indispensable degradation of the hyperkeratosicmicaceous plaque stimulated by fungal toxins at the level of dermal receptors and growth factors, Rhodotorula and Malassezia(3) being theeffectors most involved.

Antifungal agents used in the form of cream, lotion or pills are selected according to results obtained by culture and antifungal tests ofisolated species following the norms of the Clinical and Laboratory Standards Institute for antifungal tablets. The therapy was com-plemented with a diet based on fruits, vegetables, food containing n-3, n-6 and n-9 PUFA and a supplement of minerals and antioxidantsformulated to provide rapid reversal of acute cases. Some cases also required psychological support.

Compared with patients treated with placebo, patients treated with the probiotic and antifungal agent showed overall improvement intheir lesions. As the causes of immunodepression were controlled, mycotic growth could be stopped and the immunological deficitreversed by dosing with IgA. In turn, this focused treatment allowed a specific action against the invasive fungal agent that resulted infavourable responses, decreasing not only the cost but also the duration of the treatment.

1. Baroni A, Paoletti I, Ruocco E, Agozzino M, Tufano MA & Donnarumma G (2004) J Cutan Pathol 31, 35–42.2. Weston S, Halbert A, Richmond P & Prescott SL (2005) Arch Dis Child 90, 892–897.3. Baroni A, Orlando M, Donnarumma G, Farro P et al. (2006) Arch Dermatol Res 297, 280–288.



Impaired immune function in an animal model for cancer cachexia

J. Faber, D. Kegler1, P. Vos1, A. van Helvoort1 and J. Garssen1,2

1Numico Research, Wageningen, The Netherlands and 2Department of Pharmacology & Pathophysiology,

Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands

In general it is assumed that 50% of all patients with cancer have significant weight loss before treatment and many of them suffer fromcachexia(1,2). Malnutrition and inflammation occurring during cachexia directly affects the immune system. Thus, patients with cachexiahave a higher susceptibility towards infections, which significantly influences survival (3).

Several preclinical studies show the cachectic features of the colon-26 tumour model in mice in which the pro-inflammatory cytokinesIL-6 and TNFa are denoted as pivotal mediators(4). In contrast, fewer studies have investigated the effects of cachexia and nutrition onimmune function. The present study aims to develop a model to study nutritional effects on immune function in mice with cachexia.

Murine colon adenocarcinoma (C26) cells were inoculated in syngenic CD2F1 mice to induce cachexia. Body weight, skeletal-muscleweight and weight of adipose tissue were measured to assess the cachectic status of the mice. To investigate effects on the immune systemcontact hypersensitivity against oxazolone was determined, as an in vivo model for cellular (T-helper (Th) 1 dependent) immunity. Inaddition, plasma cytokines, concanavalin A (ConA)-induced splenocyte proliferation and cytokine production, and lipo-polysaccharide(LPS)-induced cytokine production in whole blood were measured.

Tumour inoculation resulted in a significant impairment of body weight and wasting of skeletal muscles and adipose tissue. In addition,pro-cachectic cytokines IL-6 and TNFa in plasma demonstrated a substantial increase, whereas the anti-cachectic cytokine IL-4 wassignificant lower in the tumour-bearing mice. Contact hypersensitivity showed a significant decrease in tumour-bearing animals, reflectinga reduced Th1 immune response.

Furthermore, proliferation capacity, but also Th1 and Th2 cytokine production after ConA stimulation by splenocytes demonstrated asignificant reduction compared with the control group. Lower capacity of immune cells in whole blood to react on LPS was reflected in adecreased production of both IL-1b and TNFa in the tumour-bearing group while IL-6 remained unchanged.

Present findings demonstrate an impaired immune function in tumour-bearing mice suffering from cachexia. Thus, it is a useful modelto study potential effects of nutritional interventions on immune function in cancer cachexia.

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Fig. 1. Effect of tumour inoculation on contact hypersensitivity in a mouse model for cancer cachexia. Values are means (mm) with their standard errors represented by verticalbars for control (C) group (n= 10) and tumour-bearing (TB) group (n= 20).

1. Tchekmedyian NS, Zahyna D, Halpert C & Heber D (1992) Oncology 49, Suppl. 2, 3–7.2. Finley JP (2000) AACN Clin Issues 11, 590–603.3. Dewys WD, Begg C, Lavin PT et al. (1980) Am J Med 69, 491–497.4. Zhou W, Jiang ZW, Tian J, Jiang J, Li N & Li JS (2003) World J Gastroenterol 9, 1567–1570.



Impact of chia (Salvia hispanica L.) on the immunesystem: preliminary study

I. Fernandez1, S. M. Vidueiros1, R. Ayerza2, W. Coates2 and A. Pallaro1

1Department of Nutrition, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina and2Office of Arid Lands Studies, University of Arizona, Tucson, Arizona 85706, USA

Chia was one of the four basic foods of Central American civilizations in pre-Columbian times. Nowadays, this crop is being reintroducedto Western diets to improve human health because it is an important source of n-3 fatty acids, antioxidants, dietary fibre, protein, vitaminsand minerals, and when added to animal diets it elicits a reduction in the SFA contents of the animal products and serum lipids(1,2). Theprotein quality of chia has been demonstrated to be higher than that of common cereals, which could be important in thymus developmentsince previous studies have shown that protein quality affects thymus status(3,4). However, adverse reaction to food is frequently observedamong populations, and its symptoms may be localized in many organs and systems(5). The aim of the present study was to analyse theeffect of chia on some aspects of immune system such as the thymus and serum IgE concentration. Weanling male Wistar rats (23 d ofage) from the Department of Nutrition at the School of Pharmacy and Biochemistry of the University of Buenos Aires, were divided inthree groups (six rats each) that received for 1 month (g/kg diet): 150 ground chia seed (T1); 50 chia oil (T2); no chia (T3; control group).Diets T1 and T2 were formulated to provide equal quantities of a-linolenic acid from the chia. All the experimental diets were iso-energetic, contained (g/kg) 200 protein and 70 oil, and were prepared according to the American Institute of Nutrition guidelines(6). Foodintake was recorded (FI; g/d). At the end of the experimental period and after 4 h of fasting body weight (BW; g) was recorded. Animalswere anaesthetized in a CO2 chamber and blood was recollected by venous puncture and used to determined serum IgE levels (ng/ml)by ELISA (Bethyl Labs, Montgomery, TX, USA). Thymuses were removed, weighed (TW; mg/P0.75) and total thymocyte number (TN;no. of cells per organ) was determined using a Newbauer chamber. Statistical analysis was performed using ANOVA t test.

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T1 T2 T3

Mean SD Mean SD Mean SD

FI (g/d) 16.03 0.99 14.64 1.24 15.46 0.95BW (g) 265.78 24.01 238.88 40.25 259.26 27.50TW (mg/P0.75) 12.16 1.53 11.12 1.16 11.07 2.42TN (cells·10 - 7/organ) 109.5 17.68 81.19 18.90 98.83 15.21IgE (ng/ml) 6.77 4.06 4.67 1.05 3.18 1.05

No significant differences were observed in FI, BW, TW, TN and IgE levels when chia was added to experimental diets as seeds (T1) oras oil (T2) when compared with the control (T3). Moreover, no symptoms such as dermatitis, diarrhoea and abnormal animal growth andbehaviour were observed. Adding chia seeds or oil to experimental diets did not produce any of the problems associated with other n-3fatty acid sources such as flaxseed or marine products, e.g. fishy flavour, weight loss, digestive problems, diarrhoea and allergies, as hasbeen proposed(1).

1. Ayerza R & Coates W (2005) Nutr Res 25, 995–1003.2. Fernandez I, Ayerza R, Coates W, Vidueiros SM, Slobodianik NH & Pallaro AN (2005) Medicina (Bs As) 65, Suppl. II, Abstr 259: 113.3.3. Pallaro A, Feliu MS., Vidueiros S, Slobodianik N, Ayerza R, Coates W & Fernandez I (2004) Congreso Internacional de Ciencia y Tecnologıa de los

Alimentos. Argentina. http://www.eatchia.com/pallarosp.htm4. Pallaro A, Roux ME & Slobodianik NH (2001) Nutrition 17, 724–728.5. Foschi FG, Marsigli L, Chiappelli F, Kung MA, Bernardi M & Stefanini GF (2000) Adverse reactions to foods. In Nutrition and Immunology,

pp. 233–246 [ME Gershwin, JB German and CL Keen, editors]. Totowa, NJ: Humana Press.6. American Institute of Nutrition (1993) J Nutr 123, 1939–1951.



In vitro and in vivo immunomodulating effects of traditionally-preparedextract and purified compounds from Cetraria islandica

J. Freysdottir1,2, S. Omarsdottir3, K. Ingolfsdottir3, A. Vikingsson4 and E. S. Olafsdottir3

1Centre for Rheumatology Research and 2Department of Immunology, Landspitali University Hospital, IS-101 Reykjavik,

Iceland, 3Faculty of Pharmacy, University of Iceland, Hagi, Hofsvallagata 53, IS-107 Reykjavik, Iceland and4Department of Rheumatology, Landspitali University Hospital, IS-108 Reykjavik, Iceland

Cetraria islandica has been used for centuries in folk medicine in many countries to treat a number of conditions, mainly as an aqueousextract(1,2). C. islandica contains many compounds, some of which have shown biological activity(3–6). However, very little is knownabout their effect on the immune system. Thus, the effect of traditionally-prepared aqueous extract and purified compounds isolated fromC. islandica (polysaccharides lichenan and isolichenan and secondary metabolites protolichesterinic acid (PLA) and fumarprotocetraricacid (FPCA)) on the maturation of dendritic cells was assessed.

Human monocytes were isolated from healthy individuals and differentiated into immature dendritic cells by culturing them in thepresence of IL-4 and granulocyte–macrophage colony-stimulating factor. They were subsequently cultured in the presence of maturationfactors (IL-1b and TNFa), either alone (Neg) or with positive controls lipopolysaccharides (LPS), PGE2, cholecalciferol (VD3) or theaqueous extract or purified compounds from C. islandica. Their effect on the maturation of the dendritic cells was assessed by measuringsecretion of IL-10 and IL-12 by ELISA and expression of the surface molecules CD86 and CD209 by flow cytometry. In addition, theeffect of the aqueous extract on antigen-induced arthritis in rats was investigated.

The aqueous extract up regulated secretion of both IL-10 and IL-12, with IL-10 secretion being more prominent (Figure). Lichenan hadsimilar effects, but not isolichenan or the secondary metabolites, suggesting that the effect observed with the aqueous extract was mainlymediated by lichenan. Significantly less arthritis was observed for rats treated with the aqueous extract compared with rats treated withsaline (9 g NaCl/l) alone (data not shown).

These results suggest that the aqueous extract of C. islandica has an anti-inflammatory effect, possibly by changing the cytokinesecretion bias from IL-12 towards IL-10.

0.1

1

10

Neg. LPS PGE2 VD3 100 10 100 10 100 10 1 0.1 0.01 1 0.1 0.01

IL-1

2p40

/IL-1

0 ra

tio

(P

I)

***

**

*

*

Extract Isolichenan Lichenan FPCA PLA

Figure. IL-12p40:IL-10 secretion by dendritic cells cultured with maturation factors alone (Neg); LPS; PGE2; VD3;extract, lichenan and isolichenan at 100 and 10mg/ml; orFPCA and PLA at 1, 0.1 and 0.01mg/ml. Values are means and standard deviation represented by vertical bars. Mean values were significantly different from that for the control(Neg): *P<0.05.

1. Ingolfsdottir K (2000) Bioactive compounds from Iceland moss. In Bioactive Carbohydrate Polymers – Occurrence, Function and Use. Proceedings ofthe Phytochemical Society of Europe, vol. 44, pp. 25–36 [BS Paulsen, editor]. Dordrecht, The Netherlands: Kluwer Academic Publishers.

2. Olafsdottir ES & Ingolfsdottir K (2001) Planta Med 67, 199–208.3. Ingolfsdottir K, Jurcic K, Fischer B & Wagner H (1994) Planta Med 60, 527–531.4. Ingolfsdottir K, Jurcic K & Wagner H (1998) Phytomedicine 5, 333–339.5. Olafsdottir ES, Ingolfsdottir K, Barsett H, Paulsen BS, Jurcic K & Wagner H (1999) Phytomedicine 6, 33–39.6. Omarsdottir S, Olafsdottir ES & Freysdottir J (2006) Int Immunopharmacol 6, 1642–1650.



Oral lactoferrin and glycine display in vivo synergisticanti-inflammatory activity

A. Hartog and J. GarssenNumico Research, Wageningen, The Netherlands and Department of Pharmacology & Pathophysiology,

Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands

There is a growing awareness of the interaction of food constituents with the immune system. The present study aims to evaluateimmunomodulatory effects of two of these nutritional components, i.e. glycine (Gly) and lactoferrin (LF)(1,2). Mice orally supplementedwith gly, LF or Gly +LF were injected intradermally in the ear with zymosan. Ear swelling, as a measure of inflammation, as well as IL-1,TNFa and IL-6 levels in the ear and the number of TNFa-producing spleen cells were analysed. Gly and LF decreased the zymosan-induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reducednumber of TNFa-producing spleen cells). Gly effects (20, 50 and 100 mg per mouse per d) were concentration dependent whereas forLF only the lowest doses (0.1 and 1 mg per mouse per d) significantly inhibited the inflammatory response. Surprisingly, higher doses ofLF (5 and 25 mg per mouse per d) failed to influence the inflammatory reaction. A combination of both nutrients (LF 0.1 mg per mouseper d + gly 20 or 50 mg per mouse per d) inhibited the zymosan-induced ear swelling synergistically (Figure). Additionally, an enhancingeffect of both components was seen on the number of TNFa-producing spleen cells.

0 2 4 6-10

20

50

80

110

Control

Gly 20

LF 0.1

23 24 25

Gly/LF

Time (hr)

Sw

ellin

g (%

of c

ontr

ol a

t 6hr

)

Figure. The effect of glycine, bovine LF and a combination of gly + LF on zymosan-induced ear inflammation. Vehicle (control), gly (20 mg per mouse per d), bovineLF (0.1 mg per mouse per d) and gly + LF (Gly/LF) were orally administered for 3 d, at day 2 the ears were injected with zymosan. Ear thickness was measured before and 3, 6and 24 h after the injection. The swelling of the ears of the vehicle-treated animals at 6 h (generally the maximal swelling) was set to 100%. Results are shown as means withtheir standard errors represented by vetical bars for six mice.

The present findings show anti-inflammatory activity of gly and LF using the zymosan-induced inflammation model. Moreover, acombination of both components demonstrated a synergistic effect on inflammation of the skin and an enhancing effect on the number ofTNFa-producing spleen cells.

1. Zhong Z, Wheeler MD, Li X et al. (2003) Curr Opin Clin Nutr Metab Care 6, 229–240.2. Legrand D, Elass E, Pierce A & Mazurier J (2004) Biometals 17, 225–229.

Proceedings of the Nutrition Society (2008), 67 (OCE), E14 doi:10.1017/S002966510800623X


Interactions between dendritic cells, probiotic bacteria andthe adipokine leptin

S. C. Knight1, N. R. English1 and A. I. F. Blakemore2

1Antigen Presentation Research Group, Imperial College London, Northwick Park & St Mark’s Campus, Watford Road,

Harrow HA1 3UJ, UK and 2Genomic Medicine Group, Imperial College London, Hammersmith Campus,

Du Cane Road, London W12 0NN, UK

Perinodal adipose tissue (PAT) has specialised properties; functional dendritic antigen-presenting cells (DC) are abundant in PAT but rarein adipose tissues distant from nodes(1), and in human subjects, as previously reported in rodents, PAT normally has tissue-specificproperties, being high in PUFA(2). Several properties of PAT are altered in the inflammatory bowel disease (IBD) Crohn’s disease. It hasbeen shown first that DC in PAT lose MHC Class II expression and capacity to stimulate T-cells(1) and second that site-specific changes infatty acids are lost or altered in Crohn’s disease(2). Finally, hypertrophy of adipose tissue and increases in adipokines including leptinoccur in Crohn’s disease(2).

DC are found between gut epithelial cells with veils protruding into the lumen making direct contact with commensal bacteria. Gut DCcan then migrate via afferent lymphatics, entering lymph nodes via a perinodal sinus immediately below and connected with PAT.Potential anti-inflammatory benefits of probiotic bacteria include the capacity to affect the function of DC. Thus, bacterial components candecrease production of IL-12 and promote production of IL-10 in DC(3). By contrast, leptin, in addition to its widely-accepted role inenergy homeostasis and reproduction, acts as a pro-inflammatory cytokine promoting development of DC. In the present study, therefore,effects of probiotic bacteria on the potential of DC to respond to stimulation with leptin have been investigated by examining theirexpression of leptin receptors.

There are multiple leptin receptor splice forms, including a trans-membrane form that signals via the JAK/STAT pathway, and shorterforms of receptor(4). DC were derived from blood monocytes and effects of probiotic bacteria on gene expression were examined fordifferent forms of receptor. DC expressed both long and short forms of leptin receptor but levels of expression varied in DC from differentindividuals. Probiotic bacteria reduced relative expression of the long signalling form of receptor (Figure). Parallel studies of formation ofintracellular cytokine production showed increases in IL-10 and additional effects of probiotic bacteria on fat uptake into lipid bodiesin DC.

C P R O C P R O0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

1 6 0

Rel

ativ

ege

neex

pres

sion

Short Longisoform isoform

Figure. Dendritic cells were grown from peripheral blood CD14 + monocytes for 7 d with granulocyte–macrophage colony-stimulating factor and IL-4. Over the last 2 d somewere exposed to a mixture of two probiotic bifidobacteria (B. longum and B. breve). Gene expression of the long and short forms of leptin receptor was measured by PCR inrelation to the housekeeping gene ubcH5B. DC expressed both long and short forms of receptor. The probiotic bacteria (PRO) caused preferential down-regulation of the longsignalling form of receptor. Values are means with their standard errors represented by vertical bars.

In conclusion, probiotic bacteria caused multiple changes in DC promoting anti-inflammatory effects; selected probiotic bacteria mightprove a beneficial therapy for IBD via modulating DC.

1. Bedford PA, TodorovicV, Westcott EDA, Windsor ACJ, English NR, Al-Hassi HO, Raju KS, Mills S & Knight SC (2006) J Leukoc Biol 80, 546–554.2. Westcott E, Windsor A, Mattacks C, Pond C & Knight S (2005) Inflamm Bowel Dis 11, 820–827.3. Hart AL, Lammers K, Brigidi P, Vitali B, Rizzello F, Gionchetti P, Campieri M, Kamm MA, Knight SC & Stagg AJ (2004) Gut 53, 1602–1609.4. Quinton ND, Laird SM, Tuckerman EM, Cork BA, Li TC & Blakemore AIF (2003) Am J Reprod Immunol 50, 224–231.



A new n-3 PUFA-enriched formula for enteral nutrition modulatesoxidative LDL and inflammatory cytokines

J. Olza1, R. Moreno2, C. M. Aguilera1, A. Perez de la Cruz2, A. Gil1 and M. D. Mesa1,1Institute of Nutrition and Food Technology, Dept of Biochemistry and Molecular Biology II, University of Granada,

Granada, Spain and 2 Universitary Hospital Virgen de las Nieves, Unit of Clinical Nutrition and Dietetic, Granada, Spain

Aging is associated with chronic low-grade increases in circulating levels of inflammatory molecules. A wide range of factors includingobesity, metabolic syndrome, CVD, infection, smoking, genetic and declining function of sex hormones may contribute to the systemiclow-grade increase in inflammatory activity in the elderly. Circulating TNFa is a good predictor of mortality in the frail elderly populationand the oxidation of LDL can be a consequence of the secretion of cytokines and adhesion molecules during the inflammatory process.Dietary interventions may be good strategies to decrease pro-inflammatory activity and improve human health. It is well known that long-chain n-3 PUFA decrease the production of inflammatory cytokines. The aim of the present study was to compare the effect of T-DietPlus1 v. a standard control diet on plasma concentrations of inflammatory markers and oxidized LDL (ox-LDL) in elderly patients feedtotal enteral nutrition (TEN) for 6 months.

Sixty-five patients aged 75 years feed TEN were divided into two groups (experimental and reference). The experimental group (n 32)was fed a new enteral formula, T-Diet Plus1 (Vegenat SA), that contained (mg/l) 75 EPA and 35 DHA. Reference group (n 33) was fed astandard enteral diet (Jevity1, Abbot Laboratories) intended for nutrition in the elderly. At the end of the experimental period only sixteenpatients from the T-Diet Plus1 group and twenty from the reference group remained in the study. The daily intake was 5459 (SE 130) kJ,with no difference between groups. Cytokines (basal, 3 months and 6 months) were measured using a human serum adipokine (panel B)kit (LINCOplexTM; Linco Research, St Charles, MO, USA) with the Luminex 200 System built on XMAP technology. Ox-LDL wasdetermined using an ELISA kit (Biomedica Medizinprodukte GmbH & Co KG, Vienna, Austria) and the ultra-sensitive C-reactive protein(PCRus) with a turbidimetric immunoassay (Dade Behring Inc., Deerfield, IL, USA). Non-parametric tests were used for statisticalanalysis: Mann-Witney test was performed to evaluate differences between independent groups (P £0.05); Wilcoxon test was used todetermine the effect of each diet after 3 or 6 months (mean values with unlike superscript letters were significantly different: P £0.05).

TNF-α

0

2

4

6

8

10

0 months 3 months 6 months

pg

/ml

T-Diet Reference

ox-LDL

0

1000

2000

3000

4000

0 months 3 months 6 months

ng

/ml

T-Diet Reference

b

bb

ab aa

T-Diet1 Reference

Initial 3 months 6 months Initial 3 months 6 months

Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

PCRus (pg/ml) 1.87 0.35 1.91 0.56 0.90 0.25 2.13 0.42 1.94 0.55 1.88 0.55IL-6 (pg/ml) 47.7 10.6 44.2 10.6 34.2 13.9 41.3 11.6 59.6 18.9 42.9 17.2IL-8 (pg/ml) 9.24 1.44 9.24 1.80 10.55 4.13 8.69 1.69 7.83 1.22 6.91 1.45

The results demonstrate that ox-LDL decreased after 6 months of monitored nutrition in both groups (P £0.05), with no differencesbetween groups. No differences were found in other inflammatory markers, although both TNFa and IL-6 showed a tendency to decreaseafter 6 months, which is important considering that the patients were frail elderly with different pathologies.

This study was financed by Vegenat SA.



Conjugated linoleic acid feeding during rat suckling period enhancesintestinal IgA production

F. J. Perez-Cano, M. Molero, C. Ramırez-Santana, M. Castell, C. Castellote and A. FranchDepartment of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Conjugated linoleic acid (CLA) is a mixture of isomers of linoleic acid, of which cis-9, trans-11 and trans-10, cis-12 are the majorconstituents. These compounds induce beneficial effects on health and it has been suggested that they may modulate immune responses.However, as in man, mucosal Ig production is poorly developed during the suckling period in rats. The aim of the present work was toestablish the effect of CLA on mucosal immunity during suckling by determining IgA levels in intestinal washes from 28-d-old Wistarrats by an ELISA technique. IgA and PPAR-g (as a possible mediator of the CLA effect) mRNA expression in small intestine and colonfrom 21- and 28-d-old Wistar rats was also evaluated by real-time PCR. Pregnant Wistar rats were obtained on day 7 of gestation. On theday of birth pups were divided into eight different groups. Four groups were analysed on day 21: litters from mothers fed with CLA (80%cis-9, trans-11, 20% trans-10, cis-12; Lipid Nutrition B. V. Wormerveer, The Netherlands;10 g/kg pellet chow) during gestation andlactation (A); CLA only during gestation but litters received a CLA isomer mixture by daily oral supplementation during suckling (B);exclusively receiving CLA by oral administration during the suckling period (C); a parallel-age reference group. Another four groups werestudied at 28 d old: litters receiving CLA from day 1 to 28 (D); from day 1 to 21 (X); from weaning to day 28 (E); a parallel-age referencegroup (Z). On days 21 and 28 rats were killed and samples from the small intestine and colon were removed for RNA extraction for laterreal-time PCR. Taqman1 specific probes and primers were used for each gene (Applied Biosystems, Foster City, CA, USA). Target geneexpression was normalised by GADH and b-actin endogenous controls in each sample. In all cases statistical analysis was performed byconventional ANOVA, and when the experimental group variable had a significant effect on the dependent variable post hoc comparisons(LSD test) were performed. Differences were considered to be significant at P<0.05. The results provided evidence that mucosal pro-duction of IgA increased in animals supplemented with CLA during suckling by increasing IgA mRNA expression in the small intestineand colon and IgA protein in 28-d-old rats (see Table). PPARg gene expression was also up regulated in animals receiving CLA duringearly life. In conclusion, dietary supplementation with CLA during suckling enhances the development of the immune system in Wistarrats. Moreover, the effect of CLA supplementation is better when supplementation is given earlier and for longer.

Age Group

IgA expression (%) (normalized by b-actin)

Small intestine Colon

Mean SE Mean SE

Day 21 A 149.1 28.15 136.1 52.94B 73.81 29.32 114.9 29.19C 179.6 80.69 206.6 62.10W 100.0 39.27 100.0 20.25

Day 28 D 545.8* 190.4 372.5* 122.2X 15.91 5.04 40.32 10.82E 49.85 23.22 79.30 15.75Z 100.1 19.53 99.85 28.24

Mean values were significantly different from those for groups E, X and Z (n 4–5): *P<0.05.



Detection of biological activities promoted by proteins from supernatantfractions of Lactobacillus plantarum in a promyelocytic cell line

E. Puertollano1, M. A. Puertollano2, L. Cruz-Chamorro1, G. Alvarez de Cienfuegos1

and M. A. de Pablo1

1Area de Microbiologıa, Universidad de Jaen, Jaen, Spain and 2Instituto del Frıo (CSIC), Madrid, Spain

Recently, probiotics have acquired considerable importance because of their beneficial properties in relation to human health(1). It is nowgenerally assumed that probiotics are capable of participating in the prevention and treatment of intestinal disorders, in the modificationof ‘immune response and in the reduction of incidence of cancer(2). In fact, lactic acid bacteria or soluble compounds synthesized by thebacteria may interact with tumour cells in culture inhibiting their growth(3). The present study examined the effects promoted by proteinsfrom supernatant fractions of Lactobacillus plantarum on HL-60 cells (a promyelocytic cell line). Proteins were concentrated by cen-trifugation (Centricon plus-20; Millipore, Madrid, Spain). After the culture of HL-60 cells for 24 h in the presence of different con-centrations (5, 50 and 100mg/ml) of protein from L. plantarum, cell viability was quantified by MTT assay, lactate dehydrogenase (LDH)release, NO production, reactive oxygen species (ROS) generation and caspase-3 activity. In addition, the haemolytic activity of super-natant fractions was determined. Cell viability was also determined visually by double staining with Hoechst 33342 and propidium iodidedyes. Results indicated that proteins from L. plantarum produced a cytotoxic effect on this cell line, as well as a loss of cell viability afterthe treatment. Similarly, a significant haemolytic effect was detected. The measurement of NO production, ROS generation and caspase-3activity showed that proteins from L. plantarum did not promote apoptosis in HL-60 cells. In conclusion, it is suggested that proteins fromL. plantarum supernatant fractions exert necrotic activity rather than an apoptotic action on HL-60 cells.

Fig. 1. Measurement of cytotoxicity by LDHrelease. Values, expressed as a percentage of cyto-toxicity, are means with their standard errors repre-sented by vertical bars. Mean values weresignificantly different from the control (CT) value:*P<0.05.

Fig. 2. Measurement of caspase-3 activity. HL-60cells were cultured in the presence (K) or absence( ) of caspase-3 inhibitor. The values, expressed asrelative units of fluorescence, are means with theirstandard errors represented by vertical bars. Meanvalues were significantly different from the control(CT) value: *P<0.05.

Fig. 3. Haemolytic activity of cell-free supernatantfractions from L. plantarum. PBS and 0.1 % (v/v) TritonX-100 were used as negative and positive controlsrespectively. The values, expressed as a percentage ofhaemolysis, are means with their standard errors repre-sented by vertical bars. Mean values were significantlydifferent from the control (CT) value: *P<0.05.

1. Lilly DM & Stillwell RH (1965) Science 147, 747–748.2. Cross ML (2002) FEMS Immunol Med Microbiol 34, 245–253.3. Hirayama K & Rafter J (2000) Microbes Infect 2, 681–686.



Oral health and nutritional biochemical variables in adult patientswith AIDS

N. Slobodianik1, P. Perris1, A. Squassi2,3, G. Sanchez2,3, M. S. Feliu1 and N. Bordoni31Department of Nutrition, School of Pharmacy and Biochemistry, 2Oral Care Clinic for High Risk Patients (CLAPAR I)

and 3School of Dentistry, University of Buenos Aires, Argentina

High prevalence of oral disease has been observed in patients with AIDS(1–3). The aim of this preliminary study was to analyse oral healthand its relationship with nutritional biochemical variables in a group of adult patients with AIDS. Twenty-eight patients between 25 and50 years old who were HIV + were included. Dental status, gingival status and presence of mucosal alterations were determined. TheDMFT index (decayed, missed and filled teeth) and its components (gingival index, bleeding sites and the presence of erosion on softtissues) were calculated and evaluated(4). Samples of whole blood and non-stimulated saliva were collected from fasting patients andserum transthyretin (TTR), C3c fraction (C3c) and total saliva IgA levels were determined by quantitative radial immunodiffusion onlayers (The Binding Site, Birmingham, UK and Diffuplate; Biocientifica, Buenos Aires, Argentina)(5–7). The results were (mg/l): IgA82 (SD 41); TTR 271 (SD 177); C3c 828 (SD 380). When results were compared with reference values (227 (SD 74), 337 (SD 92) and1350 (SD 270) respectively) a reduction in levels was observed. Moreover, also observed were: (a) a negative correlation between the Dcomponent of DMFT and the number of bleeding sites and TTR levels (r - 0.68, P<0.001); (b) a positive correlation between thepresence of lesions on soft tissues and the concentration of total IgA in saliva (r 0.84, P<0.001). No correlation between gingival indexand biochemical variables was observed. The results suggest a compromised nutritional status concomitant with alterations in oral healthin the study group.

R2 = 0,4591

0

2

4

6

8

10

12

14

16

0 50 100 150 200 250C3c (mg/dL)

CD

R2 = 0,7103

0

2

4

6

8

0 5 10 15 20

Ig A (mg/dl)

Lessions onsoft tissues

This work was partially supported by the University of Buenos Aires (Grants B060 and O750).

1. MacPhail LA & Greenspan JS (1997) Oral Dis 3, Suppl. 1, S190–S193.2. Aguirre-Urizar JM, Echebarria-Goicouria MA & Eguia del Valle A (2004) Med Oral Patol Oral Cir Bucal 9, Suppl., 148–157.3. Fidel PL (2006) Adv Dent Res 19, 80–84.4. Loe H (1967) J Periodontol 38, 610–618.5. Mancini G, Carbonara AO & Heremans GF (1965) Immunochemistry 2, 235–254.6. Feliu MS & Slobodianik N (1993) Acta Bioquim Clin Latinoam xxvii, 519–526.7. Slobodianik N & Feliu MS (1992) Acta Bioquim Clin Latinoam xxvi, 101–102.



Gut microbes and obesity in adolescents

M. Sotos1, I. Nadal1, A. Marti2*, A. Martınez2*, M. Martin-Matillas3*, C. Campoy3*,M. A. Puertollano4*, J. Warnberg4*, A. Marcos4* and Y. Sanz1 (*on behalf of the

EVASYON study group)1Instituto de Agroquımica y Tecnologıa de Alimentos (CSIC), Valencia, Spain, 2Dpto Fisiologıa y Nutricion,

Universidad de Navarra, Navarra, Spain, 3Dpto Pediatria, Universidad de Granada, Granada, Spain and4Grupo Inmunonutricion, Dpto Metabolismo y Nutricion, Instituto del Frio (CSIC), Madrid, Spain

The composition of the gut microbiota has been associated with obesity in animal models and considered to be a potential environmentalfactor involved in this disorder(1). In the present preliminary study the composition of the faecal microbiota of obese and overweightadolescents (aged 14.8 (SD 1.3) years) was investigated initially and after following a nutritional intervention strategy based on an energy-restricted diet (30–40%) and a physical activity programme (energy expenditure 3762–11 286 kJ/week) for 3 months. Eight obese andoverweight individuals were identified according to the International Obesity Task Force criteria(2) in the frame of the EVASYON project.The microbiological analyses were carried out by fluorescent in situ hybridization, using oligonucleotide probes targeting the mainmicrobial groups colonizing the human distal gut (Archaea, Bacteroides, Bifidobacterium, Clostridium coccoides–Eubacterium rectale,Clostridium leptum, Enterobacteriaceae, Enterococcus, Fusobacterium prausnitzii, Lactobacillus, Roseburia–Eubacterium, Ruminococcusand sulphate-reducing bacteria). Enterobacteriaceae and sulphate-reducing bacterial counts were significantly reduced (P<0.05) in faecalsamples of individuals (n 5) showing remarkable reductions in their weight (4–7 kg) but not in those (n 3) showing minor weight losses(<2 kg) after the intervention. In contrast, this last group of children showed significantly lower (P<0.05) counts of Roseburia–Eubac-terium populations. These gut microbes could play a role in obesity by affecting either the energy harvest from the diet(3) or the signallingpathways that link inflammation with obesity(4). Overall, the present preliminary study shows that modifications in the gut microecologyare associated with corporal weight in adolescents under a similar energy-restricted diet. Investigations are in progress to confirm thistrend and assess whether the intentional manipulation of the gut microbiota could be envisaged as a strategy to combat obesity andimmune disorders resulting from obesity.

Table 1. Microbial composition of faeces from overweight and obese adolescents

Weight reduction . . . 4–7 kg <2 kg

Microbial group

Initial 3 months Initial 3 months

No. of cells/g faeces (·108) No. of cells/g faeces (·108)

Mean SE Mean SE Mean SE Mean SE

Enterobacteriaceae 9.61 1.23 4.95* 0.96 7.44 2.97 6.78 1.28Roseburia-Eubacterium 16.5 10.23 9.88 5.76 21.3 4.39 12.4* 5.96Sulphate-reducing bacteria 3.53 3.24 1.09* 0.58 3.21 2.49 1.62 0.37

Mean values were significantly different from initial values (Student’s t test): *P< 0.05.

The EVASYON study was supported by grants from FIS (Spanish Ministry of Health) and this work was also supported by grant AGL2005–05788-C02–01from the Spanish Ministry of Science and Education.

1. Ley RE, Backhed F, Turnbaugh P et al. (2005) Proc Natl Acad Sci USA 102, 11070–11075.2. Cole TJ, Belizzy MC, Flegal KM et al. (2000) Br Med J 320, 1–6.3. Turnbaugh PJ, Ley RE & Mahowald MA et al. (2006) Nature 444, 1027–1031.4. Bleau C, Lamontagne L & Savard R (2005) Clin Exp Immuno 140, 427–435.



PUFA in the pathogenesis and treatment of patients withmultiple sclerosis

L. S. Harbige1,2, E. Pinto1, M. Xiang1, M. Leach1,2 and M. K. Sharief3

1Centre for Biosciences Research, School of Science, University of Greenwich, Kent, UK, 2Medway School of Pharmacy,

Universities of Kent and Greenwich, Kent, UK and 3Department of Neurology, King’s, Guy’s and St Thomas’ Hospital,

London, UK

Multiple sclerosis (MS) is a central nervous system-specific demyelinating disease, and most patients demonstrate a relapse–remittingform of the disease characterised by attacks (relapses) and periods of recovery (remission). Although the aetiology of MS remainsunknown, much evidence suggests the presence of autoimmune mechanisms in the disease pathogenesis. Cytokine dysregulation has beenfound in peripheral blood mononuclear cells (PBMC) during the relapse–remitting phases of MS, i.e. raised TNFa and IL-1b and lack ofregulatory transforming growth factor b (TGFb) production(1), and in an animal model of MS a protective effect of g-linolenic acid (GLA;18: 3n-6)-rich borage oil associated with raised TGFb(2). The aim of the present study was to determine the effects of a selected GLA-richoil (BGC20-884) at two doses (a low dose (5 g/d) and a high dose (14 g/d)) and a placebo control (PEGA) on the clinical course andPBMC cytokine and membrane fatty acid profiles of thirty-six patients with active MS in a randomized double-blind placebo-controlledtrial format over 18 months. Patients were diagnosed and assessed using international criteria for MS. Relapse rate and expanded disabilitystatus scale (EDSS) were assessed every 3 months and blood taken and PBMC isolated for cytokine studies and membrane fatty acids.

High-dose BGC20-884 treatment markedly and significantly reduced the annualized mean of relapses per patient per year over 18months (Figure) and disability progression (EDSS) compared with the placebo control and low-dose BGC20-884 treatment. PBMCcytokine changes ran parallel with the clinical findings, i.e. high-dose BGC20-884 showed no changes in TGFb :TNFa and TGFb : IL-1band no changes in membrane fatty acids, whilst the placebo control and low-dose BGC20-884 group showed significant decreases inTGFb :TNFa and TGFb : IL-1b and loss of n-6 fatty acids, particularly linoleic acid (18: 2n-6) and arachidonic acid (20: 4n-6), over time.It is concluded that high-dose BGC20-884 markedly affects the clinical course of MS. Furthermore, the EDSS improvement in the high-dose group suggests there maybe a beneficial effect on neuronal lipids and neural function in MS. The present study supports thehypothesis of dysregulation of fatty acid metabolism and cytokines in MS(1,2).

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1. Hollifield, R, Harbige L, Pham-Dinh D & Sharief MK (2003) Autoimmunity 36, 133–144.2. Harbige L, Layward M, Morris-Downes M, Dumonde D & Amor A (2000) Clin Exp Immunol 122, 445–452.



Potentiation of systemic humoral immune response in suckling rats byconjugated linoleic acid (CLA)

C. Ramırez-Santana, F. J. Perez-Cano, M. Castell, C. Castellote and A. FranchDepartment of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Cis-9, trans-11 (c9, t11) and trans-10, cis-12 (t10, c12) are the predominating molecules in the positional and geometrical isomers mixturetermed CLA. Although CLA has shown positive effects on human health and seems to be associated with immunomodulatory activities,its effect in the developing immune system has not been studied. Thus, the present study was designed to establish the effect of CLAsupplementation during gestation and/or lactation on humoral immune response, i.e. by analysing sera Ig levels during the suckling period.Wistar rats were allocated to four groups (A, B, C and W) on day 7 of pregnancy. From day 7 and throughout the study period group Cand W gestating mothers were fed standard pellet chow. Group A and B dams were fed 10 g CLA (80% cis-9, trans-11, 20% trans-10,cis-12; Lipid Nutrition B. V. Wormerveer, The Netherlands)/kg pellet chow during gestation. Moreover, group A mothers were also fedthe CLA-supplemented chow until the litters were 21 d old, the end of suckling period. Group B and C litters received the CLA mixture ofisomers by daily oral administration while their respective dams were fed standard pellet chow during lactation. In all cases litters wereequalised to ten rats per lactating mother. Pups from each experimental group were killed at the end of the second week of life (day 14)and at the end of the suckling period (day 21), and blood samples were collected. Serum IgA, IgG and IgM levels were quantified by theELISA sandwich technique. ANOVA and post hoc comparisons (LSD test) were performed. Differences were considered to be significantat P<0.05. Animals receiving CLA passively from their mothers (group A) during gestation and the suckling period exhibited the highestconcentrations of IgG and IgM at 14 d old (P<0.05; see Table). At the end of suckling period the serum IgG concentration in this groupwas also increased, up to three times more than in the other groups (P<0.05). Those animals supplemented with CLA only duringsuckling period (group C) showed no difference in relation to those receiving no supplement. Thus, these results demonstrate that CLAsupplementation during gestation and lactation promotes systemic humoral immune response.

Age Group

Serum IgG (mg/ml) Serum IgM (mg/ml)

Mean SE Mean SE

Day 14 A 11.5†‡ 0.84 79.8†‡ 4.67C 2.09 0.34 64.4 2.56W 1.71 0.30 59.5 4.19

Day 21 A 22.1*†‡ 2.0 88.1 7.61B 7.73 0.7 100.7 12.0C 3.94 0.28 98.7 6.70W 4.79 0.21 94.8 3.58

n 10. Mean values were significantly different from those for group B: *P<0.05. Mean values were significantly differentfrom those for group C: †P<0.05. Mean values were significantly different from those for group W: ‡P<0.05.



Effects of a diet with polyphenol-rich cereal supplementation on thefunction and redox state of peritoneal leucocytes from mice: differencesbetween a short (5 weeks) and long (20 weeks) period of supplementation

P. Alvarez, C. Alvarado, L. Jimenez and M. de la FuenteDepartment of Animal Physiology, Faculty of Biology, Complutense University of Madrid, 28040 Madrid, Spain

Although intake of natural products is a matter of great importance in relation to health, little attention is focused on the key role of cerealpolyphenols in this respect. It has been shown that many of these compounds, especially as a mixture, exert a high antioxidant activity andsynergistic effects. However, the protective role of a short-term and long-term intake of polyphenol-rich cereals on immune functionand cellular redox state is still poorly understood. Thus, the aim was to investigate the effects of 5 and 20 weeks of supplementation(200 g/kg) with four different cereal fractions on a population of female CD1 mice. Control animals received the standard diet withoutsupplementation. Several indices of the function and redox state of peritoneal leucocytes were evaluated. The cereals, designated B (wheatgerm), C (buckwheat flour), D (fine rice bran) and E (wheat middlings), contained a complex mixture of gallic acid, p-hydroxybenzoicacid, vanillic acid, sinapic acid, p-coumaric acid, ferulic acid, quercetin, catechin, rutin and oryzanol as the major polyphenolic com-pounds. In general, the polyphenol-rich cereals improved the immune function (by increasing chemotaxis capacity, lymphoproliferativeresponse to mitogens and IL-2 levels) and the redox state (by increasing catalase antioxidant activity and decreasing oxidised glutathio-ne:reduced glutathione, TNFa and malondialdehyde levels) of peritoneal leucocytes from mice. The control animals from the 20 weekssupplementation study (middle-age) showed typical age-related changes in their leucocyte functions and redox state. In several casesthe antioxidant supplement administration for 20 weeks to middle-aged mice restored values to those of the adult controls. Similar effectsof the cereal fractions were observed, but these effects were quantitatively more marked following long-term (20 weeks) compared withshort-term (5 weeks) supplementation.

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Figure. Catalase activity in peritoneal leucocytes from mice supplemented for 5 weeks and 20 weeks with polyphenol-rich cereal fractions (B, C, D or E). Values are means andstandard deviations represented by vertical bars for eight (5 weeks) and seven (20 weeks) animals. Mean values were significantly different from those for the correspondingcontrol group: *P<0.05, **P<0.01, ***P<0.001. Mean values were significantly different from those for the adult control group: ††P<0.01, †††P<0.001.

In conclusion, the present results suggest that polyphenol-rich cereal supplementation, especially designed for long-term intake, may bean important tool for optimizing the immune function and the redox state of leucocytes. Further studies are needed to determine theoptimal doses and to establish which are the key compounds involved in mediating the observed beneficial effects before their regularconsumption can be recommended.

This work was supported by MEC (BFU 2005–06777), RETICEF (RD06/0013/0003) (ISCIII) of Spain and DANONE VITAPOLE (France).



Structure and antigenicity changes in 7S soyabean allergen byenzymic deglycosylation

M. Amigo-Benavent, M. Villamiel and M. D. del CastilloInstituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain

Soyabean allergy is one of the most common food allergies. The marked rise in allergenic reactions to soyabean is related to theincreasing use of soya products in processed foods, which convert it in a major public health and soyabean industry concern. Differentprocesses have been used to mask soyabean allergenicity, including heat treatment, fermentation, enzymic proteolysis, carbohydrateconjugation, genetic modification, extrusion and agronomic practices(1). It is still unclear which of the components of soyabean causeallergenic reactions. Some authors have identified 7S as a major soybean allergen(2); 7S is a N-glycoprotein that constitutes about 25% ofthe total soyabean proteins(3). Glycans forming glycoproteins have been described as being responsible for the allergenicity of some plantsand insects(4) and their removal has been proposed to reduce allergenicity(5). PNGase F is an enzyme used to deglycosylate different N-glycans such as those present in ribonuclease B, among others. No studies focused on deglycosylation of 7S by PNGase F activity forreducing its allergenicity have been found in the literature. The aim of the present research was to assess enzymic deglycosylation asa strategy for reducing soyabean protein allergenicity.

7S was isolated by isoelectric precipitation and the protein purity checked by SDS–PAGE. Enzymic deglycosylation was carried out onthe isolated 7S (1 mg/ml) with PNGase F at pH 8 and 37�C for 24 h. Deglycosylation was followed by reversed-phase HPLC, SDS–PAGEand capillary zone electrophoresis analyses, which provided information relating to changes in protein structure. Antigenicity was assayedby immunoblotting and indirect ELISA with polyclonal rabbit anti-soyabean sera and horseradish peroxidase-labelled goat anti-rabbit IgG.

The Figure shows SDS–PAGE (A) and immunoblotting (B), corresponding to the 7S control (c) and its deglycosylated form (d). As canbe observed, the electrophoretic mobility of 7S changed as a result of deglycosylation, indicating a decrease in protein molecular mass.Immunoblotting and ELISA data suggest that the carbohydrate moiety forming 7S does not have a key role in its in vitro antigenicresponse as assayed in the present study. Further studies should be conducted in order to confirm the results by employing digestion andabsorption assays in vitro and in vivo as well as an immunological test using serum from patients allergic to soyabean.

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These studies were supported by projects AGL2004-05031 and ALIBIRD-CM-S0505/AGR-0153.

1. Wilson S, Blaschek K & de Mejia E (2005) Nut Rev 63, 47–58.2. Breiteneder H & Radauer C (2004) J Allergy Clin Immunol 113, 821–830.3. Garcıa MC, Torre M, Marina ML & Laborda F (1997) Crit Rev Food Sci Nut 37, 361–391.4. Fotisch K & Vieths S (2001) Glycoconj J 18, 373–390.5. Ahrazem O, MD, Lopez-Torrejon G, Sanchez-Monge R, Sastre J Lombardero M, Barber D & Salcedo G (2006) Int Arch Allergy Immunol 139, 96–103.



Effects of IL-6 on amylase secretion and calcium signalling in pancreaticAR42J cells: modulation by membrane fatty acid composition

N. Audi1, M. A. Martınez1, M. D. Mesa2, E. Martınez-Victoria1, M. Manas1 and M. D. Yago1

1Institute of Nutrition and Food Technology, Departments of Physiology and 2Biochemistry and Molecular Biology,

University of Granada, Spain

Oleic acid is a typical component of the Mediterranean diet. The type of dietary fat strongly influences the fatty acid (FA) composition ofrat pancreatic cell membranes, and this effect is associated with changes in the functionality of viable pancreatic acini(1,2). The AR42J cellline is a useful tool for assessing the effect of membrane compositional changes on acinar cell function(3). The aim was to study the effectsof chronic treatment with IL-6 (400 pM for 48 h) on amylase secretion and Ca2 + homeostasis in AR42J cells, and to establish whetherthese effects are influenced by different membrane FA composition.

The membrane FA composition of AR42J cells was modified by growing them in medium enriched with oleic or linoleic acid, asdescribed previously(3). Amylase activity was determined and expressed as a percentage of the initial total amylase content that wasreleased into the extracellular medium during incubation. Ca2 + mobilization (expressed as F340:F380) was determined by epifluorescencemicroscopy. Cells were loaded with the fluorescent ratiometric Ca2 + indicator fura-2. For quantification of fluorescence, cells werealternately excited at 340 and 380 nm using a high-speed monochromator. ANOVA was performed to compare amylase secretion betweengroups treated with or without IL-6. The Ca2 + decay constant for each group was calculated and mean values were comparedby ANOVA.

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Figure. Amylase release (% total content) evoked by different concentrations of cholecystokinin octapeptide (CCK-8). Values are means with their standard errors representedby vertical bars (n 16 for all groups). Mean value for the oleic acid group was significantly different from that for the oleic + IL-6 group: *P<0.05. Mean values for the linoleicacid group were significantly different from those for the linoleic acid + IL-6 group: †P<0.05.

Table. Ca2+ response evoked by perfusion with CCK-8 (1 nm) in AR42J cells with different membrane FA profiles treated for 48 h with IL-6 or vehicle

Peaks (340:380 intensity) Ca2+ decay constants (/s)

Mean SE Mean SE

Oleic acid (n 44) 2.2070 0.0861 0.0034* 0.0002Oleic acid + IL-6 (n 39) 2.1297 0.1037 0.0028 0.0002Linoleic acid (n 42) 2.1930 0.1112 0.0029 0.0003Linoleic acid + IL-6 (n 29) 2.1457 0.0978 0.0032 0.0002

n, No. of cells measured in each group. Mean value for the oleic acid group was significantly different from that for the oleic + IL-6 group: *P<0.05.

Membranes rich in oleic acid were not affected by the action of IL-6 at different concentrations of CCK-8, while membranes rich inlinoleic acid were more sensitive to the effects of IL-6 in relation to basal and stimulated amylase secretion. Dietary fat and IL-6 did notaffect Ca2 + peaks elicited by 1 nM-CCK-8. It would be useful to know whether the consumption of olive oil (rich in oleic acid) canprevent and/or attenuate the effects of pancreatic inflammatory processes compared with sunflower oil (rich in linoleic acid).

1. Martinez MA, Lajas AI, Yago MD et al. (2004) Nutrition 20, 536–541.2. Yago MD, Diaz RJ, Ramirez R, Martinez MA, Manas M & Martinez-Victoria E (2004) Br J Nutr 9, 1227–1234.3. Audi N, Mesa MD, Martinez MA, Martinez-Victoria E, Manas M & Yago MD (2007) Exp Biol Med 232, 532–541.



Effect of oil (sunflower oil) consumption with added hydroxytyrosol(natural antioxidant) on antioxidant variables in leucocytes

from healthy adults

I. Baeza1, N. M. de Castro1, L. E. Dıaz2, A. Marcos2 and M. de la Fuente1

1Departamento de Fisiologıa Animal, Facultad de Ciencias Biologicas, Universidad Complutense de Madrid, Spain and2Grupo Inmunonutricion, Departamento de Metabolismo y Nutricion, Consejo Superior de Investigaciones Cientıficas,

Madrid, Spain

Hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), also known as dihydroxyphenylethanol, is a natural phenolic antioxidant(1,2) found inolives and olive oil that is responsible for their antioxidant properties(3). Although HT is known to exert an antioxidant effect, themechanism of its action and the identity of the reactive oxygen molecule(s) targeted are not known(4). The aim of the present study was toevaluate the effect of oil (sunflower oil) consumption with ‘Oleoactive from Koipesol’ (Sos Cuetara SA, Madrid, Spain), which containsadded HT and is consumed at the level of 45–50 mg/d, on two antioxidant variables (total glutathione levels and glutathione peroxidase(GPx) activity) in leucocytes (neutrophils and lymphocytes) from healthy adults. The design was a randomized cross-over study withtwelve healthy subjects of both gender (20–45 years of age). The subjects were divided into two groups. Group A (n 6): 3 weeks of oil(sunflower oil) with added HT; 2 weeks of wash-out; 3 weeks of oil (sunflower oil) without added HT. Group B (n 6): 3 weeks of oil(sunflower oil) without added HT; 2 weeks of wash-out; 3 weeks of oil (sunflower oil) with added HT. The antioxidant variables (totalglutathione in lymphocytes and neutrophils and GPx activity in lymphocytes) were analysed before starting oil (sunflower oil) intake andat the three other time-points during the study. In both groups tested the total glutathione levels in lymphocytes did not show significantchanges. Nevertheless, in group A glutathione levels in neutrophils and GPx activity in lymphocytes increased in the subjects after theintake of oil (sunflower oil) with added HT (Figure), however no changes were found in group B. In conclusion, HT could protect againstoxidative damage since it increases the levels and the activity of two very important antioxidants in the immune cells.

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Figure. GPx activity in lymphocytes and total glutathione levels in neutrophils from subjects in group A before (basal) and after 3 weeks of oil (sunflower oil) intake with addedHT. Values are means and standard deviations represented by vertical bars. Mean values were significantly different from basal values: ***P<0.001.

This work has been supported by Sos Cuetara of Spain.

1. Papadopoulos G & Boskou D (1991) J Am Oil Chem Soc 78, 669–671.2. Baldioli M, Servili M, Perretti G & Montedoro GF (1996) J Am Oil Chem Soc 73, 1589–1593.3. Carliccio MA, Siculella L, Ancora MA, Massaro M, Scoditti E, Storelo C, Visioli F, Distante A & De Caterina R (2003) Arteroscler Thromb Vasc Biol

23, 622–629.4. ODowd Y, Driss F, Dang PM, Elbim C, Gougerot-Pocidalo MA, Pasquier C & El-Benna J (2004) Biochem Pharmacol 68, 2003–2008.



Expression of Toll-like receptors on peritoneal macrophages anddendritic cells from old mice treated with soyabean isoflavones

and green tea

I. Baeza, N. M. de Castro, L. Arranz and M. de la FuenteComplutense University of Madrid, Faculty of Biology, Department of Physiology, Madrid, Spain

Cells of the immune system recognize pathogens via Toll-like receptors (TLR), which are the basic signalling receptors of the innateimmune system and direct the course of acquired immunity by recognizing specific microbial products that activate immune cells foreffector functions(1). It is well known that ageing correlates with a decline in immunity, an increase in the inflammatory state and animpaired production of several hormones, such as oestrogens(2). In recent years it has been shown that oestrogens play an essential role inmodulating immune function and pro-inflammatory cell signalling(2,3). Soyabean isoflavones, since they show a structural similarity tooestradiol, are being investigated in order to determine whether they can mimic oestrogen actions(4). Furthermore, the possible effects ofthe green tea plant on human health, because of its content of polyphenolic antioxidants (especially catechins), has been studied withincreasing interest in recent years(5). Since the expression of TLR on cell membranes constitutes the first step in the inflammatorysignalling cascade, the aim of the present study was to determine the role of isoflavones and green tea as modulators of such TLRexpression on peritoneal macrophages and dendritic cells (those that mediate innate immunity) from old mice.

ICR-CD1 female mice aged 16 months were fed a diet supplemented with soyabean or green tea + soyabean (Diviser-Aquilea SL,Barcelona, Spain; 1 mg soyabean per mouse per d; 3.75 mg green tea per mouse per d) for 25 weeks. The control groups were fed standarddiet (A04; Panlab LS, Barcelona, Spain). Then, at the age of 22 months peritoneal leucocytes were obtained from the animals and theexpression of TLR 2 and 4 was determined by flow cytometry in macrophages and dendritic cells.

Previous studies have detected an increased expression of TLR2 and TLR4 with advancing age (L. Arranz, I. Baeza, N. M. De Castro,and M. De la Fuente, unpublished results). In the present experiment the combined diet (soyabean isoflavones + green tea) significantlydecreased the presence of TLR2 and TLR4 on cell membranes in the old animals. In conclusion, adequate treatment with isoflavones andgreen tea could be useful in slowing down the effects of the ageing process through a mechanism of control of the age-related inflam-matory state.

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Figure. Expression of TLR2 and TLR4 on macrophages and dendritic peritoneal cells. Values are means and standard deviations represented by vertical bars for groups of fouranimals. Mean values were significantly different from those for control animals: *P<0.05, **P<0.01, ***P<0.001. Mean values were significantly different from those for thesoyabean group: †P<0.05, ††P<0.01.

This work has been supported by Uriach-Aquilea OTC SL, the MEC (BFU 2005–06777) and RETICEF (RD06/0013/0003) (ISCIII) of Spain.

1. Mukhopadhyay S, Herre J, Brown GD & Gordon S (2004) Immunology 112, 521–530.2. Rehman HU & Masson EA (2005) Gend Med 2, 41–56.3. Lesmeister MJ, Jorgenson LR, Young SL & Misfeldt ML (2005) Reprod Biol Endocrinol 3, 74.4. Gris Martınez JM (2006) Med Clin (Barc) 127, 352–356.5. Friedman M (2007) Mol Nutr Food Res 51, 116–134.



The effects of organic v. conventional diets on immune variables in rats

A. M. Baranska1, E. Rembialkowska2, E. Hallmann2, L. Lueck3, J. M. Cooper3 and C. Leifert31Department of Animal Physiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02–096 Warsaw, Poland,2Division of Organic Foodstuffs, Faculty of Human Nutrition and Consumer Sciences, Warsaw Agricultural University,

Nowoursynowska 159C, 02–776 Warsaw, Poland and 3Nafferton Ecological Farming Group (NEFG), University of

Newcastle, Nafferton Farm, Stocksfield NE43 7XD, UK

Organic food production is gaining increasing interest worldwide. In Europe the regulations for organic crop cultivation prohibit the use ofchemosynthetic pesticides, mineral fertilizers, growth promoters and genetic engineering or GM organisms. Despite a small number ofstudies comparing potential effects of organic and conventional foodstuffs consumption on human and animal health, there are few reportsof in vitro immunosuppressive or no effects of the presence of chemical contaminants (e.g. pesticides) in conventional food(1). The aim ofthe present study was, therefore, to assess the effect of diets, based on organically, low-input and conventionally-grown crops on selectedimmune variables of rats in an experimental feeding trial. Experimental diets were prepared according to the nutritional recommendationsfor rats as pellets containing dried wheat, potatoes, carrots and onions and enriched with lactoalbumin, casein, rapeseed oil, minerals andvitamins, and were characterized by the content of total flavonoids, polyphenols, b-carotene and lutein, pesticides, and antioxidantactivity(2). Adult male and female Wistar rats were kept for 3 weeks under controlled conditions with free access to water and experi-mental feeds (without pesticides and mineral fertilizers, ORG-ORG; without pesticides, ORG-CV; without mineral fertilizers, CV-ORG;with pesticides and mineral fertilizers, CV-CV; control group fed Labofeed (Andrzej Morawski Feed Production Plant, Kcynia, Poland),LF), and paired and bred. Twelve young males from each experimental group were fed each of the diets for 3 and 12 weeks. Animals werekilled with Thiopental overdose, blood was collected from the heart and used for haematological analysis, spleens were isolated asepti-cally and used immediately for in vitro studies of lymphocyte proliferation.

There were no significant differences in haematological variables of rats from all dietary groups. The effect of experimental diets onspontaneous lymphocyte proliferation was similar after 3 and 12 weeks of feeding; however, the differences were significant only after 12weeks (ORG-ORG v. CV-CV, ORG-ORG v. ORG-CV, CV-ORG v. ORG-CV, CV-ORG v. CV-CV; P<0.001). A higher proliferation wasfound in rats on both diets based on products from farming systems without the use of mineral fertilizers (ORG-ORG and CV-ORG) andlower proliferation in ORG-CV and CV-CV dietary groups. These results correspond with the flavonoids and polyphenols content foundin the experimental feeds. The mitogen-stimulated proliferation index was lower in ORG-ORG and CV-ORG groups. It can only bespeculated whether the alteration in spontaneous lymphocyte proliferation can result from immunostimulatory effect of flavonoids andpolyphenols found in feed or from the immunosuppressive influence of some dietary components in products cultivated with mineralfertilizers. Further studies are in progress.

The study was supported under the Quality Low Input Food Project: FOOD-CT-2003–506358.

1. Baranska A & Skwarlo-Sonta K (2006) Pol J Food Nutr Sci 56, 13–16.2. Rembialkowska E, Hallmann E, Rusaczonek A, Bennett RN, Brandt K, Lueck L & Leifert C (2007) In Proceedings of 3rd International Congress of the

European Project Quality Low Input Food (QLIF), pp. 112–117 [U Niggli, C Leifert, T Alfoldi, L Luck and H Willer, editors]. Frankfurt, Germany:Research Institute of Organic Agriculture.



Inflammatory mediators in overweight adolescents: association withinsulin sensitivity, body composition and metabolic syndrome

R. Burrows, L. Leiva, A. M. Tong, J. Aldunate and E. DıazInstitute of Nutrition and Food Technology, University of Chile, Chile

The insulin-resistance (IR) metabolic syndrome (MetS) leads to high CVD risk in adult life and has been associated with an early state ofchronic low–grade inflammation (CLGI). The aim of the present study was to determine the association between a state of CLGI andinsulin sensitivity, body composition and MetS prevalence in overweight adolescents. The study included 158 adolescents (BMI ‡ 85thpercentile; eighty-six men) between 13 and 16 years of age, who were assessed for BMI, body composition (% body fat mass (BFM) and% free-fat mass (FFM) by pletysmography, prevalence of MetS for three or more variables (waist circumference (WC) ‡ 90th percentile;HDL-cholesterol £400 mg/l; TAG ‡ 1100 mg/l; blood arterial pressure ‡ 90th percentile; fasting glucose ‡100 mg/d), HOMA-IR(glucose · insulin/22.5) and the state of inflammation by C-reactive protein (CRP). Pearson correlation and y2 were used to studyassociations between variables, OR to calculate risk and ANOVA and Tukey test to compare averages between groups. The median andranges of CRP levels (mg/l) were 0.7 (range 0.04–9.1) in males and 0.7 (range 0.04–6.2) in females. CRP showed a correlation with BMI(P<0.05), WC (P<0.02), % BFM (P<0.05), % FFM (P<0.05), fasting insulin (P<0.001) and HOMA (P<0.001).

Table 1. Pearson correlation for CRP with variables of metabolic and cardiovascular risk

CRP

Variable r P

BMI 0.158 <0.05WC 0.199 <0.02% BFM 0.168 <0.05% FFM 0.169 <0.05Fasting insulin 0.269 <0.001HOMA 0.259 <0.001

CRP was significantly (P<0.01) associated with an anthropometric and metabolic cardiovascular risk profile. The prevalence and therisk of abdominal obesity (WC ‡ 90th percentile), IR (HOMA ‡ 3.3) and MetS were significantly higher (63%, OR 3.0; 43%, OR 4.1;26%, OR 4.1 respectively) in adolescents with CRP levels ‡1.12 mg/l (‡ tertile 2).

Table 2. Anthropometric, cardiovascular and metabolic profile across the CRP tertile distribution

CRP . . . <Tertile 1 (<0.43 mg/l) Tertile 1–2 (0.43–1.12 mg/l) >Tertile 2 (>1.12 mg/l)

Mean SD Mean SD Mean SD P

BMI (kg/m2) 2.0 0.5** 2.4 0.8 2.5 1.1 <0.01WC (cm) 88.1 6.5** 92.5 7.4 95.4 10.8 <0.01TBF (%) 35.4 6.4 37.6 7.6 40.4 7.0††‡ <0.01FFM (%) 64.3 6.9 62.5 7.9 59.7 6.9††‡ <0.01HDL (mg/l) 505 101 467 104 459 113† <0.05TAG (mg/l) 995 495 1129 687 1271 697†† <0.01HOMA 1.9 1.2 2.3 1.3† 3.4 2.6‡‡ <0.001

Mean values were significantly different from those for tertile 1–2 and> tertile 2: **P<0.05. Mean values weresignificantly different from those for< tertile 1: †P<0.05, ††P<0.01. Mean values were significantly different from thosefor tertile 1–2: ‡P<0.05, ‡‡P<0.05.

These results confirm that (1) PCR levels in overweight adolescents are associated with a greater cardiovascular and metabolic risk and(2) IR involves inflammatory processes that may play an early role in the development of cardiovascular lesions.



Immunological response in coeliac disease is age related

M. Calzado, E. Donat, B. Polo, B. Baena and C. RibesPediatric Gastroenterology and Hepatology Section, La Fe Hospital, Valencia, Spain

Anti-tissue transglutaminase antibodies (t-TG), mostly IgA class, is considered to be the most specific serological immune response incoeliac disease, and is associated with enteropathy, specific Ig A class antibody against t-TG being found at the intestinal level(1,2).

The objective was to evaluate the serological immune response in coeliac disease according to age by measuring antibodies against theautoantigen, i.e. t-TG and if t-TG can be used as a screening method. A retrospective study was done reviewing the files of all patientswho had undergone a small intestinal biopsy in the previous 3 years for diagnostic purposes. IgA t-TG (VarelisaTM, Phadia, cut off 6 IU/ml) and IgA anti-gliadin antibodies (AGA; home method, cut off 0.3 arbitrary units) were determined when the biopsy was performed. Inpatients with negative IgA t-TG, IgG t-TG (VarelisaTM; Phadia, Freiburg, Germany; cut off 10 IU/ml) was determined. A total of 280patients were included, 160 of them showed Marsh 3 lesions and one of them showed Marsh 2 lesions. All of them were consuming glutenat the time the biopsy was performed. Diagnosis of coeliac disease was established in all of them. No patient had IgA deficiency. Allchildren with t-TG>100 (upper detection limit) were classified as Marsh 3. Specificity for AGA was lower than that for t-TG. Eighty-eightpatients (55%) were <3 years of age and sixty-one were <2 years of age. Ten of the eighty-eight patients (11.4%) were IgA t-TGnegative, only one being >2 years of age. All ten subjects had elevated IgA AGA and no Ig G class antibodies against t-TG were detected.Clinical symptoms in patients who were t-TG positive and t-TG negative were the same.

<3 years old (n 88) >3 years old (n 73)

Ig A t-TG positive 78 82Ig A t-TG negative 10 1IgA AGA positive 88 75

The results show negative IgA class t-TG is expected in about 10% of patients with coeliac disease who are <2 years of age.Furthermore, no Ig G response is to be expected in those patients. It can be speculated that the immature development of the immunesystem slowly responding to gluten exposure delays the appearance of autoimmunity. This notion would be in keeping with reports of noIgA t-TG antibodies deposited at the intestinal level in younger patients(3–5). Moreover, the data confirm the response against gliadin is theimmunological trigger in coeliac disease.

1. Salmi TT, Collin P, Jarvinen O et al. (2006) Aliment Pharmacol Ther 24, 541–552.2. Salmi TT, Collin P, Korponay-Szabo IR et al. (2006) Gut 55, 1746–1753.3. LC Stene, MC Honeyman, EJ Hoffenberg et al. (2006) Am J Gastroenterol 101, 2333–2340.4. Norris JM, Barriga K, Hoffenberg EJ et al. (2005) JAMA 293, 2343–2351.5. Tosco A, Maglio M, Paparo F, Colicchio B, Sannino A, Granata V, Rapacciulo L & Troncone R (2007) Proceedings of the 40th Meeting of the

European Society of Paediatric Gastroenterology, Hepatology and Nutrition, Barcelona 2007, Abstr (In the Press).



Hormonal and immune changes with age: effect of energy restriction

P. Cano, G. Segovia, M. P. Fernandez-Mateos, A. del Arco, V. Jimenez, F. Mora and A. I. EsquifinoDepartamento de Bioquımica y Biologıa Molecular III y Fisiologıa, Facultad de Medicina, Universidad Complutense,

Madrid, Spain

The effects of chronic 40% energy-restriction on age-related changes in plasma prolactin, luteinising hormone (LH), testosterone, thyroid-stimulating hormone (TSH) and corticosterone levels as well as the distribution and activity of T and B lymphocytes in the spleen weredetermined. Male rats (3 months old) were subjected to 40% energy restriction up to the age of 6, 15 and 24 months. Hormones weremeasured by specific RIA. Lymphocyte subsets were measured by flow cytometry (FASC-Vantage; BD Biosciences, San Jose, CA,USA)(1,2). Plasma LH and testosterone levels decreased with age, and energy restriction did not change this pattern. However, plasmaprolactin and TSH levels, which increased with age in the controls, were significantly decreased in energy-restricted animals. Thepercentage of CD4+ lymphocytes, which increased with age in the controls, was further increased in energy-restricted rats. Although thepercentage of CD8 + lymphocytes decreased with age both in the control and energy-restricted rats, the values were higher in energy-restricted rats at all ages studied. The percentage of T-cells was not affected by age and was increased by energy restriction at all agesstudied. B lymphocytes decreased with age and were not modified by energy restriction. The CD4 + :CD8 + was greatly increased in theoldest control group, while energy restriction partially decreased this ratio to normal values. These data suggest that energy restriction canprevent some of the deleterious effects that aging induces on the endocrine immune system.

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Figure. Values are means with their standard errors represented by vertical bars for six animals per group.

This work was supported by SAF2003–0448 of Spain.

1. Esquifino AI, Szary A, Brown-Borg HM & Bartke A (1996) Proc Soc Exp Biol Med 211, 87–93.2. Esquifino AI, Chacon F, Cano P, Marcos A, Cutrera RA & Cardinali DP (2004) J Neuroimmunol 156, 66–73.



Use of inducible class II and MHC class I-related chain A/B expressionon T lymphocytes as an in vitro screen for the immunomodulatory

properties of dietary products

C. Carter, K. Varley and B. ClarkTransplant and Cellular Immunology, St James’ University Hospital, Beckett Street, Leeds LS9 7TF, UK

Immune-mediated rejection following renal transplantation is essentially centred around two primary mechanisms. The presence ofpreformed antibodies against donor human leucocyte antigen (HLA) in sensitised recipients results in hyperacute rejection. Non-self HLAcan also trigger a cell-mediated immune response in which a key event is the direct activation of CD4 + T-cells by non-self HLA class IImolecules expressed on antigen-presenting cells present in the donor organ. Furthermore, recent reports have highlighted an associationbetween increased MHC class I-related chain A/B (MICA/B) expression in graft tissue and immunological tissue damage and rejectionfollowing renal transplantation(1). Protocols that decrease the expression of MICA/B or class II HLA proteins either pre- or post trans-plantation would be a potentially useful approach in a live donor setting. Whilst considerable efforts are made to control the recipient’simmune system post transplant little attention is paid to this aspect in the donor pre-transplant. There are, however, a number of reportssuggesting that pharmacological agents (e.g. statins)(2)as well as dietary products (most notably fish oils)(3) reduce the expression of HLAclass II expression. Whilst administering statins to healthy donors before the transplant may be difficult to justify, the use of dietaryproducts such as fish oils may be both beneficial and appropriate. The aim of the present study was to develop an in vitro assay in whichthe effects of potential immunomodulators on MICA/B and HLA-DR expression were measured. To this end, human T lymphocytes werestimulated with phytohaemagglutinin (PHA) and HLA-DR and MICA/B expression were measured by antibody staining and two-colourflow cytometry. Incubation of peripheral blood mononuclear cells with PHA for 3–5 d led to consistently increased HLA-DR expression.This was found to be the case both in the frequency (% positive) of CD3 + T-cells expressing HLA-DR and in the mean channelfluorescence (MCF) that estimates antigen density (Table). The expression of MICA/B following PHA stimulation was found to beconsiderably lower than HLA-DR and was considered to be too inconsistent for further consideration in this assay (Table). The kinetics ofexpression and the required concentration of PHA to give optimal expression as well as alternative stimulators (anti-CD3 stimulation)were also investigated. This in vitro system is now being used to assess the ability of dietary products to down regulate the expression ofclass II HLA-DR expression on T-cells following stimulation.

Table. Expression of HLA-DR and MICA/B on T-cells following in vitro PHA stimulation

CD3/HLA-DR CD3/MICA/B

% Positive MCF % Positive MCF

Unstimulated 8.62 1.32 0.04 3.05+ PHA 64 407 16.4 8.00

1. Hankey KG, Drachenberg CB, Papadimitriou JC, Klassen DK, Philosophe B, Bartlett ST, Groh V, Spies T & Mann DL (2002). Transplantation 73,304–306.

2. Kuipers HF, Biesta PJ, Groothuis TA, Neefjes JJ, Mommaas AM & van den Elsen PJ (2005) Hum Immunol 66, 653–665.3. Hughes DA, Pinder AC, Piper Z, Johnson IT & Lund EK (2000) Am J Clin Nutr 71, Suppl., 343S–348S.



Dietary intake of nutrients of great interest in immunonutrition toprevent muscle damage in soccer players

C. Conejos1,2, A. Giner2, J. Manes1 and J. M. Soriano1

1Laboratory of Nutrition, Faculty of Pharmacy, University of Valencia, Av. Vicent Andres Estelles s/n, 46100,

Burjassot, Spain and 2Valencia C,. Ciudad Deportiva, 46980 Paterna, Spain

Soccer is a sport in which players are exposed to long-term periods of training and players need to augment strength and fat-free massgains by resistance training, minimize muscle damage and soreness and help to improve endurance. Recently, the incidence of injuries hasincreased during and preceding soccer matches, which influences the effectiveness of the team. One study(1)has reported that muscleinjury resulting from intensive exercise triggers the immune response of Ig and complement in serum, and induces the inflammatoryreaction. Although neutrophil reactive oxygen species (ROS) production is thought to play a part in clearance of phagocyte-damaged hosttissue, such as muscle tissue, by exercise(2), oxidative stress has been associated with decreased physical performance, muscular fatigue,muscle damage, and overshooting inflammation(3). In a well-trained player a good diet can enhance physical and athletic performance anddecrease the incidence of injuries during soccer matches. The repetition of intense exercise diminishes the neutrophil inflammatoryreaction, and the recovery from physical damage may be delayed(4). However, it has been observed that carbohydrate ingestion has only aminimal influence on the immune response to exercise(5). It has been suggested that 4 weeks of Fe supplementation significantly increasesbody Fe stores and inhibits the decrease in Hb concentration induced by soccer training(6). The objective of the present study was toevaluate the dietary intake of nutrients of relevance to immunonutrition in relation to muscle damage in young high-level soccer players(group A: n 20, age 17–18 years; group B: n 22, age 20–24 years). The 24 h diet-record method was used by the dietitian of the soccerclub to analyse the dietary intake of arginine, branched-chain amino acids (valine, leucine and isoleucine), Zn, Cu, Fe, Se, Mg andvitamins A, C and E.

The results showed that 65% of the players between 20 and 24 years of age had intakes below the Spanish and European recommendedintakes (SERI) for Zn and Mg, whereas approximately 60% of the youngest players were below the SERI only for Zn. Nevertheless, ifresults were compared with recommended values for sportsmen (SRV), >65% of both groups failed to achieve the SRV for Fe, Zn andvitamins A, C and E.

In conclusion, the results show that the group of players studied need to include in their diet foods containing antioxidant vitamins, Feand Zn in order to prevent possible muscle damage. Currently, a nutritional education programme is being developed to promote anappropriate food pattern to aid the reduction of lesions in soccer players.

The work was supported by University of Valencia (UV-AE-20070219).

1. Mashiko T, Umeda T, Yamamoto Y & Sugawara K (2004) Br J Sports Med 38, 617–621.2. Peake J & Suzuki K (2004) Exerc Immunol Rev 10, 129–141.3. Konig D, Wagner KH, Elmadfa I, Berg A (2001). Exercise and oxidative stress: significance of antioxidants with reference to inflammatory, muscular,

and systemic stress. Exerc Immonol Rev 7: 108–33.4. Rebelo AN, Candeias JR, Fraga MM, Duarte JA, Soares JM, Magalhaes C & Torrinha JA (1998) J Sports Med Phys Fitness 38, 258–261.5. Bishop NC, Blannin AK, Robson, PJ, Walsh NP & Gleeson M (1999) J Sports Sci 17, 787–796.6. Kang HS & Matsuo T (2004) Asia Pac J Clin Nutr 13, 353–358.



Total antioxidant capacity of refrigerated orange juice treatedwith pulsed electric fields

C. Cortes, F. Barba, M. J. Esteve, R. Gonzalez and A. FrıgolaNutrition and Food Chemistry, Faculty of Pharmacy, University of Valencia, Avd. Vicent Andres Estelles s/n, Burjassot,

Valencia, Spain

Orange juice is an important source of carotenoids and ascorbic acid, a nutrient that besides its vitamin action is valuable for itsantioxidant effect, stimulation of the immune system and other health benefits that are being actively investigated and reported, such asinhibition of the formation of cancer-causing N-nitroso compounds in the stomach(1). During processing and/or storage orange juiceundergoes an important number of undesirable effects on some nutrients, antioxidant compounds, colour, flavour and texture. The use ofpulsed electric fields (PEF) is an emerging technology in the field of food preservation(2). The aim of the present work was to study theantioxidant capacity of the orange juice treated with PEF (30 kV/cm, 100ms) in comparison with orange juice subjected to conventionalthermal treatments (90�C during 20 s), as well as changes in orange juice stored at 2 and 10�C, using a method adapted from that of Milleret al.(3). The Trolox equivalent antioxidant capacity (TEAC) of the samples was (mmol Trolox/l) 4.03 (SD 0.04), 3.51 (SD 0.04) and 2.49(SD 0.20) for untreated, PEF-treated and pasteurized orange juice respectively. Thus, TEAC decreased significantly (P<0.05) after theorange juice was processed by both types of treatment; the decrease being greater in the pasteurized juice (38.2%) than in the PEF-treatedjuice (12.9%). Thus, PEF treatment of orange juice had an antioxidant capacity more similar to that of the untreated juice. The totalantioxidant capacities of samples during refrigerated storage are shown in the Table. The results show a decrease with time at bothtemperatures, although the decrease was higher in the samples stored at 10�C. Compared with conventional pasteurization, PEF treatmentled to a higher total antioxidant activity of orange juice immediately after processing, as well as during storage at 2–10�C.

TEAC (mmol Trolox/l)

Temperature (�C) Period of storage (weeks) 0 1 2 3 4 6 7

2 PEF juice 3.51 3.22 2.98 2.94 2.33 2.23 2.21Pasteurized juice 2.49 1.93 2.10 2.40 1.83 1.80 1.80

10 PEF juice 3.51 2.375 2.10 2.37 1.54 1.53 –Pasteurized juice 2.49 2.02 2.15 2.52 1.74 1.70 –

This study was carried out with funds from the Spanish Ministry of Science and Technology and European Regional Development Funds (ERDF) (ProjectAGL-2003–05236-C02–02 and AGL-2006–13320-C03–03).

1. Valkoa M, Rhodesb CJ, Moncola J, Izakovica M & Mazur M (2006) Chem Biol Interact 160, 1–40.2. Min S, Jin ZT, Yeom H, Min SK & Zhang QH (2003) J Food Sci 68, 1265–1271.3. Miller NJ, Diplock AT, Rice-Evans C, Davies MJ, Gopinathan V & Milne A (1993) Cli Sci 84, 407–412.



Examination of host immune resistance against Listeria monocytogenesinfection in cyclophosphamide (CPA)-treated mice after

dietary lipid administration

L. Cruz-Chamorro1, M. A. Puertollano2, E. Puertollano1, C. Carazo3, G. Alvarez de Cienfuegos1

and M. A. de Pablo1

1Area de Microbiologıa, Universidad de Jaen, Jaen, Spain, 2Instituto del Frıo (CSIC), Madrid, Spain and 3Area

de Microbiologıa y Parasitologıa Clınica, Hospital Medico-Quirurgico, Jaen, Spain

Numerous investigations have clearly established that the administration of diets containing fish oil (FO) may produce important immu-nosuppressive effects in both animals and human subjects(1). In fact, this property has been utilized in the reduction of typical symptomscaused by diseases characterized by an overactivation of the immune response. Nevertheless, prolonged utilization and the administrationof high levels of n-3 PUFA may result in a severe reduction in host immune resistance to infectious micro-organisms(2). The action ofdifferent dietary lipids in severely-immunosuppressed mice was evaluated using a model of chemotherapy in which cyclophosphamide(CPA) injections, which cause a delay in the onset of acquired cellular resistance, were followed by an enhanced and slightly prolongedresponse in Listeria monocytogenes-infected mice(3). Balb/c mice were fed one of four diets, which contained olive oil (OO; 200 g/kg diet;n 5), FO (200 g/kg diet; n 5), hydrogenated coconut oil (HCO; 200 g/kg diet; n 5) or low fat (LF) for 4 weeks. After the feeding period,mice were treated with CPA or PBS (control), before infection with L. monocytogenes. Survival analysis and measurement of viablebacteria counts for spleens and livers and serum pro-inflammatory cytokine levels were carried out. The FO-rich diet reduced survival,particularly in CPA-treated mice. CPA was responsible for a significant increase in viable bacteria recovery from spleens and livers withineach group fed high-fat diets, which was exacerbated in mice fed an FO diet. In addition, significant increases in both TNFa and IL-12p70levels were detected in this group. The application of CPA moderately aggravates the immunosuppressive state in FO-fed animals.

Table. Recovery of viable bacteria from spleens and livers of mice fed dietary lipids after a 48 h challenge with L. monocytogenes

Diets

L. monocytogenes (log10 CFU )

Spleen Liver

- CPA + CPA - CPA + CPA

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LF 3.5c 1.6c 3.8c 1.1 3.0d 0.9 3.2d 1.2OO 4.3b 2.0 4.6b 1.8 4.5c 2.1 5.0c 1.8FO 6.3a 2.5 6.7b 1.8 5.9a 1.1 7.0a 2.1HCO 6.0a 2.1 6.5a 2.6 5.3b 2.0 6.2b 2.3

Values are means with their standard errors represented by vertical bars for three independent experiments after logarithmic (log10)transformation (five mice per dietary group). CFU, colony-forming units. a,b,c,dMeans within a column with unlike superscript letterswere significantly different (P<0.05; two-way ANOVA).

These results may be of crucial relevance in clinical nutrition, particularly when n-3 PUFA are administered to patients who areimmunocompromised and at risk of sepsis.

1. Calder PC (2000) Lipids 36, 1007–1024.2. de Pablo MA, Puertollano MA & Alvarez de Cienfuegos G (2002) Clin Diagn Lab Immunol 9, 945–950.3. Kerckhaert JA, Hofhuis FM & Willers JM (1977) Immunology 32, 1027–1032.



Effect of dietary oils on oxidative stress and cytokine productionby murine macrophages

R. de la Puerta Vazquez1, M. A. Fernandez Arche1, A. Marquez Martın2 and V. Ruiz-Gutierrez2

1Dpto Farmacologıa, Facultad de Farmacia, Universidad de Seville, Spain and 2Grupo de Nutricion y Metabolismo

Lipıdico, Instituto de la Grasa, (CSIC), Seville, Spain

Many studies have shown that the nature of the lipid consumed in the diet significantly affects the prevalence of coronary, inflammatory orautoimmune diseases(1,2). The present study was conducted to compare the impact of feeding mice on diets enriched with edible oils(150 g/kg diet; fish oil (FO), olive oil (OVO) and orujo olive oil (ORO)) with that of a basal diet (BD) enriched with 20 g maize oil/kg onthe ability to modulate oxidative reactive species and pro-inflammatory mediator generation by stimulated murine macrophages. Swissmale mice were fed on the different diets for 8 weeks. Diets were formulated according to American Institute of Nutrition (AIN)recommendations(3). Peritoneal macrophages were isolated from these mice and stimulated. Reactive oxygen (O2

– and H2O2) and nitrogen(NO2

–) species, PGE2, TNFa and IL-6 were measured in the supernatant fractions from 106 cells. All test diets down regulated NOgeneration compared with the BD (Fig.1); in contrast, FO increased H2O2 generation whereas OVO and ORO group diets significantlyinhibited this ROS production compared with the BD. In addition, ORO was able to decrease O2

– formation compared with the BD group(Fig.1). Finally, both OVO and FO groups significantly decreased PGE2 and cytokine production (Fig. 2). These results are in agreementwith those of other authors in that a diet enriched in olive oil was found to show a protective effect against oxidative stress andinflammation(4,5)and they confirm the preventive anti-inflammatory properties of FO(6). Moreover, the results provide important additionaldata about the ability of ORO to prevent oxidative damage to cells.

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This study is part of the project AGL2005–00572/AlI, financially supported by the Comision Interministerial de Ciencia y Tecnologia (CICYT).

1. James MJ, Gibson RA & Cleland LG (2000) Am J Clin Nutr 71, 343–348.2. Yaqoob P (2003) Curr Opin Clin Nutr Metab Care 6, 133–150.3. Bieri JG, Stoewsand GS, Briggs GM, Philips RW, Woodward JC & Knapka JJ (1977) J Nutr 107, 1340–1348.4. Moreno JJ, Carbonell T, Sanchez T, Miret S & Mitjavila MT (2001) J Nutr 31, 2145–2149.5. Perona JS, Arcemis C, Ruiz-Gutierrez V & Catala A (2005) J Agric Food Chem 53, 730–735.6. Calder PC (2006) Am J Clin Nutr 86, 1505–1519.



Seasonal changes in the neuroimmunoendocrine system activity inyoung adult male rats administered with a balanced controlled diet

from weaning

A. I. Esquifino1, M. P. Fernandez-Mateos2, V. Jimenez1, J. Rios1 and P. Cano1

Departamentos de 1Bioquimica y Biologia Molecular III and 2Biologia Celular de Medicina, Universidad Complutense,

Madrid, Spain

Temporal changes in the activity of the neuroimmunoendocrine system maintain homeostasis in living organisms(1,2). The present workanalyses the 24 h changes in the activity of the neuroimmunoendocrine system in young adult male rats (3 months old) kept understandard conditions of controlled light (fluorescent cool white bulbs providing 100 lux intensity at the level of the cages) with 12 h light- 12 h dark (light on at 08:00 hours) at 22�2�C and ad lbitum access to a balanced diet (AIN-93G; Diets Inc., Pennsylvania, USA) andwater. The conditions were identical for all seasons studied. Animals were killed by decapitation at six different time intervals (every 4 hthroughout a single 24 h period) during spring, summer, autumn or winter time periods taking as an index the regulatory mechanism ofprolactin, a hormone that is involved in the development and maintenance of immune function. The median eminence dopamine con-centration, plasma prolactin levels and submaxillary lymph node lymphocyte proliferative capacity were studied. Globally, dopamineconcentration in the median eminence showed specific 24 h variation depending on the season studied. Changes in plasma prolactin levelswere in accordance with variations in dopamine. Likewise, proliferative capacity of lymphocytes from the submaxillary lymph nodesexhibited specific 24 h variation according to the season (Figure). There were specific seasonal correlations between dopamine, prolactinand the proliferative capacity of the lymphocytes. This outcome may suggest the existence of a seasonal signal that could allow indivi-duals to adapt their physiological functions to the annual environmental changes.

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1. AI Esquifino, D Pazo, RA Cutrera & DP Cardinali (1999) Chronobiol Int 16, 451–460.2. N Vazquez, E Dıaz, C Fernandez, V Jimenez, AI Esquifino & B Dıaz (2007) Physiol Res 56, 79–88.



Effect of long-chain fatty alcohols from orujo olive oil on nitric oxideand eicosanoid generation

M. A. Fernandez Arche1, R. de la Puerta Vazquez1, A. Marquez Martın1 and V. Ruiz-Gutierrez2

1Department of Pharmacology, School of Pharmacy, University of Seville, C/ Profesor Garcıa Gonzalez No. 2

and 2Instituto de la Grasa (CSIC), Av. Padre Garcıa Tejero No. 4, 41012 Seville, Spain

Olive pomace oil (‘orujo’ oil) is an olive oil product suitable for human consumption that is traditionally produced in Spain(1). The non-acylglycerol component of this oil is a good source of interesting minor components, e.g. triterpenes(2), or fatty alcohols, derived fromwaxy materials. Tetracosanol (C24OH; 30%), hexacosanol (C26OH; 37%) and octacosanol (C28OH; 15%) are the major constituents ofthe long-chain fatty alcohol (LCFA) fraction isolated from orujo olive oil(3). A similar mixture of long-chain alcohols, termed ‘polico-sanol’ and purified from waxy materials of different sources such as sugar cane, bees wax, rice bran or spinach, have shown manybeneficial physiological activities(4,5). The present study focused on the effect of LCFA isolated from orujo olive oil on NO, PGE2 andTNFa release by a lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW-264.7) as well as the effect on thromboxaneB2 (TXB2) generation by A-23187-stimulated rat peritoneal neutrophils (PMN). Nitrite (as an index of NO generation) levels weredetermined by a fluorometric method. PGE2, TNFa and TXB2 production were quantified by sandwich immunoassay.

LCFA significantly and dose-dependently decreased the NO production in LPS-stimulated RAW-264.7 cell line macrophages (Fig. 1).Western-blot analysis for inducible NO synthase (iNOS) showed that NO reduction was a consequence of the 100% inhibition of iNOSexpression at a dose of 100mg/ml (Fig. 2). By contrast, LCFA scarcely affected PGE2 levels (Fig. 1). TNFa production was alsosignificantly decreased by LCFA at the highest dose assayed (100 mg/ml; Fig. 1). LCFA significantly reduced TXA2 production in ratPMN stimulated with A-23187 (Fig. 3).

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Fig. 2. Effect of LCFA subfraction on iNOS expressionand densitometric analysis in RAW 264.7 cells. DEX,dexamethasone; OD, optical density.

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These results showed that LCFA isolated from ‘orujo’ oil has a protective effect on some mediators implicated in the development ofinflammatory damage in these experimental models and suggest its potential value as a functional component of the olive pomace oil.

This study is part of the project AGL2005–00572/ALI, financially supported by the Comision Interministerial de Ciencia y Tecnologia (CICYT).

1. Perona JS, Aracemis C, Ruiz-Gutierrez V & Catala A (2005) J Agric Food Chem 53, 730–735.2. Perez Camino MC & Cert A (1999) J Agric Food Chem 47, 1558–1562.3. Marquez A (2007) Doctoral Thesis, Universidad de Sevilla.4. Taylor JC, Rapport L & Lockwood GB (2003) Nutrition 19, 192–195.5. Singh DK, Li L & Porter TD (2006) J Pharmacol Exp Ther 318, 1020–1026.



Fermented soyabean products as hypoallergenic food

J. Frias1, Y. S. Song2, C. Martınez-Villaluenga1, E. Gonzalez de Mejia2 and C. Vidal-Valverde1

1Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain and 2Department of Food

Science and Human Nutrition, University of Illinois Urbana-Champaign, 228 ERML, MC-051, 1201 W Gregory Drive,

Urbana, IL 61801, USA

Food allergy has become a public health problem that continues to challenge both the public and the food industry, and soyabean iscausative of food allergy. The allergenicity of food proteins has been shown to be modified by food-processing technologies such asfermentation. The objective of the present research was to ferment soyabeans as flour (liquid fermentation) or as cracked seeds (solidfermentation) using different micro-organisms in order to study the effect on immunoreactivity after fermentation. Immunochemicalmethods have been developed and validated for the detection and quantification of the major human allergenic soyabean proteins, and acomparison with fermented soyabean proteins has been carried out. ELISA and Western blot were used to quantify IgE antibody responseand HPLC to identify and quantify total amino acids. The extractable protein concentration decreased after fermentation (from 31% forcracked seeds fermented with Bacillus subtilis to 74% with Aspergillus oryzae). Lactobacillus plantarum- and naturally-fermentedsoyabean flour showed a higher reduction in IgE immunoreactivity (£92% and 97%) in comparison with the corresponding fermentedproducts of cracked seeds (92% and 88% reduction respectively). Among the solid fermented products, the lowest performance was givenby mould strains, Rhizopus oryzae and A. oryzae, which showed a reduction in allergenic values of 66% and 71% respectively. AlthoughB. subtilis showed the highest soluble protein concentration (221.5 mg protein/g product), this fermented soyabean product yielded a 75%and 86% reduction in immunoreactivity against human plasma and human pooled plasma samples respectively.

The raw soyabean flour presented the highest complexity on protein profile and immunoreactivity. Human plasma samples presented anintense immunoreactivity towards specific immunodominant proteins. Fermentation with B. subtilis or L. plantarum and natural fermen-tation yielded peptides of smaller size and less-intense immunoreactivity. Moreover, soyabeans subjected to fermentation processesshowed rises for most of the non-essential and essential amino acids (P £0.05). In conclusion, fermentation applied to soyabean induces anoticeable variation in protein profile and allergenicity and there is potential for developing nutritious hypoallergenic soya products.

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Figure 1. Effect of fermentation of soybean seeds and soya fermented products on immune response by ELISA.

This work was partly funded by AGL2004-0886ALI and by USDA-Future Foods Initiative.



Immunomodulatory properties of Lactobacillus salivarius arenot limited to the intestine

J. Galvez1, B. Arribas1, M. E. Rodrıguez-Cabezas1, E. Bailon1, M. Comalada1, D. Camuesco1,J. Xaus2 and A. Zarzuelo1

1Department of Pharmacology, University of Granada, Granada, Spain and 2Puleva Biotech, SA, Granada, Spain

Previous studies have shown the beneficial effects exerted by probiotics on inflammatory bowel disease(1), an intestinal condition char-acterized by an altered intestinal immune response(2). However, it would be interesting to know whether the immunomodulatory prop-erties of probiotics are restricted to a local effect in the intestine or whether their effect can also be extrapolated to other systemic immunealterations. The aim of the present study was to test the effect of a probiotic, Lactobacillus salivarius CECT5713, in two experimentalmodels of local or systemic altered immune response, i.e. the trinitrobenzenesulfonic acid (TNBS) model of rat colitis and the lipopo-lysaccharide (LPS)-induced septic shock in mice. For this purpose, mice or rats (n 10) were given the probiotic (5 · 108 colony-formingunits/ml drinking water), starting 2 weeks before damage induction. A control group (n 10) without probiotic was also used for reference.Colitis was induced in rats by intracolonic administration of TNBS (10 mg) and after 1 week was evaluated both histologically andbiochemically (myeloperoxidase activity, glutathione content, inducible NO synthase (iNOS) expression)(3). Septic shock was induced inmice by administering LPS (40 mg/kg, intraperitoneally) and the mice killed 24 h later, when colon and spleen were removed. ColoniciNOS expression was determined by Western blot, and activated T-cells were obtained from spleens by concanavalin A incubation and theimmune response evaluated by RT–PCR or ELISA for different cytokines (IL-2, -5, -6 and -10). The results showed that L. salivarius wasable to ameliorate both the local and systemic altered immune response. The probiotic exerted intestinal anti-inflammatory activity, sinceit significantly reduced the extension of the colonic damage induced by TNBS in comparison with non-treated colitic rats; this effect wasaccompanied by a 42% reduction in myeloperoxidase activity (P<0.05), a 44% increase in glutathione content (P<0.05) and a reductionin colonic iNOS. Moreover, the probiotic treatment significantly prevented the increase in colonic weight (mg/cm) induced by septicshock (264 (SE 15) v. 322 (SE 15); P<0.05), without showing differences from normal mice (246 (SE 14)). Similarly, the LPS-inducedcolonic iNOS expression was lower in the probiotic-treated mice (30%). LPS also stimulated the expression of different cytokines assayedin the splenocytes, while the probiotic-treated mice showed a reduction in cytokine expression of 80% for IL-5 and 100% for both IL-2and IL-6 (Figure 1). IL-10 secretion was reduced in control mice (603 (SE 102) pg/ml; P<0.05) in comparison with the normal group(1064 (SE 80) pg/ml), which was increased in probiotic-treated mice (1034 (SE 150 pg/ml; P<0.05) when compared with the LPS controlgroup. In conclusion, the immunomodulatory properties of the probiotic L. salivarius are not restricted to the intestine, since it is also ableto ameliorate the alteration in the systemic immune response derived from LPS administration to mice.

Figure 1. Lactobacillus salivarius administration reduced cytokine expression (RT-PCR) in LPS-induced septic shock in mice.

1. Ewaschuk JB & Dieleman LA (2006) World J Gastroenterol 12, 5941–5950.2. Hanauer SB (2006) Inflamm Bowel Dis 12, S3–S9.3. Peran L, Sierra S, Comalada M et al. (2007) Br J Nutr 97, 96–103.



Vitamin E as an IgE inhibitor: stability during cold storage ofhuman milk

G. Garcıa-Llatas1, R. Lacomba1, F. Ortega1, A. Alegrıa1, R. Barbera1 and D. Silvestre2

1Nutrition and Food Chemistry, University of Valencia, Avda. Vicente Andres Estelles s/n, 46100 Burjassot, Valencia, Spain

and 2Food Science, Cardenal Herrera-CEU University, Avda. Seminario s/n, 46113 Moncada, Valencia, Spain

Human breast milk is recommended as the unique food for neonates based on its known properties. When the production of milk by themother is not sufficient or the mother is not able to feed her child for professional reasons, milk banks or the mothers’ practice ofcollecting their own milk are the existing alternatives for breast-feeding. In both situations cold storage (refrigeration or freezing) can beused in neonatal units, at home and in human milk banks(1,2).

Many micronutrients with immunological properties are found in breast milk. In addition to its antioxidant activity, vitamin E has beenshown to be associated with a decrease in the frequency of allergen sensitisation by the inhibition of IgE responses to allergic stimuli;asthma, rhinitis and hayfever are atopic disorders characterised by raised serum IgE and skin sensitisation to common environmentalallergens(3).

The stability of vitamin E contents can be influenced by temperature and time of storage. Thus, the aim of the present study was theevaluation of vitamin E contents during cold storage of human milk. Fourteen samples of mature human milk from healthy and well-nourished women were collected from a breast-feeding workshop. Each sample was divided in three aliquots, one of which was analysedjust after collection (F) and the others were stored under the conditions used at home (refrigerating at 2–4�C for 48 h; R) and at milk banks(freezing at - 18�C for 15 d; Fz).a-, b-, g- and d-Tocopherols were quantified simultaneously in the milk samples by a normal-phase HPLC method with fluorescence

detection(4) (four determinations per sample). The results (ranges) are shown in the Table.

Tocopherols(mg/100 ml human milk) F R Fz

a 209.9–264.0 169.3–223.4 193.2–247.3b 34.8–57.5 44.1–68.1 25.0–47.7g 54.2–66.0 49.6–61.4 55.9–67.8d 9.25–18.0 14.1–22.8 9.43–18.2

One-way (storage temperature) ANOVA applied to the results showed no significant differences (P<0.05) between the treatmentsstudied. Thus, the inhibition of IgE responses provided by vitamin E from human milk is maintained after it is refrigerated or storedfrozen.

This study is part of the project PRUCH 03/36 (2003–2004) supported financially by Cardenal Herrera-CEU University.

1. Human Milk Banking Association of North America (2006) Best Practice for Expressing, Storing and Handling Human Milk in Hospitals, Homes andChild Care Settings, 2nd ed. [F Jones and MR Tully, editors]. Raleigh, NC: HMBANA.

2. Lindemann PC, Foshaugen I & Lindemann R (2004) Arch Dis Child Fetal Neonatal Ed 89, F440–F441.3. Fogarty A, Lewis DS, Weiss S & Britton J (2000) Lancet 356, 1573–1574.4. Rodrigo N, Alegrıa A, Barbera R & Farre R (2002) J Chromatogr 947A, 97–102.



Melatonin supplementation modifies experimental chronic colitis in mice

M. Golab, M. Dudziak and K. Skwarlo-SontaDepartment of Animal Physiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland

Melatonin (Mel) has been adopted by most living organisms as a circadian timing signal, but is also known to be a potent free radicalscavenger and immunomodulatory agent(1). Mel is not only synthesized in the pineal gland, but also in the gastrointestinal tract (GIT),where it does not depend on the light–dark cycle and may be induced by food intake or withdrawal. GIT-derived Mel may also haveparacrine and autocrine functions, modifying the intestinal blood flow or mucosal regeneration(2). The potent free radical scavengingactivity of Mel may give rise to its protective and therapeutic application in chronic inflammation of the GIT(3).

The aim of the present study was to examine the immunomodulatory properties of Mel when administered differently in experimentalcolitis in mice. Experiments were conducted on 6–8-week-old male BALB/c mice (five to six individuals per group) housed in standardlaboratory conditions (22�C, 12 h light–12 h dark). Chronic colitis was induced by administration of dextran sodium sulfate (DSS; 0.06 gper mouse per d) in the drinking water for 2 or 3 weeks. From the beginning of the experiment some groups of mice were simultaneously(preventively) treated with Mel (10 mg/kg body weight) by oral administration (Expt 1) or in the drinking water during the night (Expt 2).Another group of mice received Mel therapeutically after showing the first clinical symptoms of colitis (positive occult blood tests, rectalbleeding). Mice were killed by CO2 asphyxia, colons were isolated and proximal and distal fragments used for histological tests.Additionally, spleens were isolated and splenocytes cultured in the presence of mitogens. Colon mucosa was gently scraped into aprotease-inhibitor solution, centrifuged and supernatant fractions used for the measurement of cytokine concentrations using flow cyto-metry with a mouse T-helper (Th1)/Th2 cytokine cytometric bead array kit (Becton Dickinson, Franklin Lakes, NJ, USA).

In control mice (without colitis) Mel caused immune cell proliferation in gut lymphatic nodules without any change in cytokineconcentration and crypt structure. DSS administration resulted in an elevation in mucosal TNFa, a decrease in IL-2, subsequent shorteningand crypt loss as well as intensive mononuclear infiltration to the mucosa. Preventive Mel supplementation caused no changes in TNFaconcentration in DSS-treated mice, but completely reversed the DSS-induced decrease in mucosal IL-2 content. Mel did not preventinflammatory infiltration, but crypt loss was located either in the proximal or distal part of the colon, depending on the period of Meltreatment. Therapeutic Mel administration did not change IL-2 concentration but caused a significant decrease in TNFa and interferon-gconcentrations. No significant changes in Th2-specific cytokines (IL-4, IL-5) after preventive or therapeutic administration of Mel wereobserved. In Expt 1 possible Mel-induced systemic changes in immunity were perturbed by the stress associated with oral administration.Splenocyte proliferation was changed by colitis and Mel in Expt 2, as a result of the less-invasive method of Mel treatment. Both B- andT-cell proliferation were decreased by Mel in mice without colitis. Splenocyte proliferation was elevated in the DSS-treated animals anddecreased by Mel only if administered preventively.

These results show a possible protective action of Mel in the GIT, but its effects are dependent on the method and period ofadministration. Further experiments are in progress.

Supported by MNiN grant 2P04C 117 29 and by Faculty of Biology, University of Warsaw intramural grant BW #1720/20 to M.G.

1. Altun A, Ugur-Altun B (2007) Int J Clin Pract 61, 835–845.2. Bubenik GA (2002) Dig Dis Sci 47, 2336–2348.3. Nosal’ova V, Zeman M, Cerna S, Navarova J, Zakalova (2007) J Pineal Res 42, 364–370.



Effect of dietary supplementation on lymphocytes subsets andliver-related variables in hospitalised patients with anorexia nervosa

S. Gomez1, L. E. Dıaz1, E. Nova1, G. Morande2 and A. Marcos1

1Departamento de Nutricion y Bromatologıa, Instituto del Frıo (CSIC) 28040 Madrid, Spain and2Servicio de Psiquiatrıa, Hospital Nino Jesus, 28009 Madrid, Spain

Medical complications are common and often serious in patients with eating disorders such as anorexia nervosa (AN)(1). Several studieshave described an increase in serum liver enzymes in severely-malnourished patients who are affected by AN(2)and in the refeeding phaseof therapeutic intervention(3,4). It is also considered that, the lymphocyte subset ratios CD4:CD8 and CD2:CD19 are indexes of mal-nutrition(5). Thus, a study was carried out that aimed to evaluate the effect of a dietary supplement (Pentadrink fibra; Nutricia SA, Madrid,Spain; energy density 6.3 kJ/ml) on some liver-related and immunological variables. The supplement was used in order to increase theenergy content of the refeeding diet administered to the patients with AN during their admission to the hospital. Sixty patients with ANwere divided into three groups: (1) without oral supplementation (ANWP; n 21); (2) with 200 ml oral supplementation (ANP; n 19); (3)with 400 ml oral supplementation (ANPP; n 19). The results were compared with those obtained from a control group matched for gender,age and socio-economic status. Energy intake and energy profile were evaluated for each group. Liver-related variables (glutamic–oxaloacetic transaminase (GOT), glutamic–pyruvic transaminase (GPT), g-glutamyltransferase (GGT), alkaline phosphatase (AP) andbilirubin) and immunological variables (CD4 + , CD8+ , CD2+ , CD19+ cell numbers and ratios) were determined. All variables wereassessed on admission to (time 0) and discharge from hospital (time 1).

The mean initial energy intake (kJ/d) of the patients was 6145 (SD 560, 6947 (SD 594), 11520 (SD 878) in the ANWP, ANP and ANPPgroups respectively. At discharge the energy intake (kJ/d) was: ANWP, 12218 (SD 1150); ANP, 13188 (SD 1283); ANPP, 14154 (SD 1317).CD8+ in ANPP, CD2+ in ANP and ANPP, and CD19 + in all three groups showed a significant decrease during the study. Thesechanges led to an increase in the CD2:CD19; however, the increase was only significant in the ANPP group (see Figure). Both CD4:CD8and CD2:CD19 were always within normal values. Although GOT and AP showed a significant increase in the ANPP group, the levelswere also maintained within normal values. The results show that the inclusion of the energy supplement in the refeeding therapy ofpatients with AN does not produce adverse effects on the liver enzymes or immunological variables measured. However, special careshould be taken with those patients with AN at risk of suffering hepatic disease, particularly if the treatment is prolonged.

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Figure. Mean values were significantly different from those for the controls (C) (Student’s t test): *P<0.05. Mean values were significantly different from those for time 1(Student’s t test): †P<0.05. (0), admission to hospital; (1), discharge from hospital.

This work has been supported by Nutricia (Spain).

1 Mitchell JE & Crow S (2006) Curr Opin Psychiatry 19, 438–443.2 Jones SC, Pieri LF & Losowsky MS (1999) Eur Eat Disord Rev 7, 28–36.3 Ozawa Y, Shimizu T & Shishiba Y (1998) Intern Med 37, 32–39.4 De Caprio C, Alfano A, Senatore I, Zarrella L, Pasanisi F & Contaldo F (2006) Nutrition 22, 572–575.5 Marcos A (2007) Nutrition 13, 853–862.



Borage (Borago officinalis) oil supplementation in relation to monocytechemoattractant protein 1 expression in healthy subjects

M. Xiang1, E. Pinto1, M. A. Rahman1, M. Leach1,2 and L. S. Harbige1,2

1Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB, UK and 2Medway School of

Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK

Essential fatty acids (EFA) have unique roles as precursor molecules of chemical regulators of inflammatory cell function(1–3). Incomparison with linoleic acid (18: 2n-6), g-linolenic acid (GLA; 18: 3n-6) may have superior biopotency because the GLA bypasses theD6 desaturation, which is regarded as a key regulatory rate-limiting enzymic step controlling the formation of long-chain (LC) PUFA(4,5).The present study investigated GLA-rich borage oil supplementation in relation to the monocyte chemoattractant protein 1 (MCP-1;CCL2) expression from peripheral blood mononuclear cells (PBMC) at the gene and protein levels in human subjects. Seven healthyvolunteers who ingested 14 g borage oil/d consecutively for 13 weeks were studied. It was found that the MCP-1 production from bothunstimulated and phytohaemagglutinin (PHA)-stimulated PBMC was reduced during the time-course of the intervention. Furthermore,MCP-1 from the PHA-stimulated PBMC decreased significantly during the 13 weeks of the intervention period. In addition, the level ofPBMC MCP-1 gene expression was reduced significantly during the supplementation. A significant positive correlation was foundbetween the expression of MCP-1 gene and MCP-1 production from both unstimulated (r 0.40, P<0.05; Figure (A)) and PHA-stimulatedPBMCs (r 0.66, P<0.001; Figure (B)).

The study has, for the first time, revealed that GLA-rich borage oil supplementation in human subjects results in the inhibition of PBMCMCP-1 expression at the gene and protein levels. The suppressive effect of GLA-rich borage oil on PBMC MCP-1 expression may bebeneficial to chronic inflammatory diseases.

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1. Harbige LS (2003) Lipids 38, 323–341.2. Kast RE (2001) Int Immunopharmacol 1, 2197–2199.3. Belch JJ & Hill A (2000) Am J Clin Nutr 71, Suppl., 352S–356S.4. Xiang M, Harbige LS & Zetterstrom R (2007) Acta Paediatr 96, 387–390.5. Xiang M, Rahman MA, Ai H, Li X & Harbige LS (2006) Ann Nutr Metab 50, 492–498.



Vitamin D deficiency among older adults in England remainsa cause for concern!

V. Hirani, A. Ali and K. TullDepartment of Epidemiology and Public Health, Royal Free and University College London Medical School,

University College London, London, UK

The importance of vitamin D in Ca absorption and metabolism for bone health is well known. Vitamin D deficiency in adults clinicallymanifests as osteomalacia and osteoporosis. There is also now emerging evidence on how vitamin D may modify the risk of severalchronic diseases, such as cancers and CVD and is protective against type 1 diabetes mellitus, multiple sclerosis and rheumatoid arthritis.These effects may be related to newly-proposed biological roles for vitamin D such as having a modulating function for immunity(1) andregulating the growth of many tissues other than bone(2). Concerns about vitamin D deficiency, its role and importance, have been raisedby the WHO. In the UK since 1998 government agencies have emphasised the importance of vitamin D in the elderly(3). In 2000 theHealth Survey for England (HSE) showed the prevalence of vitamin D deficiency (serum concentrations of £25 nmol 25-hydro-xycholecalciferol/l) to be 15% in women and 10% in men living in private households.The aim of the present study was to assess vitaminD status of individuals aged ‡65 years, and make comparisons with earlier surveys (HSE 2000 and the National Diet and Nutrition survey(NDNS) data collected in 1994) and examine associations between vitamin D deficiency and risk factors.

A valid vitamin D sample was obtained from 2070 informants (950 men and 1120 women) as part of the HSE 2005, a nationally-representative survey of individuals aged ‡65 years living in private households in England. Analysis included 1160 informants (524 menand 636 women), excluding those on medication that would affect the vitamin D status and those taking vitamin supplements.

The prevalence of vitamin D deficiency (£25 nmol 25-hydroxycholecalciferol/l) was 20% in women and 12% in men. When a higherthreshold of <50 nmol/l was used (defined as vitamin D insufficiency) 62% of women and 57% of men were vitamin D insufficient.Regression analyses showed that women were more vitamin D deficient than men (OR 1.7) and that vitamin D deficiency increased withage (OR 1.7 for those aged 70–74 years and OR 3.2 for those aged ‡85 years), was more likely for those who smoked cigarettes (OR3.1), was more prevalent in the winter and autumn (OR 2.7) and was associated with poor general health (OR 1.9). Separate analysis foreach gender showed that among men vitamin D deficiency was also associated with vitamin B12 deficiency (OR 1.9) and was more likelyin those with cancer (OR 2.2). In women deficiency was also 50% more likely among those in manual social classes and 90% more likelyin obese women (BMI>30 kg/m2).

Overall, the results show no significant improvements in vitamin D status in comparison with earlier HSE 2000 results or the NDNSresults. Low vitamin D status shows an association with many risk factors and poor general health outcomes. Further action and guidanceis required to actively address vitamin D deficiency among the elderly.

1. Hayes CE, Nashold FE, Spach KM & Pedersen LB (2003) Cell Mol Biol (Noisy-le-Grand) 49, 277–300.2. Moan J, Lagunova Z & Porojnicu A (2005) Sunlight, Vitamin D and Health, p. 38 [O Gillie, editor]. http://www.healthresearchforum.org.uk/reports/

sunbook.pdf3. Department of Health (1998) Nutrition and Bone Health with Particular Reference to Calcium and Vitamin D. Report on Health and Social Subjects

no. 49. London: The Stationery Office.



Evaluation of diet, anthropometry and immunocompetence ofyoung male athletes

D. Lacerda1, V. Cotta-de-Almeida2, A. Montero3, W. Savino1 and A. Marcos4

1Laboratory on Thymus Research, Dept of Immunology and 2Dept of Ultrastructure and Cell Biology, Institute Oswaldo

Cruz–Fiocruz, Rio de Janeiro, Brazil, 3Dpto Nutricion, Bromatologıa y Tecnologıa de los Alimentos, Facultad de Farmacia,

Universidad San Pablo-CEU, Madrid, Spain and 4Grupo inmunonutricion, Dpto Metabolismo y Nutricion, Instituto Frıo,

Consejo Superior de Investigaciones Cientıficas, Madrid, Spain

Currently, it is well accepted that nutrition is an important factor for the development of the immune response. Epidemiological andclinical results suggest that any nutritional deficiency alters immunocompetence and increases the susceptibility to infections(1,2). thus, thepurpose of the present study was to compare the nutritional status of ten young male elite track and field athletes (20–48 h training/week)aged 13–18 years at a baseline state (48 h relaxation after training) with a control group consisting of ten volunteer students, matched byage and socio-economic status, who were doing<8 h physical exercise/week. Nutritional status was assessed by dietary intake for 3 d,anthropometry (BMI and triceps skinfold thickness) and immunocompetence (total counts of leucocytes, lymphocytes and lymphocytesubsets (CD3, CD4, CD8, CD19 and CD16 + 56)).

The energy intake was similar for athletes and controls (8327 v. 8235 kJ/d respectively), and lower than that recommended by the Foodand Nutrition Board, Institute of Medicine(3), the American Dietetic Association(4)and the American Academy of Pediatrics(5). BMI andtriceps skinfold thickness were significantly lower for the athletes than for the controls. Total leucocyte and total lymphocyte counts weresimilar for both groups. However, a decrease in CD3, CD8, CD19 and CD16 + 56 lymphocyte subset counts was found for athletes incomparison with controls.

Controls (n 10) Athletes (n 10)

Mean SD Mean SD

Age (years) 16.3 1.38 17.1 0.80Energy intake (kJ) 8235 672 8327 584

Anthropometric variablesWeight (kg) 68.7 6.63 63.7 7.63Height (m) 1.75 0.68 1.76 0.77BMI (kg/m2) 22.5 1.18 20.7* 2.00Triceps skinfold thickness (mm) 11.9 2.78 5.67* 1.1

Immunological variablesTotal leucocytes (cells/mm3) 6287 1533 5786 1026Total lymphocytes (cells/mm3) 3413 1003 3527 918Lymphocyte subsets (cells/mm3):

CD3 1380 418 919* 380CD8 659 224 432* 151CD19 248 54 179* 60CD16 + 56 (NK cells) 257 73 185* 59

NK, natural killer. Mean values were significantly different from those of the controls (Student’s t test): *P<0.05.

These results suggest that the young athletes assessed in the current study could show subclinical malnutrition, with a leaner body andan impaired immunocompetence compared with the control group, although the dietary intake was similar for both groups but belowrecommendations. A higher-energy diet should be recommended for these high-performance athletes in order to avoid further risk ofmalnutrition and to avoid more serious complications, such as infections.

1. Gleeson M, Nieman DC & Pedersen BK (2004) J Sport Sci 22, 115–125.2. Moreira A, Kekkonen RA, Delgado L, Fonseca J, Korpela R & Haahtela T (2007) Eur J Clin Nutr 61, 443–460.3. Food and Nutrition Board, Institute of Medicine (2002) Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol,

Protein and Amino Acids (Macronutrients). Washington, DC: National Academy Press.4. American Dietetic Association (2000) J Am Diet Assoc 100, 1543–1556.5. American Academy of Pediatrics (2006) Pediatrics 11, 544–559.



Effect of inulin and probiotic bacteria on iron availability inbeans (Phaseolus vulgaris L.)

J. M. Laparra1, R. P. Glahn2 and D. Miller1

1Department of Food Science, Cornell University, Ithaca, NY 14853, USA and 2US Plant, Soil, and Nutrition Laboratory,

Agricultural Research Service, US Department of Agriculture, Ithaca, NY 14853, USA

Anaemia is the most common nutritional disorder in the world and Fe deficiency (ID) is implicated in a majority of these cases. Clinicalstudies have emphasized the importance of Fe in the integrity of the immune system(1). One of the keys is poor bioavailability of Fe in thediet. Beans constitute a good source of protein for large groups of the population around the world, and can also be a good source ofessential minerals such as Fe. Nevertheless, legumes also contain phytates, polyphenols, etc., which impair Fe bioavailability. Among thephytochemicals, polyphenols have been suggested to have a beneficial effect on immune cell function(2). Prebiotics such as inulin havebeen suggested to have an enhancing effect on Fe absorption(3), and can also modulate the immune system(4). The objectives were toevaluate the effects of inulin and probiotic bacteria on Fe availability from white and red beans.

White and red beans, with and without supplemental 40 ginulin/kg, were subjected to an in vitro gastrointestinal digestion (pepsin,pH 2; pancreatin, pH 7.2). The digests were incubated overnight with Bifidobacterium infantis (ATCC 15697) or Lactobacillus acid-ophilus (ATCC 11974). Aliquots were then placed in the upper chamber of cell-culture plates containing monolayers of Caco-2 cells(5).Cell associated Fe and ferritin formation were used as estimates of Fe bioavailability.

Inulin by itself increased white-bean Fe uptake in Caco-2 cultures; however, no differences in ferritin formation were detected. Thisobservation may be explained by the capacity of each ferritin molecule to bind 4000 Fe atoms. Fermentation of both white and red beans,with or without inulin, with probiotic bacteria caused differences in Fe availability only as a function of the bacteria tested. In fermentedsamples higher growth rates for B. infantis than L. acidophilus were quantified, probably as a result of a faster bacteria metabolicadaptation to the media and use of nutrients in the digests. Following fermentation with B. infantis Fe uptake from both white and redbeans was decreased in Caco-2 cells. This observation correlated with an increased total phenolic content in the digests. However, thefermentation with L. acidophilus resulted in a lower total phenolic content and subsequent higher Fe uptake values than controls.

These results show that inulin may enhance Fe bioavailability, and suggest that probiotic bacteria caused differences in solublepolyphenols in the digests that may explain the contrasting results from B. infantis and L. acidophilus.

Beansample

Iron in theupper chamber (mg)

ferritin 1

(ng mg- 1 protein)Total phenols

(mg Gallic acid g- 1)

No treatment with bacteriaWhite 2.07�0.02 62.88�2.29 3.03�0.08Red 2.65�0.11 3.43�1.33 4.17�0.33White + Inulin 2.08�0.29 61.43�4.17 3.08�0.24Red + Inulin 2.79�0.19 3.56�1.09 4.05�0.48

Incubated with B. infantisWhite 2.07�0.01 44.15�5.21 3.48�0.12Red 2.72�0.10 1.75�1.99 5.45�0.33White + Inulin 2.10�0.05 50.80�8.56 4.25�0.19Red + Inulin 2.63�0.03 1.61�0.41 5.35�0.12

Incubated with L. acidophilusWhite 2.04�0.01 75.55�6.11 1.85�0.28Red 2.75�0.04 6.31�1.89 3.04�0.25White + Inulin 2.13�0.03 86.50�7.38 1.80�0.14Red + Inulin 2.64�0.01 6.15�0.51 2.95�0.32

1. Mullick S, Rusia U, Sikka M & Faridi MA (2006) Indian J Med Res 124, 647–654.2. Alvarez P, Alvarado C, Puerto M, Schlumberger A, Jimenez L & De la Fuente M (2006) Nutrition 22, 913–921.3. Yeung CK, Glahn RE, Welch RM & Miller DD (2005) J Food Sci 70, R88–R92.4. Watzl B, Girrbach S & Roller M (2005) Br J Nutr 93, S49–S55.5. Glahn RP, Lee OA, Yeung A, Goldman MI & Miller DD (1998) J Nutr 128, 1555–1561.



Effect of different doses of creatine supplementation onendogenous creatine synthesis

B. Li1, M. Xiang2, L. S. Harbige2,3, X. Li1 and H. Ai11Division of Nutrition and Biochemistry, Institute of Sports Medicine, The Third Hospital, Peking University, Beijing,

People’s Republic of China, 2Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB,

UK and 3Medway School of Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK

Creatine (Cre) has been widely used as a nutritional ergogenic aid among athletes since the middle of the 1990s(1) but has not beencategorised as a stimulant. Athletes often take a greater dose of Cre for a longer period in order to obtain a rapid, high and longerergogenic effect(2). L-Arginine:glycine amidinotransferase (L-AGAT) in the kidney is regarded as a key enzyme in endogenous Cresynthesis in mammals, and guanidinoacetic acid (GAA) is the precursor in this synthesis(3). The present study has investigated the effectof supplementation with 0, 0.75, 1.5, 3.0 and 6.0 g Cre/kg body weight per d for 4 weeks on L-AGAT activity and GAA concentration inthe kidneys and total Cre in the gastrocnemius muscle in adult male Sprague-Dawley rats. L-AGAT activity and GAA concentration in thekidneys decreased (%) by 19.5, 42.6, 61.9 and 66.5, and 5.9, 13.7, 23.8 and 24.5 respectively in the groups supplemented with 0.75, 1.5,3.0 and 6.0 g Cre/kg per d for 4 weeks. The total Cre in the gastrocnemius in the supplemented groups increased (%) by 7.1, 8.5, 12.6 and13.7 respectively. The weight of the gastrocnemius in the groups receiving 0.75, 1.5 and 3.0 g Cre/kg per d also increased significantly (seeTable). No significant differences were found in the body weights among the groups supplemented with 0, 0.75, 1.5 and 3.0 g Cre/kg per dfor 4 weeks (Table). However, the body weight of the group receiving 6.0 g Cre/kg per d was significantly less than that of the controlgroup (Table). After 3 d of supplementation diarrhoea was observed in all the rats in the group receiving 6.0 g Cre/kg per d, and persistedfor the remainder of the supplementation period, which probably explains the loss in body weight. No significant differences were foundin the serum Cre kinase activity and serum creatinine among the five groups. L-AGAT activity was positively correlated with theconcentration of GAA (n 50, r 0.80, P<0.001). The Cre intake was, however, negatively correlated with the L-AGAT activity (n 50,r 0.87, P<0.001) and the concentration of GAA (n 50, r 0.75, P<0.001).

Cre (g/kg per d) n

Body weight (g) Gastrocnemius weight (g)

Mean SD Mean SD

0 10 365.2 36.0 2.02 0.170.75 10 379.4 23.7 2.33** 0.241.5 10 374.8 19.6 2.19* 0.103.0 10 371.8 21.8 2.27** 0.206.0 10 340.6** 31.0 2.05 0.19

Mean values were significantly different from that for the control group (without Cre supplementation): *P<0.05,**P<0.01.

These results indicate that in rats L-AGAT activity and GAA concentration could be rapidly reduced by supplementation with 0.75–3.0 g Cre/kg per d, which is equivalent to 10–40 g Cre/d (67 kg body weight) in human subjects(4), suggesting that high-dose Cre sup-plementation may result in depression of endogenous Cre metabolism and may have potential adverse effects on the body. Little is knownabout the effect of high-dose Cre supplementation on the immune system, the results of a follow-up study of immune function will be ofinterest.

1. Bemben MG & Lamont HS (2005) Sports Med 35, 107–125.2. Brudnak MA (2004) Toxicol Lett 150, 123–130.3. Wyss M & Kaddurah-Daouk R (2000) Physiol Rev 80, 1107–1213.4. Calabrese EJ (1991) Principles of Animal Extrapolation, pp. 499–527. Chelsea, MI: Lewis Publishers Inc.



Effect of cessation of creatine supplementation onendogenous creatine synthesis

X. Li1, M. Xiang2, L. S. Harbige2,3 and H. Ai11Division of Nutrition and Biochemistry, Institute of Sports Medicine, The Third Hospital, Peking University, Beijing,



Creatine (Cre) can be obtained exogenously from either the diet or supplementation(1–3). It can also be endogenously synthesised in manand other mammals(4,5). One of the adverse effects of Cre supplementation is that L-arginine:glycine amidinotransferase (L-AGAT), a keyenzyme in the endogenous Cre synthesis in mammals, may be severely inhibited(6). The present study investigated the time-course ofrecovery of L-AGAT activity and guanidinoacetic acid (GAA) concentration after 1 week of supplementation with 3.0 g Cre/kg bodyweight per d Cre in adult male Sprague-Dawley rats. On days 1, 2, 4, 8, 16 and 32 (ten treatment and ten control rats per time point) afterthe 7 d supplementation period L-AGAT activity and GAA concentration in the kidney recovered (%) by 32.8, 51.5, 76.1, 94.4, 100.2 and102.0 (Figure (A)) and 77.9, 86.2, 96.8, 100.6, 101.1 and 108.0 (Figure (B)) respectively when compared with their respective controls.The total Cre in the gastrocnemius muscle decreased rapidly and reached its lowest level on day 4 after the supplementation period, andthen increased gradually. No significant differences were found in serum Cre kinase activity and serum creatinine in the experimentalgroups when compared with their respective control groups.

These findings indicate that in rats the reduction in L-AGAT activity and GAA concentration associated with supplementation with 3.0 gCre/kg per d for 1 week could be reversed, suggesting that short-term and high-dose Cre supplementation does not result in depression ofendogenous Cre metabolism. As little is known about the effect of Cre supplementation on the immune system, investigation of the effectof long-term and high-dose Cre supplementation on endogenous Cre metabolism in the immune cells of rats will be of interest.

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1. Pan JW & Takahashi K (2007) Am J Physiol 292, R1745–R1750.2. Candow DG & Chilibeck PD (2007) J Nutr Health Aging 11, 185–188.3. Silva AJ, Machado Reis V, Guidetti L, Bessone Alves F, Mota P, Freitas J & Baldari C (2007) J Sports Med Phys Fitness 47, 58–64.4. Derave W, Marescau B, Vanden Eede E, Eijnde BO, De Deyn PP & Hespel P (2004) J Appl Physiol 97, 852–857.5. Braissant O, Henry H, Loup M, Eilers B & Bachmann C (2001) Brain Res Mol Brain Res 86, 193–201.6. Wyss M & Kaddurah-Daouk R (2000) Physiol Rev 80, 1107–1213.



Activation of innate immunity by a probiotic bacterium anda fermented milk containing this micro-organism

C. Maldonado Galdeano1,2, A. de Moreno de Leblanc1, C. Dogi1, S. Chaves1, E. Carmuega3,R. Weill4 and G. Perdigon1,2

1CERELA, Chacabuco 145, 4000 Tucuma, Argentina, 2Cat Inmunologıa, Fac Bqca, Qca y Fcia, Universidad Nacional

de Tucuman, Argentina, 3Nutritia, Buenos Aires, Argentina and 4Dto Investigacion y Desarrollo, Danone SA, Argentina

Improvement of the immune status of the host is one of the beneficial properties attributed to probiotics. Previous results have shown thatthe effect induced by a probiotic strain Lactobacillus casei CRL 431 is through innate immunity(1,2,3). The present work investigated thebehaviour of another probiotic bacterium Lactobacillus casei DN 114001 in relation to the interaction between the epithelial and immunecells, using mice as the experimental model. Using electron microscopy and fluorescent bacteria it was demonstrated that this bacteriuminteracts with the intestinal epithelial cells and the small fragments of the bacterium can internalize and make contact with the immunecells (macrophages and dendritic cells) present in Peyer patches and in the lamina propria of the small and large intestine. These antigenicparticles remain in the gut for 72 h, similar to other particulate antigens. A subsequent study investigated the effect of the inclusion of thisprobiotic bacterium in fermented milk on the mucosal innate immune system of the gut in BALBc mice. The fermented milk wasadministered to the mice for five consecutive days, which was previously determined to be the optimal dose to stimulate the immuneresponse. At the end of the administration period the animals were killed and the small and large intestine was removed for thedetermination of: (a) the number of IgA- and cytokine (IL-10, interferon g (IFN-g), TNFa)-producing cells in histological slices of bothintestines; (b) the expression in the lamina propria of the small intestine of the different receptors present in the immune cells involved inthe innate immunity, e.g. mannose receptor CD206 present in macrophages or on the dendritic cell surface (this receptor participatesmainly in the internalization process and in antigen clearance); (c) Toll-like receptor 4 (TLR4), which is involved in the adhesion and pro-inflammatory signals induced by pathogenic bacteria, as a measure of possible adverse effects. Increases were found in the number ofIgA-, TNFa- and IFNg-producing cells in both intestines. The anti-inflammatory cytokine IL-10 also showed a significant increase inrelation to the control (without fermented milk). This increase may have been induced to down regulate the mucosal response. Thereceptor CD206 was slightly increased, but TLR4 was unchanged. These results showed that innate immunity is involved in the gutmucosal immune activation observed, and led to an investigation of the effect of the consumption of this fermented milk in early period ofthe life, when the maturation of the innate immune cells occurs. After weaning, newborn mice received the fermented milk until theadulthood (45 d of age). The number of cells positive for maturation markers, such as F4/80+ cells (macrophages) and 33D1 + cells(dendritic cells) were determined in the small intestine. The expression of these markers increased in treated animals compared with thecontrol group (newborn mice not receiving fermented milk).

These results demonstrate that the probiotic bacterium or its fragments interact with epithelial and phagocytic immune cells associatedwith the gut. This observation is reflected in the results obtained with the fermented milk, which show an increase in the number ofCD206 receptors and activation of the immune cells associated with the gut, with an increase in cytokine- and IgA+ -producing cells. Theresults for the specific markers of maturation of the macrophages and dendritic cells in adult mice (45 d of age) that received thefermented milk after weaning are in agreement with those of previous studies.

It has been demonstrated that fermented milk containing the probiotic strain L. casei DN 114001 can modulated the gut immuneresponse mainly through the innate immune system by increasing the receptors related to maturation of the macrophages and dendriticcells associated with the gut, by activating these cells and by increasing the number of IgA + B-cells.

1. Galdeano CM & Perdigon G (2004) J Appl Microbiol 97, 673–681.2. Maldonado Galdeano C, de Moreno de Leblanc A, Vinderola G, Bibas Bonet ME & Perdigon G (2007) Clin Vaccine Immunol 14, 485–492.3. Maldonado Galdeano C & Perdigon G (2006) Clin Vaccine Immunol 13, 219–226.



Milk-derived supplement inhibits in vitro lymphocyte proliferationand IL-2 production

S. Marın-Gallen, F. J. Perez-Cano, M. Castell, C. Castellote and A. FranchDepartment of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Recently, significant progress has been made in the characterisation of milk components affecting the function of the immune system. Theaim of the present report was to study the effect of a premium ultra-grade freeze-dried bovine whey-protein concentrate (WPC) on thefunctional capacity of lymphocytes. Thus, the immunomodulating properties of this dairy extract were evaluated in relation to theproliferative response of spleen T and B lymphocytes. Splenocytes from adult Wistar rats were incubated in a medium supplemented withWPC (60–480mg protein/ml), which contains (g/kg)<0.1 fat, 750–900 proteins and 35–50 carbohydrates, and also lactoferrin 9.2; IgG300–600, IgA 50–70 and IgM 70–90 and high proportions of active compounds, e.g. natural growth factors and hormones, vitamins andamino acids. Standard commercial infant formula (SIF; 40–415mg protein/ml) and BSA (250–500mg protein/ml) were also added as milk-derived and inert control proteins respectively. Concanavalin A (ConA; specific stimulus for T-cells) or pokeweed (Phytolacca amer-icana) mitogen (specific for B-cells) were added to the cell culture for 72 h. Proliferating cells were quantified by means of the BioTrakTM

cell proliferation system (BioTrak, Carlsbad, CA, USA) based on 5-bromo-20-deoxyuridine incorporation. IL-2 levels were quantified byELISA in 24 h-culture supernatant fractions obtained from ConA-stimulated lymphocytes. Statistical analysis was performed by con-ventional ANOVA and when an experimental group variable had a significant effect on the dependent variable post hoc comparisons(LSD test) were performed. Significant differences were accepted at P<0.05.

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Results showed a dose–dependent inhibitory effect of WPC on both spleen B- and T-lymphocyte proliferative response induced bymitogen stimulation (see Figure). The inhibitory effect on T- and B-cell proliferation was approximately 55% and approximately 40%respectively. In parallel, a WPC dose-dependent inhibition of IL-2 production was also found (approximately 80%). Cell viability was notmodified by WPC addition. SIF produced similar inhibitory effects. However, non-milk-derived proteins such as BSA did not modifythese lymphocyte responses. WPC and SIF, both milk-derived components, inhibited lymphocyte proliferation and IL-2 productionin vitro. This immunomodulatory effect may prevent the newborn from over-reacting immunologically to the environmental antigensduring early life.



Influence of fruit intake and antioxidants on the prevention ofopacities of the lens

M. I. Martınez1, J. Martınez-Raga1,2, J. Raga1, A. Alegre3, M. C. Gimeno1, E. Llusar1

and I. Alfonso-Sanchez4

1Instituto CEU Sobre Drogas y Conductas Adictivas (IDYCA), Universidad Cardenal Herrera – CEU, Moncada,

Valencia, Spain, 2Unidad de Conductas Adictivas de Gandıa, Valencia, Spain, 3IES Pobla de Vallbona (Valencia),

Conselleria d’Educacio, Generalitat Valenciana, Spain and 4Consorcio Hospital General Universitario and

Facultat de Medicina, Universitat de Valencia, Spain

The WHO considers cataracts to be the leading cause of avoidable blindness worldwide. Cataracts develop as a result of protein glycation,which takes place when proteins react with sugars, by cross-link formation and by oxidative processes(1). Such end products of glycationalter proteins, DNA and lipids, modifying their chemical properties and causing a colour loss of the lens to yellow or brown, whichimpairs vision. Given the importance of oxidative processes in the development of cataracts, the putative role of antioxidant agents in itsdevelopment have been investigated.

The aim of the present study was to assess the possible protective effect of the different antioxidant agents present in fruit on thedevelopment of opacities of the lens relative to age.

The subjects were a random sample (one in every three individuals attending a community pharmacy) of seventy-four male and femalepatients with a mean age of 59 (range 45–80) years. Subjects completed a self-administered questionnaire to assess lifestyle status, as wellnutritional habits with a particular focus on the fruit intake.

N-acetyl-serotonin, a precursor of melatonin, appears to be effective in preventing damage to the lens caused by UVA radiation, bothfor its ability to capture free radicals and for its antioxidant properties. Vitamin E also has a protective effect against radiation-inducedcataracts by reducing oxidative stress. In addition, a similar effect has also been observed in relation to cataracts induced by tobaccosmoking. N-acetyl-carnosine plays an important role as an antioxidant against free radicals (superoxide, hydroxide) both in the lipid phaseof the cell membranes of the lens and in the aqueous membrane. It has also been suggested that vitamin C decreases cataract risk relativeto age among middle-aged Japanese. After oral administration of a combination of antioxidant micronutrients (b-carotene, vitamin C andvitamin E) for a 3-year period a slight deceleration of cataract progression relative to age was observed. A majority of patients wereconsuming fruit regularly (93.2% v. 6.8%; Table). Of those consuming fruit, 18.8% showed opacities of the lens (P=0.949), with arelative risk of 0.94. Thus, it could be considered that consuming fruit may provide a mild protective effect against developing opacities ofthe lens. To date, there are very few studies showing the protective effect of antioxidants on the development of cataracts in humansubjects(2). However, it has been suggested that both natural and artificial antioxidants could have a protective effect in the prevention ofcataracts in the early stages of oxidative damage to the lens.

Table. Relationship between fruit intake and development of opacities of the lens

Lens opacity

No Yes Total

Fruit intake No n 4 1 5% No-fruit consumers 80.0 20.0 100% Total sample 5.4 1.4 6.8

Yes n 56 13 69% Fruit consumers 81.2 18.8 100% Total sample 75.6 17.6 93.2

The present results suggest that antioxidants from fruit could provide some protective effects in the prevention of opacities of the lens.

1. Moeller SM, Taylor A, Tucker KL, McCullough ML, Chylack LT, Hankinson SE, Willett WC & Jacques PF (2004) J Nutr 134, 1812–1819.2. Gritz DC, Srinivasan M, Smith SD, Kim U, Lietman TM, Wilkins JH et al. (2006) Br J Ophthalmol 90, 847–851.



Alcohol consumption in secondary-school students: effects onplasma total antioxidant capacity

M. I. Martınez1, J. Martınez-Raga1,2, A. Alegre3, J. A. Dominguez1 and I. Alfonso-Sanchez4

1Instituto CEU sobre Drogas y Conductas Adictivas (IDYCA), Universidad Cardenal Herrera – CEU, Moncada, Valencia,

Spain, 2Unidad de Conductas Adictivas de Gandıa, Valencia, Spain, 3IES Pobla de Vallbona (Valencia), Conselleria

d’Educacio, Generalitat Valenciana, Spain and 4Consorcio Hospital General Universitario and Facultat de Medicina,

Universitat de Valencia, Spain

National Household Surveys have been carried out biannually in Spain since 1994 to establish the patterns and trends of substance useamong students in secondary education (age 14–18 years). Alcohol has been consistently, although to a different extent, the psycho-activesubstance of abuse most commonly used by students. In 2004 42.7% of 14–18 year olds had had a drink at least once in their lives, theprevalence being higher among boys than among girls (45.3% and 40.2% respectively).

The aim of the present study was to compare the prevalence of alcohol consumption in a random sample of students in secondaryeducation (age 14–18 years) from Valencia (Spain) with that of the National Household Survey, and to relate alcohol use to antioxidantstatus.

An anonymous self-administered questionnaire designed to collect information on alcohol consumption was completed during schoolhours by a sample of 291 Valencian secondary school students between January and June 2006. A blood sample was collected from asubgroup of volunteers (n 53) and plasma total antioxidant capacity (TAC) was determined in 1 ml serum using spectrophotometry(1) inorder to establish its relationship with alcohol consumption. Both TAC and alcohol consumption could have a negative impact on thepresent and future health of the subjects since excessive production of free radicals and lipid peroxidation may be implicated in thedevelopment of chronic diseases, such as atherosclerosis and cancer, and are responsible for cellular aging(2).

Among this sample of adolescents the percentage of students surveyed in 2006 that reported using alcohol and cannabis was muchlower (36.7%) than that in the 2004 Spanish National Household Survey (65.6%) and showed no significant gender differences. Referencevalues for plasma TAC were in the range 1.30–1.77 mmol/l, and did not appear to be affected by alcohol use (1.37 (SD 0.20) mmol/l v.1.37 (SD 0.18) mmol/l). The Figure shows the percentage distribution of values for plasma TAC and alcohol use according to age.

12 13 14 15 16 17 18 19 AGE

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Alcohol consumption does not significantly affect plasma TAC, probably because of its cardioprotective effects at moderate doses.Within this age-group no significant differences were observed.

1. Rice-Evans C & Miller NJ (1994) Methods Enzymol 234, 279–293.2. Soardo G, Donnini D, Varutti R et al. (2005) Alcohol Clin Exp Res 29, 1889–1898.



Synthesis of galactooligosaccharides with prebiotic potential duringhydrolysis of lactose by Lactozym 3000 L HP G

C. Martınez-Villaluenga, A. Cardelle-Cobas, N. Corzo, A. Olano and M. VillamielInstituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain

Galactooligosaccharides (GOS) are non-digestible oligosaccharides consisting of two to twenty-20 molecules of galactose and glucose thatare recognized as prebiotics because they can stimulate the proliferation of lactic bacteria and bifidobacteria in the human intestine(1). Thestimulation of the immune system is among the possible benefits of prebiotics that are being investigated(2). Although no definitiveconclusions have been reached, the possibility that prebiotic oligosaccharides could influence the immune response would make prebioticdietary intervention a more attractive choice than probiotics in the reduction of atopic diseases in infants(3) and the chronic inflammatorybowel disease in human subjects(4).

The role of human milk in the stimulation of the immune response in newborn babies during the first months of life is well known. Oneof the carbohydrates in human milk for which the immune effect has been established is 60-galactosyl lactose. This trisaccharide and othercarbohydrates have potential use as ingredients in the development of different infant formulas. Recently, considerable attention has beenpaid to improving GOS production; enzymic transgalactosylation from lactose being one of the most promising alternatives. Lactozym3000 L HP G is a commercial preparation in which b-galactosidase from Kluyveromyces lactis is the most active enzyme, althoughtransgalactosylation can also occur at the same time; the transferase:b-galactosidase activity depends on the different reaction conditionsused. The present study is an exhaustive investigation of the optimal conditions for GOS (60-galactosyl lactose, galactobiose and allo-lactose) formation during the hydrolysis of lactose with Lactozym 3000 L HP G. The effect of the reaction conditions (temperature, pH,time, substrate and enzyme concentrations) was different for the formation of di- and trisaccharides. Thus, the best conditions forproducing galactobiose and allolactose were 50�C, pH 6.5, 250 mg lactose/ml, 3 U enzyme/ml and 300 min, while the optimal conditionsfor the synthesis of 60-galactosyl lactose were 40�C, pH 7.5, 250 mg lactose/ml, 3 U enzyme/ml and 120 min.

This work has been financed under an R & D programme of the Spanish Ministry of Education and Science, project AGL-2004–07227-C02, and projectALIBIRD S-0505/AGR/000153 from the Comunidad de Madrid. A.C.-C. thanks MEC for an FPU grant and C.M.-V. is the recipient of a I3P-CSICcontract.

1. Sako T, Matsumoto K & Tanaka R (1999) Int Dairy J 9, 69–80.2. Maning TS & Gibson GR (2004) Best Pract Res Clin Gastroenterol 18, 287–298.3. Moro M, Arslanoglu S, Stahl B, Jelinek J, Wahn U & Boehm GA (2006) Arch Dis Child 91, 814–819.4. Macfarlane S, Macfarlane GT & Cummings JH (2006) Aliment Pharmacol Ther 24, 701–714.



Raffinose family oligosaccharides of lupin(Lupinus albus L. cv multolupa) as a potential prebiotic

C. Martınez-Villaluenga1, J. Chicholoska2, A. Kliber2 and K. Gulewicz2

1Instituto de Fermentaciones Industriales, CSIC, C/ Juan de la Cierva 3, 28006 Madrid, Spain and 2Department of

Animal Physiology and Biochemistry, Agricultural University, Wolynska 35, Poznan 60-637, Poland

The beneficial effects on health attributed to prebiotics are directly related to the stimulation of the growth and activities of lactic bacteriaand bifidobacteria in the human intestine(1). Moreover, proliferation of these beneficial bacteria contributes indirectly to the stimulation ofthe immune system through IgA and IL (IL1, IL6 and g-IL) production(2–6).

Raffinose family oligosaccharides (a-galactosides) are non-digestible oligosaccharides with an extent of polymerization of three to sixmolecules. These compounds are a(1!6)galactosides linked to the C-6 of the glucose moiety of sucrose, which are abundant in legumeseeds of which lupins are the richest source (120 g/kg seed dry weight)(7). These oligosaccharides can be easily extracted from the seedsand further purified(8) to be used as ingredients during the manufacture of different functional foods. In ovo studies have demonstrated thepotential prebiotic effect of a-galactosides derived from lupin seeds(9).

In the present work the evaluation of a-galactosides as prebiotics was carried out in vivo using an animal model. These results werecompared with those obtained for pure raffinose, and also commercial fructo-oligosaccharides (FOS) and inulin. Doses of 15 mg/100 gbody weight were administered orally once daily to Wistar rats for 23 d. The numbers of faecal bifidobacteria were estimated at days 0, 10and 23. Oligosaccharide administration for all experimental groups showed gradual increases (P £0.05) in faecal bifidobacteria with theduration of the experiment, reaching the highest value after the longest time period. The numbers of faecal bifidobacteria in rats afteradministration of raffinose family oligosaccharides (RFOS) from lupin seeds were comparable with those found with pure raffinose andcommercial fructo-oligosaccharides and inulin (Figure).

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Figure. Effect of oligosaccharide administration (15 mg/100 g body weight) on numbers of faecal bifidobacteria.

Thus, this in vivo study has shown that a-galactosides from lupin stimulate bifidobacterial growth and indirectly immune response.Nevertheless, subsequent intervention studies are needed to establish definitive conclusions.

This work is a result of a bilateral scientific cooperation joint project between Poland (Polish Academy of Sciences) and Spain (CSIC).

1. Gibson GR & Roberfroid MB (1995) Br J Nutr 125, 1401–1412.2. McCraken VJ & Gaskins HR (1997) In Probiotics: A Critical Review, pp. 85–111 [GW Tannock, editor]. Norwich, Norfolk: Horizon Scientific Press.3. Kato I (1997) Healthist Special anniversary ed., 60–66.4. Ishikawa H. (1997) Healthist Special anniversary ed. 787–789.5. Ballongue J (1993) Bifidobacteria and Probiotic Action, pp. 357–428 [S Salminen and A von Wright, editors] New York: Marcel Dekker.6. Isoulari E (2000) Hosp Med 61, 6–7.7. Martınez-Villaluenga C, Frıas J & Vidal-Valverde C (2005) Food Chem 91, 645–649.8. Martınez-Villaluenga C, Frıas J, Gulewicz K & Vidal-Valverde C (2004) J Agric Food Chem 52, 6920–6922.9. Martınez-Villaluenga C, Wardenska M, Pilarski R, Bednarczyk M & Gulewicz K (2004) Folia Biol (Krakow) 52, 135–142.



Antioxidant and anti-inflammatory potency of different wheat varietiesand fractions

N. Mateo Anson1,2, R. v. d. Berg1, R. Havenaar1, G. Haenen2 and A. Bast21TNO Quality of Life, PO Box 360, 3700 AJ Zeist, The Netherlands and 2University of Maastricht, PO Box 616,

6200 MD Maastricht, The Netherlands

Risk factors for diet-related disorders are oxidative stress and chronic inflammation. Wheat is a source of phytochemicals with antioxidantactivity that might play a role in the observed protection of whole-grain diets against metabolic disorders.

The aims of the present study were first to investigate the bioaccessibility of antioxidant and anti-inflammatory activity from differentwheat fractions during gastrointestinal (GI) transit and second to demonstrate the contribution of wheat compounds to these health factors.

Experiments were performed in the TIM system, which is a dynamic computer-controlled model consisting of gastric, duodenal, jejunaland ileal compartments simulating conditions in the human GI tract(1,2). Samples were collected in 1 h aliquots for 6 h from the dialysatesof the jejunal and ileal compartments to measure the kinetics of bioaccessibility of antioxidant capacity and anti-inflammatory responses.Antioxidant capacity (Trolox equivalent antioxidant capacity assay) and ferulic acid, polyphenol and protein contents were determined.Anti-inflammatory effects were measured in extracts and TIM samples using a human macrophage cell system with lipopolysaccharide(LPS)-induced TNFa and IL-6 secretion.

Antioxidant capacity was unevenly distributed within the wheat fractions (aleurone fractions>bran fractions>flour fractions), withoutdifferences between cultivars (Tiger and Crousty). Antioxidant capacity was correlated with the ferulic acid content (R 0.96, P<0.00001).Ferulic acid was the major contributor to the antioxidant capacity in bran and aleurone fractions (50–60%). However, ferulic acid did notreduce TNFa and IL-6 levels in a dose-dependent manner. The TIM experiments showed that the antioxidant capacity was bioaccessibleduring GI digestion. In the case of the bran and aleurone fractions the bioaccessibility was significantly lower (20–30% initial antioxidantcapacity) than that from the flour fraction. The bioaccessibility of ferulic acid was similarly low from the bran and aleurone fractions. Inexperiments with flour ferulic acid was below the detection limit in the dialysate samples, because of the low initial level at oral intake.The dialysates samples collected from TIM showed reduced levels of TNFa for the bran and aleurone fractions, reaching maximalinhibition at the 2nd hour and 3rd hour (Figure). These data corresponded with the maximum levels of the bioaccessibility of theantioxidant capacity from these fractions.

0

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LPS 1 2 3 4

% T

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-α (

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Figure. Variations in TNFa induced by dialysate samples from the jejunal compartment from 1st hour to 4th hour (1–4). The dialysate samples represent the absorption from thein vitro digestion of starch and the wheat fractions of the variety Tiger (flour, bran and aleurone). Values are means and standard deviations represented by vertical bars for threedifferent days.

This publication is financially supported by the European Commission in the Communities 6th Framework Programme, Project HEALTHGRAIN(FOOD-CT-2005–514008). It reflects the authors’ views and the EC is not liable for any use that may be made of the information contained in thispublication.

1. Minekus M & Havenaar R (1998) Reactor system. European Patent no. 0642382. Eur Patent Bull 98/07, Art. 97(4) and (5) EPC, dated 11.02.98.2. Minekus M, Marteau P, Havenaar R & Huis in ’t Veld JHJ (1995) Altern Lab Anim 23, 197–209.



Elicitation of an allergic reaction in mice orally sensitized to whey orcasein proteins

A. J. Nauta1, B. Schouten2, B. C. A. M. van Esch2, S. van Doorn1, G. A. Hofman2,L. W. J. van den Elsen2, L. E. M. Willemsen2, L. M. J. Knippels1 and J. Garssen1,2

1Immunology, Numico-Research BV, Wageningen, The Netherlands and 2Pharmacology and Pathophysiology,

UIPS, Utrecht University, The Netherlands

Cow’s-milk allergy (CMA) is the most common food allergy in children, affecting 1–2% of all infants. So far, the pathogenesis of thedisease is incompletely understood, and no effective treatment is available to cure or actively prevent food allergy. Animal models mayprovide a useful tool to explore the mechanisms underlying the development of CMA and to identify new therapeutic strategies. Thepurpose of the present study was to develop a murine model of IgE-mediated cow’s milk hypersensitivity that closely mimics the clinicalfeatures of human CMA.

Female C3H/HeOuJ mice (5 weeks old; n 6) were sensitized by intragastric administration of casein or whey, using cholera toxin (CT)as an adjuvant (methods adapted from Li et al. 1999 and Frossard et al. 2004(1,2)) and boosted five times at weekly intervals. At week 7the mice were challenged subcutaneously (ear) and orally. Serum levels of mouse mast cell protease-1 (mMCP-1), total IgE and allergen-specific IgE, IgG1 and IgG2a were measured. The acute allergic skin reaction was determined by measuring ear swelling.

An antigen-specific acute allergic skin response was induced in casein- and whey-sensitized mice (mm; 71.2 (SD 8.4) and 137.9 (SD 21.7)respectively v. - 4.6 (SD 4.7) for CT controls; P<0.01). Total IgE and mMCP-1 serum concentrations were enhanced in both the casein-and whey-sensitized mice. In whey-sensitized mice whey-specific IgE, IgG1 and IgG2a serum levels were enhanced, while in casein-sensitized mice only casein-specific IgG1 was increased (Table). In casein-sensitized mice the number of mast cells per villus–crypt unitwas enhanced compared with whey-sensitized and CT control mice (1.0 (SD 0.2) v. 0.1 (SD 0.1) v. 0.3 (SD 0.0); P<0.01).

Taken together these results suggest that the oral administration of whey and casein elicit an allergic reaction in mice that mimicsimmediate CMA in human subjects. Differential pathophysiological changes were observed in whey-sensitized and casein-sensitizedmice. The serology of whey-sensitized mice more closely resembles the human situation, whereas casein-sensitized mice develop gas-trointestinal symptoms similar to the clinical features of human CMA. These mouse models of CMA provide a useful tool to examine themechanism underlying the development of CMA and to explore new therapeutic strategies for the treatment of food allergy.

Sensitized

Whey Ig Casein Ig

Control* SD Whey* SD Control* SD Casein* SD

IgE 0.28 0.02 1.67 0.33 0.11 0.01 0.26 0.32IgG1 0.22 0.09 0.99 0.17 0.09 0.04 0.54 0.33IgG2a 0.25 0.07 0.59 0.20 0.13 0.01 0.23 0.23

* Presented as OD values.

1. Frossard CP, Hauser C & Eigenmann PA (2004) J Allergy Clin Immunol 114, 377–382.2. Li XM, Schofield BH, Huang CK, Kleiner GI & Sampson HA (1999) J Allergy Clin Immunol 103, 206–214.



Effect of enteral nutrition on plasma soluble adhesion molecules inan elderly population

J. Olza1, M. D. Mesa1, R. Moreno2, A. Perez de la Cruz2, A. Gil1 and C. M. Aguilera1

1Institute of Nutrition and Food Technology, Dept Biochemistry and Molecular Biology II, University of Granada, Granada,

Spain and 2Universitary Hospital Virgen de las Nieves, Unit of Clinical Nutrition and Dietetic, Granada, Spain

The entry of inflammatory cells into the arterial wall is mediated by adhesion molecules expressed on the endothelial surface by binding totheir counter-ligands on the leucocytes. The origin of soluble adhesion molecules such as endothelial selectin (sE-selectin), intercellularadhesion molecule 1 (sICAM-1) and vascular cell adhesion molecule 1 (sVCAM-1) is mainly the activated endothelium. Thus, theyare considered to be biomarkers of the inflammatory state of the arterial wall(1). sICAM-1 appears to be a general marker of a pro-inflammatory status, whereas sVCAM-1 emerges as a predictor for future cardiovascular events in patients with pre-existing disease. BothDHA and EPA have been found to decrease the expression of soluble adhesion molecules in studies of the cytokine-activated endotheliumin vitro(2). Enteral nutrition may influence inflammatory molecule secretion in patients under pathological conditions. Feeding long-chainn-3 PUFA results in partial replacement of arachidonic acid in cell membranes by EPA. A number of experimental and human trials havedemonstrated immunomodulatory properties of n-3 PUFA, by suppressing different variables of immune function, including the produc-tion of inflammatory cytokines and eicosanoids(3). There are no reports of studies that have evaluated the influence of dietary EPA andDHA on soluble adhesion molecules in elderly patients fed an enteral diet, which is the aim of the present study.

Thirty-two patients aged 75 years were fed a complete formula for enteral nutrition (T-Diet Plus1; Vegenat SA, Madrid, Spain) withadded EPA (75 mg/l) and DHA (35 mg/l), as compared with standard diets. Patients were monitored for 6 months, and blood samples weretaken at the beginning and after 3 and 6 months of feeding. At the end of the experimental period only sixteen patients remained in thestudy. Mean daily intake was 5459 (SE 130) kJ/d. Plasma EPA and DHA were measured by GC in order to establish fatty acid incor-poration; EPA increased by approximately 41% while DHA increased by 6%. The plasma soluble adhesion molecules sE-selectin,sICAM-1 and sVCAM-1 were measured by immunoassay with a LINCOplexTM kit (Millipore Corp., Billerica, MA, USA) using theLuminex 200 System built on XMAP technology (Luminex Corp., Austin, TX, USA). A non-parametric Wilcoxon test was used todetermine statistical differences between dietary interventions after 3 or 6 months (P £0.05).

T-Diet Plus1sE-selectin (ng/ml) sICAM-1 (ng/ml) sVCAM-1 (ng/ml)

Mean SE Mean SE Mean SE

Initial 35.0 3.37 238.9 29.0 1161 91.83 month 34.0 2.34 254.6 34.8 1134 70.26 month 29.5 3.87 286.6 59.3 1288 90.4

sE-selectin tended to decrease over the 6-month period after dietary intervention, although differences were not significant, probablybecause of the great variation between patients with a number of underlying pathologies. sICAM-1 and sVCAM were not modified afterthe experimental period. The decrease in some plasma adhesion molecules may indicate an improved outcome for elderly patients fedenteral diets, which may have the potential to be complementary to pharmacological treatments.

This study was financed by Vegenat SA.

1. Paoletti R, Gotto AM Jr & Hajjar DP (2004) Circulation 109, 20–26.2. Yaqoob P & Calder P (2003) Eur J Med Res 8, 337–354.3. Mesa MD, Aguilera CM & Gil A (2006) Nutr Hosp 21, 105–118.



Serum C3c and C4c concentrations and adenosine deaminase activity ofchildren with cystic fibrosis: preliminary study

P. Perris1, M. S. Feliu1, S. Barbeito2, I. Strasnoy2, M. Ferraro2 and N. Slobodianik2

1Department of Nutrition, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina and2Nutrition Service, Pedro Elizalde Hospital, Buenos Aires, Argentina

Nutrition is a critical component of the management of cystic fibrosis (CF), and nutritional status is directly associated with bothpulmonary status and survival(1). Previous results have shown that several serum fractions (apoB, transferrin) related to the nutritionalstatus of children with CF are diminished(2). The present preliminary study measured in the same group of children specific serum proteinsassociated with the immune system and the activity of adenosine deaminase (ADA), an enzyme associated with T lymphocytes(3,4).Sixteen children of both genders with CF, between 5 months and 11 years of age, were evaluated between September 2005 and February2007,with assistance from the Nutrition Service. Samples of whole blood were collected from fasting patients. C3 and C4 complementfractions (C3c, C4c) were measured by single radial immunodiffusion techniques using commercially-available kits (Diffuplate; Bio-cientifica, Buenos Aires, Argentina)(5). The activity of ADA was determined by the method of Giusti & Galanti(6). The results werecompared with reference values obtained from healthy children matched for age and gender.

Group

C3c (mg/l) C4c (mg/l) ADA (U/l)

Mean SD Mean SD Mean SD

CF 882* 356 183* 42 36.3* 15.8Reference 1263 455 275 77 23.0 5.6

Mean values were significantly different from those for the reference group: *0.001<P<0.02.

Significantly decreased C3c and C4c values with a concomitant increase in the activity of ADA were observed in patients with CF. Theincrease in the activity of ADA would be an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, whichwould be toxic for T-cell lymphocytes(4). These preliminary results suggest that the immune system, evaluated using serum levels of C3cand C4c and the activity of ADA, is altered. Specific nutritional support should be established and adjusted to individual needs.

Partially supported by University of Buenos Aires (Grant B-060).

1. Dodge JA & Turck D (2006) Best Pract Res Clin Gastroenterol 20, 531–556.2. Perris P, Barbeito S, Strasnoy I, Ferraro M, Ramos O & Slobodianik N (2007) Proceedings of the AACC Annual Meeting. Poster-B-56.

http://www.aacc.org/AACC/events/ann_meet/annual2007/3. Kurashige S, Akusawa X, Yoshida T, Teshima C, Kodama K & Mitsuhashi S (1982) Microbiol Immunol 26, 87–92.4. Feliu MS & Slobodianik NH (2000) Nutrition 16, 1082–1083.5. Mancini G, Carbonara AO & Heremans GF (1965) Immunochemistry 2, 235–254.6. Bergmeyer HU (1965) Methods of Enzymatic Analysis. New York: Academic Press.



Introduction of complementary foods to the infant diet within thefirst year of life: evaluation of general recommendations using

Achievable Benchmarks of Care�

N. Pastor1, B. Soler2, C. Lifschitz1,3 and The Generacion Study Group1Mead Johnson Nutritionals, Madrid, Spain, 2E-C-BIO, SL Estudios Cientıficos, Spain and

3Division of Gastroenterology and Nutrition, Texas Children’s Hospital and Children’s Nutrition Research Center,

Baylor College of Medicine, TX, USA

The primary objective of the present study was to assess the effect of DHA and arachidonic acid (ARA) supplementation of infantformulas on the incidence of respiratory illnesses during the first year of life. Enrolment for this multicentre prospective open-label12-month observational study was conducted from 2002 to 2003 and included 1392 children from 357 Spanish paediatricians (theGENERACION Study Group). Infants were assigned in the proportion 4.4:1 to receive a formula supplemented with 3.2 g DHA and 6.4 gARA/kg or a low or non-supplemented control formula. Eligible infants were healthy, born at a gestational age that exceeded 36 weeksand non-breast-fed. Infants were to visit the paediatrician at baseline and months 1, 3, 5, 7, 9, and 12. At each subsequent visit recordswere taken of: anthropometric measurements; month of introduction for gluten-free cereal, gluten, fruits, vegetables, meat, fish, egg yolk,whole egg, cow’s milk and legumes; the occurrence of clinical symptoms associated with common ailments in infancy. Results of themain objective have been published elsewhere(1,2), and showed a significantly lower incidence of bronchitis or bronchiolitis inDHA +ARA-fed children. The secondary objective was to determine the adherence to paediatricians’ recommendations to guidelines(3,4)

on the introduction of complementary foods to the infant’s diet. Achievable Benchmarks of Care (ABC)1 ratios were also calculated todetermine the standards of excellence attained by the 10% of top performers(5) and identify areas for improving adherence to guidelineson the introduction of complementary foods(6). The Table summarizes the findings from the study. Overall, the adherence was appropriatefor most recommendations, but some recommendations need to be reinforced, such as the introduction of fruits, fish, cow’s milk andlegumes.

Recommendation n % Adherence ABC1 (%)

Cereal without gluten from 4 months of age 999 97.2 98.9Cereal with gluten from 6 months of age 829 94.5 100Fruits from 6 to 7 months of age 1001 16.1 30.3Vegetables from 6 to 7 months of age 989 82.9 94.3Meat from 6 to 7 months of age 974 92.4 100Fish from 9 to 10 months of age 921 75.5 97.8Egg yolk from 9 to 10 month of age 910 91.1 100Whole egg from 12 months of age 790 84.4 100Cow’s milk from 12 months of age 530 55.5 79.4Legumes from 12 months 679 59.6 83.1

1. Pastor N, Soler B, Mitmesser S, Ferguson P & Lifschitz C (2006) Clin Pediatr 45, 850–855.2. Pastor N, Soler B, Ferguson P & Lifschitz C (2005) J Pediatr Gastroenterol Nutr 40, 698–699.3. Ministry of Health (2001) Consejo de buen Provecho para tus Hijos. Alimentacion infantil (Guide to Recommended Intakes. Infant Nutrition). Direccion

General de Salud Publica M-52.662–2001; www.msc.es4. European Society of Paediatric Gastroenterology, Hepatology and Nutrition Committee on Nutrition (1982) Acta Paediatr Scand Suppl 302, 61–95.5. Weissman NW, Allison JJ, Kiefe CI et al. (1999) J Eval Clin Pract 5, 269–281.6. Kiefe CI, Allison JJ, Williams OD, Person SD, Weaver MT & Weissman NW (2001) JAMA 285, 2871–2879.



Plasma protein supplements modulate the activation of gut-associatedimmune system induced by Staphylococcus aureus enterotoxin B in rats

A. Perez-Bosque1, L. L. Miro1, J. Polo2, L. Russell3, J. Campbell3, E. Weaver3, J. Crenshaw3

and M. Moreto1

1Departament de Fisiologia, Facultat de Farmacia, Universitat de Barcelona, Spain, 2APC Europe, Granollers,

Spain and 3APC Inc, Ankeny, IA 50021, USA

Supplementation of diets with plasma protein has been shown to prevent the activation of lymphocyte populations of Peyer’s patches andmesenteric lymph nodes(1) and improve the intestinal epithelial barrier function in a rat model of intestinal inflammation(2). The presentstudy investigated the effects of porcine plasma proteins (SDAP) and Ig concentrate (IC) supplements on diffuse gut-associated lymphoidtissue in a model of mild intestinal inflammation. The different populations of lamina propria and intraepithelial lymphocytes, as well asmucosal expression of cytokines (interferon-g (IFN-g), TNFa, IL-6 and IL-10) and pro-inflammatory mediators (inducible NO synthase(iNOS) and leukotriene B4 (LTB4)), were investigated. Wistar-Lewis rats were fed diets supplemented with SDAP (80 g/kg; n 9), IC (15 g/kg; n 9) or milk proteins (control die; n 9) from weaning (day 21) to day 33 or 34 after birth. On days 30 and 33 rats were administered S.aureus enterotoxin B (SEB; 0.5 mg/kg). Experimental groups were control, SEB, SEB-SDAP and SEB-IC. Lymphocyte populations wereanalysed by immunohistochemistry on day 34 (i.e. 24 h after SEB administration). The markers used were: CD3 (T lymphocytes), CD25(activated T lymphocytes), CD4 (T-helper lymphocytes), CD8 (T-suppressor/cytotoxic lymphocytes), TCRgd (T-gd lymphocytes) andNKPR1A (NK cells). Cytokines were determined by a cytometric bead array assay, LTB4 by commercial ELISA and iNOS by real-timePCR in mucosal homogenates, all at 6 h after SEB administration.

In both lamina propria and epithelium compartments SEB increased the lymphocyte cytotoxic populations (T-gd 40% and 70%; NKcells 60% and 75% respectively, all P<0.05) and doubled the number of activated T lymphocytes (P<0.001). Both SDAP and ICprevented the SEB effects on the lamina propria, while in the epithelium only SDAP reduced the extent of T-cell activation (P<0.05).SEB increased mucosal iNOS expression by 28% (P<0.05) and both plasma protein supplements prevented SEB effects on iNOSexpression in the intestinal mucosa (both P<0.05).

In the mucosa SEB doubled IFN-g and LTB4 concentrations and increased TNFa and IL-6 concentrations by 20–30%; P<0.05). SDAPpartially prevented these effects on IFN-g , IL-6 and LTB4 (P<0.05). IC was also effective in reducing the expression of TNFa and LTB4

in the mucosa (P<0.05). It is concluded that dietary supplementation with plasma proteins can attenuate the intestinal inflammatoryeffects induced by SEB.

1. Perez-Bosque A, Pelegrı C, Vicario M et al. (2004) J Nutr 134, 2667–2672.2. Perez-Bosque A, Amat C, Polo J, Campbell JM, Crenshaw J, Russell L & Moreto M (2006) J Nutr 136, 2838–2843.



Dietary fish oil increases IL-4 secretion by murine splenocytes byan effect on accessory cells

D. H. Petursdottir and I. HardardottirDepartment of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland

Dietary fish oil has immunomodulatory effects(1). It decreases T-cell proliferation, reduces macrophage eicosanoid synthesis and hasdifferent effects on cytokine secretion by T-cells and macrophages. The present study examined the effects of dietary fish oil on secretionof T-helper (Th) 1 and Th2 type cytokines by splenocytes and the involvement of accessory cells in the effects of dietary fish oil oncytokine secretion.

Mice were fed diets supplemented with (g/kg) 180 fish oil+ 20 maize oil or 200 maize oil for 6 weeks (n 10). Spleen cells, isolatedT-cells, and splenocytes depleted of T-cells were stimulated with concanavalin A (ConA), anti-CD3 or anti-CD3/anti-CD28. Secretion ofthe Th1 and Th2 cytokines interferon-g (IFN-g) and IL-4 and the pro- and anti-inflammatory cytokines TNFa and IL-10 were measuredby ELISA.

Dietary fish oil decreased ConA-, anti-CD3-, and anti-CD3/anti-CD28-induced secretion of IFN-g and TNFa by total splenocytes andisolated T-cells (P<0.05). On the other hand, dietary fish oil increased secretion of IL-4 by total splenocytes (P<0.05) without an effecton IL-4 secretion by isolated T-cells (Figure). When isolated T-cells were cultured with CD11b + cells, T-cells from mice fed the fish oildiet secreted more IL-4 than T-cells from mice fed the maize oil diet (P<0.05; Figure).

Total T cells T + CD11b+ cells

IL-4 secretion following stimulationwith anti-CD3/anti-CD28

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The results from the present study demonstrate that dietary fish oil directs the immune response of splenocytes towards a Th2 phenotypeand that the effects of dietary fish oil on secretion of Th2 type cytokines are mediated by its effect on CD11b+ accessory cells.

1. Calder PC (1997) Ann Nutr Metab 41, 203–234.



Effects of borage (Borago officinalis) oil supplementation on theexpression of monocyte and T-cell adhesion molecules in

healthy volunteers

E. Pinto1 and L. S. Harbige1,2

1Centre for Biosciences Research, School of Science, University of Greenwich, Kent, UK and 2Medway School of

Pharmacy, University of Kent and University of Greenwich, Kent, UK

Essential fatty acids (EFA) have unique roles in that they can affect inflammatory cell functions directly or as precursors to molecules thatcan regulate their functions(1). In comparison with linoleic acid (LA; 18: 2n-6), g-linolenic acid (GLA; 18: 3n-6) has superior biopotencybecause it bypasses D6 desaturation, which is regarded as a key regulatory rate-limiting enzymic step controlling the formation of long-chain PUFA and is beneficial in some inflammatory disorders(1,2). In the present study seven healthy volunteers ingested 14 g GLA-richborage oil/d consecutively for 13 weeks. Peripheral blood was obtained at 0, 4, 7 and 13 weeks and the expression of the adhesionmolecules CD11a + , CD11b+ , CD36 + , CD54+ and CD62L + on monocytes (CD14 + ) and CD11a + , CD54 + and CD62L + on T-cells(CD3 + ) was investigated using specific conjugated monoclonal antibodies and flow cytometry. Cell surface expression of CD62L + ,CD36+ and CD54 + on monocytes after 4, 7 and 13 weeks was significantly lower (P<0.001, P<0.005 and P<0.01 respectively)compared with baseline expression. CD62L+ and CD54 + expression on T-cells after 4 and 7 weeks of supplementation was alsosignificantly lower (P<0.05 and P<0.01 respectively) compared with baseline expression. The Figure illustrates flow cytometric analysisof CD14 +CD62L + expression before and after 13 weeks of borage oil supplementation.

(A) Before (B) After

These results demonstrate that GLA can significantly decrease cell surface expression of certain adhesion molecules particularly onmonocytes, suggesting that this may be one of the mechanisms by which it is beneficial in some human and experimental autoimmuneinflammatory disease states(1,2).

1. Harbige LS (2003) Lipids 38, 323–341.2. Harbige LS, Yeatman N, Amor S & Crawford MA (1995) Br J Nutr 74, 701–715.



Effect on lymphoproliferation and in vitro Ig production of splenocytesfrom suckling rats when supplemented with conjugated linoleic acid

C. Ramırez-Santana, F. J. Perez-Cano, M. Castell, A. Franch and C. CastelloteDepartment of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of linoleic acid that contain two conjugated doublebonds, of which cis-9, trans-11 and trans-10, cis-12 predominate. Besides the health effects described in human subjects, it has beenreported that CLA modulates immune cell functions in animal models, although there are no reports on this effect during early life. Thus,the objective of the present work was to establish the effect of CLA supplementation during gestation and/or lactation on ability of spleenlymphocytes to produce Ig and proliferate under ex vivo conditions. Pups were divided into four groups (A, B, C and W), with each groupcomprising ten rats per lactating mother. Mothers of C and W group pups were fed standard pellet chow from day 7 of gestation andthroughout the study period. Mothers of group A and B pups were fed pelleted chow with 10 g CLA (80% cis-9, trans-11, 20% trans-10,cis-12; Lipid Nutrition B.V. Wormerveer, The Netherlands)/kg during gestation and mothers of group A pups continued with thissupplemented chow until weaning. Groups B and C received the CLA isomer mixture by daily oral supplementation while dams were fedstandard pelleted chow during lactation. Animals were killed and their spleens were removed at day 21, the end of suckling period.Splenocytes were isolated and cultured. After incubation for 7 d, in vitro IgG and IgM production was quantified using the ELISAsandwich technique. Cells stimulated with phorbyl myristate acetate–ionomycin (250 ng/ml) were used to: (1) quantify IL-2 and inter-feron-g (IFN-g) levels in culture supernatant fractions after 24 h incubation using ELISA; (2) to measure cell proliferation after incubationfor 72 h (see Table). Cell viability was also evaluated. Conventional ANOVA and post hoc comparisons (LSD test) were performed.Significant differences were accepted at P<0.05. No differences among groups were seen in in vitro IL-2 and IFN-g production orlymphocyte proliferation (see Table), irrespective of life period (gestation or suckling), duration and route of supplementation. However,spontaneous IgM production by splenocytes from group B animals was higher compared with that of groups C and W (P<0.05;see Table). These results suggest that supplementation with CLA during the gestation and suckling periods increases spontaneous Igproduction by immunocompetent cells.

Group

IgM production (ng/ml) Proliferation (%)

Mean SE Mean SE

A 937.9 144.0 196.9 37.60B 1225.6*† 193.5 257.2 29.33C 539.5 139.7 234.5 57.89W 534.9 157.3 191.5 33.23

n 10. Mean value was significantly different from that for group C: *P<0.05. Mean valuewas significantly different from that for group W: †P<0.05.



Anti-inflammatory effects of cocoa in rat carrageenin-inducedpaw oedema

S. Ramos-Romero, E. Ramiro-Puig, F. J. Perez-Cano, C. Castellote, A. Franch and M. CastellDepartment of Physiology, Faculty of Pharmacy, University of Barcelona, Spain

Acute inflammation is the fastest immune system response to damage. Normally, short-lasting and limited inflammation develops beneficialeffects. However, permanent inflammation becomes harmful and painful. Carrageenin-induced paw oedema is a reproducible acuteinflammatory model used in the screening of anti-inflammatory drugs. Cocoa, a product from Theobroma cocoa seeds, contains flavonoidswith potential anti-inflammatory properties. Previous studies have shown that in vitro cocoa extract inhibits some pro-inflammatorycytokines (TNFa, monocyte chemotactic protein 1 (MCP-1) and IL-6)(1). The aim of the present study was to establish the in vivo anti-inflammatory effect of cocoa. Female Wistar rats (Harlan Iberica SA, Barcelona, Spain) received water (reference animals; n 10), quercetin(3.1 g/kg body weight orally; n 10) or cocoa at doses of 4.6 (n 10) or 9.6 (n 10) g/kg body weight orally for 7 d. On day 8 some referenceanimals were injected with quercetin (10 mg/kg rat intraperitoneally (ip)). After 1 h all animals were injected with 0.1 ml carrageenin l(1%, w/v) into subplantar area of the right hind paw. Paw volume was measured by plethysmometry at 30 min post carrageenin injectionand every hour for 6 h. Peritoneal macrophages (PM) and paw inflammatory exudates were obtained at the end of the study. PM werestimulated with lipopolysaccharide (LPS; 10mg/ml) and supernatant fractions were obtained after 24 h incubation. TNFa levels in exudatesand supernatant fractions were determined by an ELISA technique. A significant reduction in paw oedema (P<0.05) was observed in bothgroups treated with cocoa from 4 h after carrageenin injection (Figure). At 6 h paw volumes from cocoa-treated animals were about 35%lower than that of the reference group. Quercetin treatment did not modify paw volume. TNFa levels in paw inflammatory exudates werelower in animals treated with the highest cocoa dose (Table). However, TNFa secreted by PM was reduced in the group treated with 4.6 gcocoa/kg and in animals injected with quercetin ip. In summary, a cocoa-rich diet could play a role as an anti-inflammatory adjuvant. Thisimmunomodulatory action can be partially attributed to a reduction in cytokine release by pro-inflammatory cells such as macrophages.

time from induction (h)

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*

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*

TNFa (pg/ml )

Inflammatory exudate Non-stimulated PM LPS-stimulated PM

Mean SE Mean SE Mean SE

Reference 82.4 12.5 5991.3 685.2 11780.9 1444.6

Quercetin: ip 106.6 18.3 327.6** 87.7 1810.3** 410.6Orally 63.4 15.1 4395.6 1147.3 7925.2 1954.4

Cocoa: 4.8 g/kg 65.4 10.1 1801.2* 1037.7 2444.0** 1217.79.6 g/kg 38.5* 12.2 6719.7 2896.3 12309.7 4489.7

Mean values were significantly different from those for the reference group: *P<0.05, **P<0.001.

1. Ramiro E, Franch A, Castellote C, Perez-Cano F, Permanyer J, Izquierdo-Pulido M & Castell M (2005) J Agric Food Chem 53, 8506–8511.



Lactobacillus fermentum exerts a beneficial effect inan experimental model of rheumatoid arthritis in mice

M. E. Rodrıguez-Cabezas1, F. Fisac1, E. Bailon1, M. Comalada1, D. Camuesco1, J. Xaus2,A. Concha3, P. Talavera3, A. Nieto4, A. Zarzuelo1 and J. Galvez1

1Department of Pharmacology, University of Granada, Spain, 2Puleva Biotech SA, Granada, Spain, 3Department of

Pathology, Hospital Universitario ‘Virgen de las Nieves’, Granada, Spain and 4Health and Progress Foundation,

Granada, Spain

Previous studies have shown the intestinal anti-inflammatory activity of the probiotic Lactobacillus fermentum CECT 5716 acting as amodulator of the intestinal immune response(1). The aim of the present study was to determine the efficacy of this probiotic as a systemicimmune modulator in the experimental model of rheumatoid arthritis induced by Mycobacterium butyricum–Freund’s adjuvant in mice(2).Male Balb/C mice (n 10) were given the probiotic in the drinking water (108 colony-forming units (CFU)/ml) for 2 weeks and a controlgroup (n 20) not receiving probiotic was also used for reference. After this period 0.1 ml of a suspension of 10 mg Mycobacteriumbutyricum in 10 ml Freund’s adjuvant was administered subcutaneously in the right paw in treated mice and in half the control group,whereas Freund’s adjuvant was administered in the left paw of the mice; normal mice (n 10) only received Freund’s adjuvant in bothpaws. Animals were assessed for clinical arthritis for 4 weeks, and after this period all mice were killed and their arthritic status evaluatedby histological analysis of the paws. In addition, the spleens were removed and T-cells were obtained from spleens and activated withconcanavalin A and the lymphocyte response evaluated by RT–PCR for the cytokines IL-2 and IL-10.

The results showed that the probiotic treatment ameliorated the severity of the disease since it significantly improved the clinicalarthritis score (6.0 (SE 0.7) in control mice with arthritis v. 3.3 (SE 0.3) in probiotic-treated mice; P<0.05) (Figure 1). Similar findings wereobtained by histological analysis. Mycobacterium-induced arthritis also stimulated the expression of IL-2 in the splenocytes, which wasalso reduced in the probiotic-treated mice. Although IL-10 expression by splenic T-cells was not modified during the course of arthritis incontrol mice, treatment with the probiotic stimulated the expression of this regulatory cytokine in mice with arthritis. In conclusion,probiotic treatment ameliorated the disease outcome in the Mycobacterium butyricum model of rheumatoid arthritis in mice, showingsystemic immunomodulatory properties.

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Figure 1: CLINICAL ARTHRITIS SCORE

Non arthritic Control arthritic L.fermentum

1. Peran L, Camuesco D, Comalada M et al. (2006) Int J Colorectal Dis 21, 737–746.2. Mihara M, Nishimoto N, Yoshizaki K & Suzuki T (2002) Immunol Lett 84, 223–229.



Immunological changes after dehydration resulting from physical effortin a hot environment

J. Romeo1, D. Jimenez-Pavon2,3, M. Cervantes-Borunda4, J. Warnberg1, L. E. Dıaz1 and A. Marcos1

1Immunonutrition Research Group, Department of Metabolism and Nutrition, Consejo Superior de Investigaciones

Cientıficas, Madrid, Spain, 2Research Group EFFECTS 262, Department of Medical Physiology, School of Medicine,

University of Granada, Granada, Spain, 3Facultad de Ciencias de la Actividad Fısica y del Deporte, Universidad

Politecnica de Madrid, Madrid, Spain and 4Facultad de Educacion Fısica y Ciencias del Deporte, Universidad

de Chihuahua, Mexico

Intensive exercise is well known to affect the immune function, leading to higher risk of infections and inflammatory processes insportsmen(1–3). Several factors are involved in the immune modulation, such as the intensity and duration of the physical activity as wellas inter-individual variations(4,5). Thus, the aim of the present study was to confirm the effects of intensive physical effort in a relativelyshort time period and high environmental temperature on variables of immune function and inflammatory markers. A total of twenty-twoyoung male adults (age 21.2 (SD 1.7) years; VO2max 55.4 (SD 3.6) ml/kg per min) volunteered to participate in an exercise session of 60 minon a treadmill ergometer at moderate speed (60% maximum aerobic speed) in hot environmental conditions (35�C and humidity 65%).Total leucocyte and lymphocyte counts, absolute values of T-lymphocyte CD8+ , CD4+ , CD3+ , natural killer and CD19 + subsets andthe capacity of cytokine production (IL-2, IL-4, IL-5, IL-10, interferon-g and TNFa) were evaluated before and after exercise withoutrehydration. Several inflammation-related proteins (caeruloplasmin, C-reactive protein, C3 and C4) were also measured. The results showthat absolute numbers of leucocytes and neutrophils increased while eosinophils decreased after the intensive exercise. In addition,caeruloplasmin, C3 and C4 levels also increased after exercise. In contrast, no changes in T-lymphocyte subsets or cytokine productionwere observed.

Before exercise After exercise

Mean SD Mean SD

Leucocytes (·109/l) 5.73 1.48 8.84* 2.39Neutrophils (·109/l) 3.56 1.10 6.83* 2.17Lymphocytes (·109/l) 1.68 0.44 1.59 0.30Eosinophils (·109/l) 0.17 0.13 0.08* 0.05Caeruloplasmin (mg/l) 325.8 49.6 342.8* 56.3C3 (g/l) 0.99 0.14 1.04* 0.13C4 (g/l) 0.23 0.07 0.25* 0.07

Mean values were significantly different from those before exercise (Student’s t test): *P<0.05.

Since prolonged bouts of intensive exercise cause a temporary reduction in various aspects of immune function that usually lasts 3–24 hafter exercise, the results confirm those from previous studies and suggest that 60 min exercise in a hot environment is enough to cause animmune system adaptation to these conditions leading to an increase in some immune cells and promoting inflammatory processes.Interestingly, these changes in immune cells have not altered the T-lymphocyte subsets and the capacity for cytokine production;therefore, immune cell function seems to be preserved in relatively-short-term acute exercise. Further research is necessary to determine towhat extent habitual intensive exercise under high environmental temperature can impair the immune system.

1. Gleeson M (2007) J Appl Physiol 15 (Epublication ahead of print version).2. Nieman DC (1997) J Appl Physiol 82, 1385–1394.3. Hoffman-Goetz L & Pedersen BK (1994) Immunol Today 15, 382–387.4. Shephard RJ (1997) Physical Activity, Training and the Immune Response. Carmel, IN: Cooper Publishing Group.5. Nieman DC & Pedersen BK (1999) Sports Med 27, 73–80.



Intake of some immunonutrients and ecoimmunonutrients byyoung high-level basketball players

G. Santos1,2, M. A. Buil2, J. A. Ferrero2, J. J. Delgado2, J. Silva2 and J. M. Soriano1

1Laboratory of Nutrition, Faculty of Pharmacy, University of Valencia, Av. Vicent Andres Estelles s/n 46100,

Burjassot Spain and 2Pamesa Valencia Basketball Club, Av. Germans Maristes s/n 46013, Valencia, Spain

An adequate nutritional intake is essential to preserve the health of basketball players. Several studies have demonstrated a low energyintake by student basketball players on training days, as well as a daily diet with an unbalanced composition of nutrients (too much fat anda low intake of Ca and Fe)(1–3). A new approach to the nutrition of basketball players is being developed, according to a project of theUniversity of Valencia (Project UV-AE-20070219), that aims to study immunonutrition and ecoimmunonutrition as the key factors inorder to enhance physical and athletic performance and to decrease the incidence of physical damage.

The aim of the present study was to determine the consumption of ecoimmunonutritional nutrients (fibre) and some immunonutritionalnutrients (Zn, Fe, Mg, vitamins A, C and E) by young basketball players.

The diet of ten male basketball players from Pamesa Valencia’s 12–13-year-old team (weight range 47.4–81.5 kg; height range 1.63–1.95 m) and eight male basketball players of Pamesa Valencia’s 16–17-year-old team (weight range 75.6–103.0 kg; height range 1.79–1.995 m) was studied. Three 24 h recalls per player (two weekdays and one day of a weekend) were used for data collection. DIAL version1.01 (Alce Ingenierıa, Madrid, Spain) was used to evaluate the dietary intake of the ecoimmunonutrients and immunonutrients investi-gated.

The youngest basketball players had an intake of Mg, Fe and vitamin C that exceeded the Spanish recommended daily intakes (RDI)(4)

by 20.7, 81.6 and 84.3% respectively. The consumption of vitamin A and vitamin E was lower than the RDI (12.3 and 28%) and the Znintake was adequate. The intake of fibre was lower than the Spanish final nutritional objective(5). The older group of basketball players hadan intake of Mg, Fe, Zn, vitamin C and vitamin E that exceeded the RDI by 38.6, 65.3, 29.3, 186.5 and 26.6% respectively. Theconsumption of vitamin A was 10.9% lower than the RDI and the intake of fibre was the adequate.

In view of the inadequate intake for some of the ecoimmunonutrients and immunonutrients, the basketball players, their relatives andtheir trainers and condition coaches are being educated to improve their knowledge of nutrition.

1. Dubnov G & Constantini NW (2004) Int J Sport Nutr Exerc Metab 14, 30–37.2. Leinus K & Oopik V (1998) Nutr Res 18, 683–691.3. Martinchik AN, Baturin AK, Petukhov AB, Baeva VS, Zemlianskaia TA, Sokolov AI, Peskova EV & Tysiachnaia EM (2003) Vopr Pitan 72, 35–40.4. Instituto de Nutricion (1994) Ingestas de Energıa y Nutrientes Recomendadas para la Poblacion Espanola (Recommended Energy and Nutrient Intakes

for the Spanish Population). Madrid, Spain: Departamento de Nutricion, Universidad Complutense.5. Serra Majem L & Aranceta Bartrina J (2001) Sociedad Espanola de Nutricion Comunitaria. Guıas Alimentarias para la Poblacion Espanola (Spanish

Society of Community Nutrition. Nutrition Guidelines for the Spanish Population). Madrid, Spain: SENC.



Moderate ingestion of beer reduces inflammatory and oxidativebrain events induced by aluminium in mice

A. Schultz1, R. Oliver1, M. Bautista1, M. J. Gonzalez-Munoz4, I. Meseguer4, A. Pena4,M. I. Sanchez-Reus2, J. Benedı3 and F. J. Sanchez-Muniz1

1Dpto Nutricion, 2Dpto Bioquımica y Biologıa Molecular II, 3Dpto Farmacologıa, Facultad de Farmacia,

Universidad Complutense, Madrid, Spain and 4Dpto Nutricion, Bromatologıa y Toxicologıa, Facultad de Farmacia,

Universidad de Alcala, Madrid, Spain

Al is a highly neurotoxic element and has been suggested to participate in the degeneration of cells in the brain of human subjects andexperimental animals. Although the exact mechanism by which the metal may influence degenerative brain processes in unknown, there isevidence that exposure to Al causes an increase in both oxidative stress and inflammatory events(1). On the other hand, Si intake affectsthe bioavailability of Al at the gastrointestinal level, and therefore the Al accessibility to the brain. The aim of the present study was toexamine the effect of supplementing Si in the diet, as silicic acid or by drinking beer, to prevent inflammation and oxidative stress in brainof mice exposed to oral Al. Four groups of male adult NMRI mice with an initial body weight of 30 g were studied: (1) negative controladministered with deionised water; (2) positive control receiving 450 mg Al(NO3)3/kg body weight per d; (3) positive treatment groupwith Si (diet 2 plus 50 mg silicic acid/l; (4) positive treatment group with beer (diet 2 plus beer, equivalent to 1 litre/d for human subjects).The Al and Si contents of beer and brain tissue were measured by inductively-coupled plasma MS and atomic emission spectrometryrespectively following wet ashing of the organic matter. Whole right hemibrains were dissected, frozen, homogenized and analysed formineral content and mRNA for superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and TNFa.

Brain Al levels were significantly lower (P<0.01) for the negative control animals than for those of groups 2–4. Si intake in the form ofbeer lowered, although not significantly (2.45mg/g v. 3.85mg/g), the brain Al levels of intoxicated mice (group 2). Quantitative RT–PCRshowed that administration of Al significantly decreased levels of both SOD and catalase mRNA for group 2, which were normal for thebrains of mice receiving silicic acid or beer. Furthermore, for group 2 TNFa and GPx mRNA levels were significantly increased (P<0.05;Figure) suggesting that Al induced inflammation and brain damage. These levels were approximately normal for groups 3 and 4 afterconsuming Si as silicic acid and beer respectively (P<0.05). There was a significant correlation between brain Si and Al levels and theexpression of some brain enzymes.

The results suggest that moderate beer consumption, by means of its Si content, effectively protects against the neurotoxic effects of Al.However, consumption of strong beers should be avoided because of the well-known negative effect of alcohol on the brain.

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Figure. The GPx values, expressed in arbitrary units, are means with their standard errors represented by vertical bars for determinations for twelve mice. a,b,cMeans with unlikesuperscript letters were significantly different (P<0.05; Kruskal-Wallis and non-parametric multiple comparison test).

This work has been supported by the Asociacion de Cerveceros Espanoles and project AGL-2005–07204-C02–01.

1. Campbell A (2006) J Alzheimers Dis 10, 165–172.



Impact of glycation on duodenal digestibility of Bowman-Birk inhibitors

J. M. Silvan and M. D. del CastilloInstituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain

Adverse effects of soyabean-containing diets include poor digestibility and allergy to soyabean proteins. To improve the nutritionalquality of soya foods inhibitors are generally inactivated by heating or eliminated by fractionation during food processing(1). Theseinhibitors could generate an immune response(2). Bowman-Birk (BBI) and Kuntiz (KTI) inhibitors are heat stable and they may beinactivated by glycation(3), which also reduces soyabean-protein allergenicity(4). The aim of the present study was to investigate theimpact of the BBI glycation on the activity of duodenal digestive enzymes in order to optimise the beneficial effects of soyabean proteins.

The BBI was glycated with glucose, fructose and fructooligosaccharides (FOS; Raftilose P95; Orafti Espana SL, Barcelona, Spain) inKOH (0.2%, w/v) at 60�C for 20 min. The protein glycation was confirmed by isoelectric focusing (IEF) and SDS–PAGE gel electro-phoresis. In vitro duodenal digestion was performed using the method described by Moreno et al.(5). Samples were incubated withtrypsin–chymotrypsin for 120 min.

Fig. 1 shows the IEF gel (pH 3–10) stained with Comassie Blue. Different electrophoretic profiles were obtained for native and heatedBBI samples (lanes 1 and 2) compared with BBI glycated with glucose, fructose and FOS (lanes 3–5). A decrease in positive chargeswas observed as a result of the involvement of basic amino acids (Lys and Arg) in the Maillard reaction (glycation). This analysisalso confirmed BBI stability to heat treatment at 60�C for 20 min. Heating had no effect on the isoelectric point of BBI. Fig. 2 shows theSDS–PAGE gel (10%, w/v) stained with Comassie Blue. The molecular mass (8 kDa) of native BBI (lane 1) was increased as aconsequence of glycation with glucose, fructose and FOS (lanes 2–4; Fig. 2(a)). All samples inhibited trypsin and chymotrypsin activities.Samples did not change their electrophoretic profiles after enzymic digestion (Fig. 2(b); lane 1, native BBI; lane 2, heated BBI; lanes 3–5BBI glycated with glucose, fructose and FOS respectively). Further studies are needed to establish the glycation conditions that cause areduction in BBI inhibitory activity against digestive enzymes and to identify the glycation product responsible for this change. Both IEFand SDS–PAGE analyses are suitable for following glycation of BBI and other soyabean proteins.

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Authors thank ORAFTI for kindly supplying commercial FOS. These studies were funded by projects AGL2004-05031 and ALIBIRD-CM-S0505/AGR-0153.

1. Friedman M & Brandon DL (2001) J Agric Food Chem 49, 1069–1086.2. Kaayla TD (2004) Nexus Magazine 11; available at www.nexusmagazine.com3. Kato,Y & Matsuda T (1997) J Agric Food Chem 45, 3826–3831.4. van de Lagemaat J, Silvan JM, Moreno J, Olano A & Del Castillo MD (2006) Food Res Int 40, 153–160.5. Moreno FJ, Mellon FA, Wickham MS. Bottrill AR & Mills EN (2005) FEBS J 272, 341–352.



Lymphocyte subpopulations in Venezuelan preschool childrenof high socio-economic status

L. Solano and D. LloveraCentro de Investigaciones en Nutricion, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Edo,

Carabobo 2001, Venezuela

Nutrition is a critical determinant of the immune response, thus malnutrition is the most common cause of immunodeficiency in the world.Micronutrient deficiencies, especially Zn deficiency, have already been shown to be associated with modification of the immune responsein undernourished children, mostly those living in poverty; however, data on children of high socio-economic classes is lacking. The aimof the present study was to establish lymphocyte population values and to investigate possible interactions with nutritional status,Zn deficiency and socio-economic conditions in a group of children from high socio-economic classes.

The subjects were 104 preschool children attending a private nursery. Their nutritional status was measured by the weight-for-heightanthropometric indicator(1), lymphocyte subpopulations (CD3 + , CD4 + , CD8 + , CD20) by flow cytometry and serum Zn by atomicabsorption spectrophotometry. Socio-economic data were collected using the Graffar-Mendez method(2). Data are presented as means andstandard deviations, frequency and percentile distribution. Statistical analysis was by Student’s t test, ANOVA and Bonferroni test, with asignificance level of P<0.05.

A normal nutritional status was found in 74.3% of the children, 9.5% were undernourished and 16.2% were overweight. Lymphocytesubpopulations (CD3 + 65 (SD 6)%, CD4 + 34 (SD 6)%, CD8 + 28 (SD 6)%, CD20 18 (SD 5)%) and serum Zn (889 (SD 150)mg/l) werewithin normal values. There was no significant difference by gender, age or nutritional status. Low concentrations of serum Zn werepresent in 6.7% of the children. There was a tendency to lower (NS) values for the T lymphocyte population for those children with serumZn<730mg/l (10th percentile for the population), as shown in the Table.

Lymphocytes

Serum Zn £730mg/l Serum Zn >730mg/l

Mean SD Mean SD

T CD3 (%) 62 5 65 6T CD4 (%) 33 5 35 6T CD8 (%) 25 6 28 6T CD4:CD8 1.34 0.68 1.30 0.39B CD20 (%) 19 7 18 6

Since there are no reference values for the lymphocyte subpopulation in Venezuelan children and these results are in agreement withprevious reports relating to other population groups(3), the up-to-date data from the present study could be used as a reference for futurestudies.

These children from high socio-economic classes showed lymphocyte populations within the normal range and a low prevalence of Zndeficiency. Although a significant association between the lymphocyte population and serum Zn was not observed, the data provideinformation on the association between Zn concentrations and cell-mediated immunity expressed by T lymphocytes, as previouslyreported(4).

Poverty, infectious diseases and poor sanitation contribute to the development of undernutrition and specific micronutrient deficiencies,and to modifications of the cellular immune response. Maintenance of an adequate nutritional and immune status in the children evaluatedcould be associated with a protective environment related to their socio-economic status.

Project Sponsor: Consejo de Desarrollo Cientıfico y Humanıstico de la Universidad de Carabobo, Venezuela.

1. World Health Organization (1995) Physical Status: The Use and Interpretation of Anthropometry. Technical Report Series no. 854. Geneva: WHO.2. Mendez-Castellano H & Mendez MC (1994) Sociedad y Estratificacion. Metodo Graffar-Mendez-Castellano (Society and Stratification. The Graffar-

Mendez Method). Caracas, Venezuela: Fundacredesa.3. Comans-Bitter WM, de Groot R, van den Beernd R & Neijens HJ (1997) J Paediatr 130, 388–393.4. Long KZ & Nanthakumar N (2004) Am J Hum Biol 16, 499–507.



Timing of gluten introduction and quantity and nature ofgluten-containing foods consumed by infants in Valencia, Spain

B. Soto, A. Lopez and C. RibesPediatric Gastroenterology and Hepatology Section, La Fe Hospital, Valencia, Spain

Currently, there is no information on the level of gluten intake for Spanish children. Recent studies have shown that the timing of glutenintroduction in relation to breast-feeding as well as the amount of gluten could be related to a higher incidence of coeliac disease(1,2). Thefood questionnaire estimates how frequently certain foods are eaten in a specific period of time. The aim of the present study was todevelop and validate a food questionnaire for the assessment of gluten consumption of children aged 3–12 months in the Valencia area.

Information on the gluten-containing food intake of healthy children aged 3–12 months was collected prospectively, including the brandnames. For the study the children were assigned to two groups: 3–6 months; 7–12 months. Information was compiled by interviewingparents to establish how and when foods other than breast milk or formula milk were introduced into their children’s diet. The most-frequently-used gluten-containing food products were selected and included in the FFQ, which covered a period of 7 d. To calculate theamount of gluten in these products the vegetable protein content was multiplied by 0.8(3). To validate the FFQ the results of this FFQ werecompared with those from a 7 d food record(4). If gluten had been introduced before the study was conducted, the exact timing ofintroduction was recorded.

A total of 100 children were included in the study. In the age-category 3–6 months none of the thirty babies had started consuminggluten. Moreover, when asked about the timing of gluten introduction the thirty mothers with children aged between 7 and 24 months allreported that gluten had been introduced after the sixth month of life.

Age (Months) <7 7–8 8–9 >9

% children consuming gluten 0 60 70 100

In the 7–12 months age-group (n 70) the most important gluten-containing food products consumed were muffins and croissants,cereals, chocolate, ready-to-eat fruit and ready-to-eat meals, biscuits, bread, pasta (macaroni, spaghetti etc.) and crumbed products. Allchildren >9 months were consuming gluten-containing food products. In a preliminary study of the gluten intake in this group the meangluten intake obtained from the FFQ was 2.55 mg/d.

The current amount of gluten consumed by children in Spain, and more specifically in Valencia, has been established. The variety ofgluten-containing food is limited and controlled, and the mean daily gluten intake in this preliminary study is 2.55 mg/d. Moreover, thetiming of gluten introduction for infants in Valencia is between 7 and 9 months of age. It has been shown previously that glutenintroduction after 7 months of age in at-risk infants is associated with a higher risk of coeliac disease, i.e. 1.87 as compared with glutenintroduction between 4 and 6 months (OD 1)(2). It is possible that this late introduction of gluten could be related to the higher prevalenceof coeliac disease observed in the Valencia area in the last 5 years.

1. Ivarsson A, Persson LA, Nystrom L et al. (2000) Acta Paediatr 89, 165–171.2. Norris JM, Barriga K & Hoffenberg EJ (2005) JAMA 293, 2343–2351.3. Overbeek FM, Uil-Dieterman IG, Mol IW, Kohler-Brands L, Heymans HS & Mulder CJ (1997) Eur J Gastroenterol Hepatol 9, 1097–1099.4. Hopman EG, Kiefte-de Jong JC, le Cessie S, Moll H, Witteman J, Bleeker S & Mearin M (2007) Clin Nutr 26, 264–271.



Selenium deficiency in adults infected with HIV in the era ofhighly-active antiretroviral therapy

M. Stambullian1, M. S. Feliu2,4, C. M. Lopez3,4, A. E. Pineiro3,4, I. Cassetti1 and N. Slobodianik2,4

1Helios Salud Health Center, 2Department of Nutrition and Food Science, 3Department of Toxicology and 4School of

Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

The introduction of highly-active antiretroviral therapy (HAART) has produced a minor incidence of malnutrition and an improvement inthe survival and immunological functions of patients infected with HIV. On the other hand, there are still reports of micronutrientdeficiency in the early stage of the disease(1,2,3). Se is particularly relevant in HIV because it may modulates immune cells(1,2,3). The aimof the present study was to evaluate Se status in adults infected with HIV. The cross-sectional study included fifty-one adults infected withHIV: 72.5% males; mean age 37.1 (SD 6.9) years; 80.4% under HAART treatment; median CD4 count 426 (20–1029) cells/mm3; medianviral load <50 (<50-273000) copies/ml. Samples of whole blood were collected from fasting patients. Plasma Se was determined inhaemolysis-free plasma by flame atomic absorption spectrometry. A calibration curve was established using commercial standards.Reference values were taken from the literature (60–160mg/l)(4–6). The Ethics Committee of the University of Buenos Aires approved thestudy. All participants gave informed consent before recruitment. Statistical analysis was by Student’s t test, with significance at P<0.05,using SPSS version 13.0 (SPSS, Chicago, IL, USA). Although nutritional status evaluated by BMI was adequate (median 24.02 (inter-quartile range 22.3–25.5) kg/m2), 82.4% of the patients presented with Se below reference levels (median 42.4 (SD 17.9; 95% CI 37.5,47.3)mg). Plasma Se levels were not significantly different between subjects grouped according to the stage of the disease (HIV or AIDS);the same outcome was observed with and without HAART (P=0.35 and P=0.83 respectively). The results showed a high proportionof patients with Se deficiency, which could reduce the number of immune cells and/or their function. These results are in agreement witha previous study performed in children(7). Supplementation with Se may delay the development of the disease and may improve theprospects of survival and quality of life.

The research was partially supported by University of Buenos Aires (Grant B-060).

1. Carr A & Cooper D (2000) Lancet 356, 1423–1429.2. Lai H, Lai S, Shor-Posner G, Ma F, Trapido E & Baum MK (2001) J Acquir Immune Defic Syndr 27, 56–62.3. Baum MK, Shor-Posner G, Lai S, Zhang G, Lai H, Fletcher MA, Sauberlich H & Page JB (1997) J Acquir Immune Defic Syndr Hum Retrovirol 15,

370–374.4. Varian (1989) Analytical Methods. Flame Atomic Absorption Spectrometry. Publication no. 85-100009-00. Mulgrave, Victoria: Varian Australia Pty

Ltd.5. Varian (1988) Analytical Methods. Graphite Tube Atomizers. Publication no. 85-100848-00. Mulgrave, Victoria: Varian Australia Pty Ltd.6. Lockitch G, Halstead A & Wadsworth L (1988) Clin Chem 34, 1625–1627.7. Pallaro A, Barbeito S, Strasnoy I, Franchello A, Giraudi V, Ramos O & Slobodianik NH (1998) Acta Bioquim Clin Latinoam XXXII, 555–557.



Can dietary supplements reduce cell-surface expression ofmolecules involved in organ transplantation?

K. Varley, B. Clark and C. CarterTransplant Immunology Department, St James Hospital, Leeds LS9 7TF, UK

Preformed donor-relevant antibodies are a contraindication to successful renal transplantation, causing irreversible damage to theendothelial cells on the donor organ that leads to vessel obstruction and swift rejection. Such sensitised patients represent a challenge tosuccessful renal transplantation and individual treatment plans have become necessary in order to obtain a negative cross-match. Currentmethods used to treat the recipient before transplant are based on the removal of donor-relevant antibodies and include the use ofhigh-dose intravenous Ig, immunoadsorption and plasmapheresis. All methods of reducing antibody levels require immunosuppressiontherapies in order to halt antibody re-synthesis. Whilst significant efforts are made to control the recipient’s immune system, scant regardis given to direct modulation of the donor antigens present on the transplanted organ.

In the present study a strategy was explored in which pre-conditioning of the donor, in a live donor situation, could lead to a transientreduction of human leucocyte antigen (HLA) expression at the time of transplant, thus reducing organ antigenicity. Several studies haveshown that specific compounds can act as immunomodulatory agents. Whilst the use of prescription drugs in a healthy donor would beobviously inappropriate, n-3 fish oil (a widely-used food supplement) has been shown to have a direct effect on peripheral monocytes byinhibiting the expression of surface molecules involved in antigen presentation(1). Although controversial, the relatively innocuous natureof fish oils indicates their potential as an acceptable immune-response modifier for use in the diet of the organ donor before transplant.

In order to quickly screen a number of potential compounds, work was undertaken to establish an in vitro screen for HLA class IIexpression and other relevant cell-surface molecules on human monocytic cell lines. This procedure will be used to test potentialcompounds (health food or dietary supplements) to assess their ability to down regulate molecules involved in antigen presentation. Thepre-monocytic cell line THP-1 was analysed by two-colour flow cytometry to measure baseline expression of CD14/HLA-DR. Cells weresubsequently stimulated with lipopolysaccharide (LPS) for 24 h or 48 h and analysed for CD14/HLA-DR up-regulation. A representativeexample showing CD14 (FL-1)/HLA-DR(FL-2) expression from unstimulated and LPS-stimulated THP-1 cells is shown in the Figure.

Unstimulated Stimulated

It was found that resting (unstimulated) THP-1 cells did not express CD14, although they expressed variable levels of HLA class IIHLA-DR (Figure). The levels of class II showed a marginal increase following stimulation with LPS. CD14 expression was also detectedon significant numbers of THP-1 cells following stimulation with LPS (Figure).

1. Hughes DA & Pinder AC (2000) Am J Clin Nutr. 71, Suppl., 357S–760S.



Psychological stress and immune function: establishing both acuteand chronic psychological stress models in balb/c mice

E. Vazquez, A. Barranco, M. Manzano, M. L. Jimenez and R. RuedaScience & Technology, R&D Dept, Abbott Nutrition, Granada, Spain

Stress-induced immunological alterations have been considered to be a major cause of increased risk for immune-related diseases suchas cancer, autoimmune disorders, food allergy, alteration of mucosal barrier function and susceptibility to infection. Stressors triggerphysiological responses involving the hypothalamic–pituitary–adrenal axis (HPA), which is closely associated with the immune system.It is well known that some foods, in addition to contributing essential nutrients, also contain additional components such as prebiotic,probiotic and other functional ingredients that induce a balanced immune system and improve disease resistance and general healthystatus. There is a need for appropriate animal models as a tool for testing the potential benefit of some of these functional foods on theHPA–immune system axis.

The aim of the present work was to establish both acute and chronic mouse models for the study of cellular immunity modulation bypsychological stress. Female 6-week-old Balb/c mice were used in both stress models (n=12 for each experimental group). The acutestress model comprised for four consecutive days overnight 16 h-restraint with food and water deprivation (RST model). On the otherhand, the chronic stressor was based on the natural panic of mice in relation to water; thus, animals were placed on a very small water-surrounded platform for 1 h/d for 10 d (the water-avoidance stress model; WAS). At the end of both experimental schedules mice werekilled and serum, small intestine, spleen and thymus were removed. Physiological variables such as body weight, intestinal length andweight, intestinal mucosa and serum corticosterone levels were recorded. The immunological variables determined were spleen andthymus weights, intestinal secretory IgA content, serum ovoalbumin-specific antibody production, number of peritoneal macrophages,number of spleen cells, number of spleen natural killer cells and the expression of the surface spleen cell markers CD45, CD3, CD4, CD8,CD45R/B220, CD16/CD32 and Ia/Ie. Both stress models were found to impair the normal animal status, affecting most of the selectedphysiological and immunological variables (table 1). In the RST model the body, thymus and small intestine weights, the levels ofcorticosterone and ovoalbumin-specific antibody in serum and the number of spleen B-, T-, T-helper, CD3–CD4 + and CD3–CD8 + cellswere significantly lower in the stressed animals than in the controls (P<0.05). Similarly, in the WAS model the stressed animals showeda lower numbers of spleen T-helper, T-cytotoxic and total T-cells, although the numbers of CD3–CD4 + and CD3–CD8 + cells weresignificantly higher. In addition, the level of serum corticosterone and thymus weight were significantly lower in stressed animals than incontrols.

In conclusion, both psychological stress models significantly affected the normal status of the mice, the RST model (acute) being muchmore detrimental to the animals than the WAS model, which seems to be an appropriate tool to mimic the psychological stress damageexperienced by populations in Western countries.

Table 1. Data of anthropometric measurements and cell counts

WAS model RST model

Control Stress Control Stress

Body weight (g) 19,76�1.07 19,86�1,28 18,95�0,85 15,91�1,24*Thymus weight (g) 0,059�0,011 0,039�0,006* 0,038�0,009 0,016�0,006*Spleen weight (g) 0,131�0,02 0,135�0,017 0,118�0,02 0,090�0,0180Spleenocytes (n� cells) 93,11 · 106�15,91 · 106 98,54 · 106�16,35 · 106 87,87 · 106�25,5 · 106 50,53 · 106�25,83 · 106*NK (n� cells) 1,68 · 106�0,37 · 106 2,703 · 106�0,82 · 106* 3,05 · 106�0,71 · 106 1,26 · 106�0,22 · 106*Small intestine mucose weight (g) 0,662�0,092 0,659�0,071 0,759�0,078 0,562�0,091*

Mean�SD. *P<0.001.



Effects of a synbiotic on intestinal and immune functions ofhealthy adults

B. Viadel1, E. Nova2, J. E. Carreres1 and A. Marcos2

1Department of New Products, AINIA, Technologic Center, Valencia, Spain and 2Immunonutrition Group,

Department of Metabolism and Nutrition, Instituto del Frıo, CSIC, Madrid, Spain

Synbiotic preparations contain a combination of probiotics and prebiotics (fermentable fibre). The principal purported health-promotingeffect of probiotics is the enhancement of mucosal immune defences, while fructose oligosaccharides have a bifidogenic effect on humancolonic endogenous flora. The main objective of the present study was to evaluate the effects of a 6-week intervention with a synbioticproduct (Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei ssp. paracasei, Streptococcusthermophilus, Bifidobacterium sp. (2.4 · 109 colony-forming units/d) and fructo-oligosaccharides; SYMBYO1: Laboratorios Phergal SA,Madrid, Spain) on the intestinal and immune functions of thirty-six healthy adults aged 25–45 years (sixteen males, twenty females).Subjects were randomly assigned to the symbiotic (S; seven males, eleven females) or placebo (C; nine males, nine females) groups.Faecal and blood samples were collected at baseline and on completion of the study. Total aerobic and anaerobic micro-organisms,enterobacteria, enterococci, pseudomonas, yeasts, lactobacilli, bacteroids, Clostridium spp. and bifidobacteria were quantified in faeces.Phagocyte function was quantitatively assessed (Phagotest; BD Biosciences, Madrid, Spain) and highly-sensitive C-reactive protein,caeruloplasmin (immunoturbidimetry) and soluble adhesion molecules intercellular adhesion molecule-1 (sICAM-1), vascular cell adhe-sion molecule-1 (sVCAM-1) and sL-selectin (ELISA) were determined in serum. Intestinal motility and improvement in any previousgastrointestinal symptoms were assessed through a self-administered questionnaire. A non-parametric Wilcoxon test was used to assesschanges in faecal flora after treatment, repeated-measures ANOVA for the effect of treatment on immune variables and Monte Carlo exacttest for differences in symptom improvement.

The improvement in intestinal motility was significantly more reported in the S group than in the C group (P<0.003). Also, thecumulative self-reported improvement in any symptom was significantly more frequent in the S group than in the C group (P<0.001),although asymptomatic volunteers were significantly more frequent in the S group in the baseline evaluation (P<0.01). No significantinteraction of time · group was observed for any immunological variable except sL-selectin, which showed a significant decrease in theS group (see Table). When correlations were analysed in the groups defined by treatment, it was observed that in the S group baseline L-selectin levels were correlated with final sICAM-1 levels (r 0.468; P=0.050) and baseline sICAM-1 levels were negatively correlated withfinal L-selectin concentration (r - 0.457; P=0.056). In contrast, no significant correlations were observed between sL-selectin andsICAM-1 in the C group. The microbiological analysis of faecal samples showed no differences between the C and S groups, probablybecause a low bacterial load was administered.

C group (n 18) S group (n 18)

T1 T2 T1 T2

P (ANOVA)Mean SD Mean SD Mean SD Mean SD

sICAM-1 (ng/ml) 469 85 433 82 434 78 406 50 0.674sVCAM-1 (ng/ml) 615 212 575 198 545 126 521 148 0.732sL-selectin (ng/ml) 2586 555 2560 626 2724 600 2389* 420 0.050

T1, baseline; T2, after 6 weeks of treatment. Mean value was significantly different from that for T1 (Student’s test):*P<0.05.

The present study shows that the daily intake of the synbiotic supplement can have beneficial effects on the intestinal motility of healthysubjects and might improve the serum levels of L-selectin, possibly leading to a more beneficial profile of the inflammatory markers inrelation to the prevention of atherosclerosis and CVD.

This work has been supported by LABORATORIOS PHERGAL SA.



Changes in nutritional status and their effects on serum andthymus zinc levels and serum copper:zinc

S. M. Vidueiros1, I. Fernandez1, A. E. Pineiro2, N. Slobodianik1 and A. Pallaro1

1Department of Nutrition and 2Department of Toxicology, School of Pharmacy and Biochemistry,

University of Buenos Aires, Argentina

Previous studies have shown that the intake of low-quality dietary protein causes a decrease in plasma Zn levels and an uptake by otherorgans(1,2). It is known that nutritional disorders affect the immune system and that Cu and Zn play a critical role in its integrity.Moreover, an increase in Cu:Zn is associated with a higher risk of morbidity and mortality in patients who are immunodeficient(3,4). In thepresent work the effect of feeding a cereal-based diet and a recovery diet on serum Cu and Zn levels, Cu:Zn and thymus Zn concentrationwas investigated in weaning rats.

Wistar rats at weaning (21–23 d) were fed a diet containing 65 g precooked maize protein (M) or casein (Cas)/kg for18 d. Group M wasrefed with a 200 g casein/kg diet (MC) for 20 d. Age-matched control groups received stock diet (C1 (40 d) and C2 (60 d)). Body weight(BW; g) and body-weight gain (BWG; g/d per 100 g BW) were determined. Serum Cu (mg/ml) and Zn (mg/ml) concentrations weredetermined by atomic absorption spectrophotometry and Cu:Zn was calculated. Thymuses were removed and weighed (TW; mg) and theZn content (ng/ g organ) was determined by atomic absorption spectrophotometry.

Serum Zn was decreased in M and Cas groups compare with the age-matched controls (group C1); thus, Cu:Zn was higher in theseexperimental groups. This ratio, as well as serum Zn level, returned to normal in group MC. Thymus Zn level in group M was highercompared with groups Cas and C1, which had similar values. Thymus Zn was normal in group MC and no significant differences werefound between groups MC and C2.

Group (n 6–10)

Serum Zn (mg/ml) Serum Cu (mg/ml) Cu:Zn Thymus Zn (mg/g organ)

Mean SD Mean SD Mean SD Mean SD

M (40 d) 1.22a 0.4 1.32a 0.3 1.25b 0.4 65.7a 18.8Cas (40 d) 1.32a 0.3 1.37a 0.1 1.07b 0.2 21.4b 1.3C1 (40 d) 2.18b 0.7 1.30a 0.1 0.65a 0.2 30.1b 4.3C2 (60 d) 2.18b 0.6 1.57b 0.2 0.75a 0.2 25.6b 2.0MC (60 d) 1.94b 0.3 1.16a 0.2 0.61a 0.1 26.3b 4.9

a,bMean values within columns with unlike superscript letters were significantly different (0.05>P>0.001).

The increase in serum Zn level and the reduction in Cu:Zn during recovery was concomitant with a higher BWG (MC 5.48 (SD 0.66) v.M - 0.15 (SD 0.37); P<0.001) and TW (MC 625.4 (SD 157.2) v. M 88.6 (SD 26.2); P<0.001).

These results indicate that serum Zn and Cu:Zn are dependent on the quantity and/or quality of dietary protein. The reduced serum Znconcentration could be caused by a higher uptake of this mineral by the thymus, which was observed to have an increased Zn content.This outcome may represent a compensatory mechanism to overcome the low concentration and/or activity of thymic hormones describedin malnourished status, as it is known that Zn could be an immunomodulator of thymocyte proliferation and maturation(4). These datasuggest that it would be not necessary to determine the Cu:Zn, since data for serum Zn and for Cu:Zn lead to the same conclusion.

1. Pallaro A & Slobodianik N (1999) Nut Res 19, 1089–1095.2. Pallaro A, Roux ME & Slobodianik N (2001) Nutrition 17, 724–728.3. Fraker P (2000) Nutritional Immunology: Principles and Practice, pp. 147–156 [M Gershwin, JB German and CL Keen, editors], Totowa, NJ; Humana

Press Inc.4. Dardenne M, Pleau JM, Nabarm B et al. (1982) Proc Natl Acad Sci USA 79, 5370–5373.



Impact of a cereal-based diet on intestinal villi of growing rats

S. M. Vidueiros, I. Fernandez, N. Slobodianik, M. E. Roux and A. PallaroDepartment of Nutrition, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina

Malnutrition has an adverse impact on cell-mediated, secretory and humoural immunity, as well as on non-specific host defences.Although generalized malnutrition can affect all aspects of host immunity, the impact is greater on T-cell functions and cell-mediatedimmunity and smaller on B-cell functions as well as humoural immunity. Effects on secretory immunity are intermediate, mainly affectingIgA B-cells. Recent studies have shown that certain chemokines play an important role in regulating homeostatic lymphocyte recirculationthrough secondary lymphoid organs. The chemokine thymus-expressed chemokine (TECK) is a chemoattractant involved in the ability oflymphocytes to localize in the gastrointestinal tract(1).

The aim of the present work was to study the effect on B and T lymphocytes as well as the TECK + cell population on the intestinalvilli of growing rats after the administration of a cereal-based diet as the only source of protein.

Wistar rats were fed a 65 g precooked maize protein/kg diet for 18 d (M). An age-matched control group received a stock diet (C). Bodyweight (BW; g) was determined, ponderal growth rate (PGR; g/d per 100 g) was calculated and intestines were removed and processed bythe Saint Marie technique. Tissue sections were studied by an indirect immunofluorescence technique. CD5 + T-cells and the subsetsTCRab+ , TCRgd+ , CD4+ , CD8aa+ , CD8ab+ in the lamina propria (LP) and intraepithelium (iIEL) and IgA-B+ cells in LP weredetermined (number of cells per thirty fields). Also, the presence of TECK+ cell population was assessed.

There were significant differences between the M and C groups in BW (44.7 (SD 7.83) v. 149.1 (SD 17.7); P<0.0001) and PGR (–0.30(SD 0.39) v. 5.27 (SD 0.53); P<0.0001). As shown in the Table, the numbers of IgA + B-cells in the LP and CD5+ T-cells and CD4+ ,CD8aa+ , CD8ab+ , TCRab+ , and TCRgd+ T subpopulations in the LP and iIEL of the gut villi for the M group were significantlydecreased (P<0.001).

Phenotye . . . IgA B+ T CD5+ TCRab+ TCRgd+ CD8aa+ CD8ab+ CD4+Group (n 6) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Gut LPMaize 57.5 16.5 39.3* 13.4 35.7* 7.6 49.7* 15.2 46.9* 13.8 52.6* 17.6 59.5* 20.47Control 123.5 18.7 107.8 16.2 106.7 37.5 100.9 17.8 110.1 25.1 131.7 20.8 97.4 19.3

Gut iIELMaize 2.2† 1.6 1.6† 1.4 2.8† 1.3 0.4† 0.6 0.4† 0.7 0.5† 1.1Control 7.6 4.3 3.3 2.3 5.4 3.8 2.8 2.2 2.0 2.0 1.7 1.9

Mean values were significantly different from those for corresponding age-matched C group: *P<0.0001.Mean values were significantly different from those for corresponding age-matched C group: †0.03>P>0.001.

Differences in the size and cellularity of the gut villi and in the distribution of TECK were also observed in the experimental groups.When histological slides were analysed, the C group showed TECK+ cells in the villi and crypt epithelium, while the M group showed adecreased intensity.

The results show that the intake of a low-quality dietary protein as the only source of protein produces an important disorder in mucosalimmunity that could explain the incidence of gastrointestinal infections observed in malnourished children.

1. Kunkel EJ, Campbell JJ, Haraldsen G et al. (2000) J Exp Med 192, 761–767.



Breast-milk levels of long-chain PUFA in Kazakhstan and Sweden

M. Xiang1, L. S. Harbige1,2 and R. Zetterstrom3

1Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB, UK, 2Medway School of

Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK and 3Department of Paediatrics,

Karolinska Hospital, Karolinska Institute, Stockholm, Sweden

The deterioration of human health with increasing infant mortality rate, declining life expectancy at birth and increasing prevalence ofserious infectious diseases in Russia and other former Soviet Republics is thought to be the result of a combination of several factors suchas inadequate nutrition, poor sanitation, collapse of the healthcare system and pollution from Soviet agriculture and industries(1,2).Environmental pollutants, which may occur in breast milk and in various food products and drinking water, and which are also transferredto the foetus, constitute a severe threat to the health of infants and children. In Kazakhstan, which is the second largest area of the formerSoviet Republics with only eighteen million inhabitants, the situation is particularly alarming. Long-chain (LC) PUFA are essential dietarynutrients required for the optimal growth and development of infants, particularly of the brain and retina. It is important for exclusively-breast-fed infants to receive milk of a correct balance between n-6 and n-3 fatty acids(3–5). In the present study the composition of LCPUFA in the colostrum of Kazakh and Swedish mothers and the growth rate of their foetuses were compared. Ten and nineteenmother - term infant pairs from a countryside located about 600 km from Almaty, Kazakstan and Stockholm, Sweden, who were 1- 5 dold and exclusively breast-fed, were studied. The colostrum of the Kazakh mothers had significantly higher linoleic acid (LA; 18: 2n-6)and lower a-linolenic acid (LNA; 18: 3n-3), arachidonic acid (AA; 20: 4n-6), EPA (20: 5n-3) and DHA (22: 6n-3) levels than those of theSwedish mothers. The recommended ranges for LA:LNA and AA:DHA in human milk are 5- 10 and 0.5 - 1 compared with 32.4 and3.22 in the Kazakh breast milk, and 8.98 and 1.35 in the Swedish breast milk respectively (Table).

Ratio

Kazakh (n 10) Swedish (n 19)

Mean SE Mean SE P value

LA:LNA 32.4 2.01 8.98 0.47 <0.001AA:DHA 3.22 0.24 1.35 0.08 <0.001Sn-6/Sn-3 PUFA 24.6 1.16 6.13 0.26 <0.001Sn-6/Sn-3 LC PUFA 9.78 0.60 3.12 0.14 <0.001

The milk of the Kazakh mothers is less balanced in relation to the levels of n-6 and n-3 PUFA than that of the Swedish mothers.Whether environmental pollutants and/or PUFA might affect the developing immune and nervous systems in this Kazakh populationcould be followed up by a study of immune and neuro-functions of Kazakh and Swedish infants.

1. Zetterstrom R (1999) Acta Paediatr 429, 49–54.2. Jensen S, Mazhitova Z & Zetterstrom R (1997) Sci Total Environ 206, 187–193.3. Xiang M, Harbige LS & Zetterstrom R (2007) Acta Paediatr 96, 387–390.4. Xiang M, Rahman MA, Ai H, Li X & Harbige LS (2006) Ann Nutr Metab 50, 492–498.5. Xiang M, Harbige LS & Zetterstrom R (2005) Acta Paediatr 94, 1543–1549.



Time-course study of high-dose creatine supplementation forendogenous creatine synthesis

M. Xiang2, L. S. Harbige2,3, X. Li1, B. Li1 and H. Ai11Division of Nutrition and Biochemistry, Institute of Sports Medicine, The Third Hospital, Peking University, Beijing,



Creatine (Cre) is a popular nutritional supplement in many population groups, and has potential therapeutic effects in some diseases(1–4).Cre supplementation may increase the Cre content of muscle(5), and improve muscle strength(6). L-Arginine:glycine amidinotransferase(L-AGAT) in the kidney is the rate-limiting enzyme of endogenous Cre synthesis in mammals, and guanidinoacetic acid (GAA) isthe precursor in this synthesis. The aim of the present study was to determine the effect of high-dose Cre supplementation on L-AGATactivity and endogenous Cre synthesis. A time-course of repression of L-AGAT activity and GAA concentration by 3.0 g supplementedCre /kg per d was studied in adult male Sprague-Dawley rats. In the comparison between groups supplemented with 3.0 g Cre/kg bodyweight per d for 0.5, 1, 2, 4 and 8 d respectively and the control group (without Cre supplementation; n 10), the L-AGAT activity in thekidney in the supplemented groups decreased (%) by 31.7, 41.4, 48.9, 61.4 and 73.5 respectively (Fig. 1(A)). Furthermore, the kidneyconcentration of GAA for the groups receiving 3.0 g Cre/kg per d for 0.5, 1, 2, 4 and 8 d decreased (%) by 4.9, 20.4, 31.7, 37.9 and 40.8respectively (Fig. 1(B)). The total Cre in gastrocnemius muscle increased gradually with time and was 109.3% above the control levelsafter 8 d. In the groups receiving 3.0 g Cre/kg per d for 0.5, 1 and 2 d the serum creatinine increased significantly and, then declinedgradually until day 8. No significant differences were found in the serum creatine kinase activity among the six groups.

These results indicate that for rats L-AGAT activity and GAA concentration could be repressed rapidly by 3.0 g Cre/kg per d in 8 d,suggesting that high-dose Cre supplementation may quickly result in the depression of endogenous Cre metabolism. These results may berelevant to the immune system and this aspect is currently being investigated in lymphocytes, as little is known about the effect of Cresupplementation on immune cells.

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1. Prass K, Royl G, Lindauer U, Freyer D, Megow D, Dirnagl U, Stockler-Ipsiroglu G, Wallimann T & Priller J (2007) J Cereb Blood Flow Metab 27, 452–459.2. Kley RA, Vorgerd M & Tarnopolsky MA (2007) Cochrane Database Syst Rev 1, CD004760.3. McMorris T, Harris RC, Swain J, Corbett J, Collard K, Dyson RJ, Dye L, Hodgson C & Draper N (2006) Psychopharmacology (Berl) 17, 1–11.4. Bender A, Auer DP, Merl T et al. (2005) J Neurol 252, 36–41.5. Rockwell JA, Rankin JW & Toderico B (2001) Med Sci Sports Exerc 33, 61–68.6. Becque MD, Lochmann JD & Melrose DR (2000) Med Sci Sports Exerc 32, 654–658.



The effect of moderate dose of EPA+DHA on lipid profileand inflammatory markers in middle-aged men

H. M. Yusof and P. C. CalderInstitute of Human Nutrition, School of Medicine, University of Southampton, Developmental Sciences Building,

Mailpoint 887, Southampton General Hospital, Tremona Road, Southampton SO16 6 YD, UK

The aim of the present study was to investigate the effects of a moderate dose of long-chain (LC) n-3 PUFA (2.3 g EPA +DHA/d; 6:1)in the form of fish oil capsules on cardiovascular risk factors, particularly the plasma lipid profile and plasma inflammatory markers(C-reactive protein, IL-6, soluble adhesion molecules). In a double-blind placebo-controlled study twenty healthy middle-aged men wererandomised to fish oil providing 2.3 g/d EPA+DHA or medium-chain TAG-rich oil (placebo) for 8 weeks. Blood was collected after anovernight fast.

Plasma total cholesterol (TC) concentration was significantly increased in the fish oil group but HDL-cholesterol and LDL-cholesterolconcentrations were not significantly affected following supplementation with fish oil, although they were influenced by the placebo oil(see Table). Fish oil supplementation did not exhibit a favourable TAG-lowering effect previously shown(1). Likewise, positive effectsof LC n-3 PUFA on inflammatory markers were not observed, except that there was a significant difference between fish oil and placeboin terms of percentage change from baseline for soluble intercellular adhesion molecule 1 (sICAM-1; P=0.05). It was demonstrated that inthe fish oil group plasma sICAM-1 concentration decreased by 9.5% compared with a 9.9% increase in the placebo group. Correlationanalyses, however, did not show any relationships between the changes in the fatty acid profiles, particularly of LC n-3 PUFA, and thereduction in sICAM-1.

Outcome

Fish oil (n 9) Placebo (n 11)

Baseline 8 weeks

P

Baseline 8 weeks

PMean SE Mean SE Mean SE Mean SE

TC (mmol/l) 5.2 0.4 5.6 0.4 <0.05 4.8 0.3 5.6 0.3 <0.001LDL-cholesterol (mmol/l) 3.1 0.3 3.3 0.3 NS 3.0 0.3 3.5 0.3 <0.001HDL-cholesterol (mmol/l) 1.4 0.1 1.5 0.2 NS 1.1 0.1 1.3 0.1 <0.001TAG (mmmol/l) 1.3 0.2 1.7 0.3 NS 1.1 0.1 1.4 0.2 NSTC:HDL 4.1 0.5 4.2 0.5 NS 4.5 0.3 4.5 0.3 NSLDL:HDL 2.5 0.4 2.6 0.4 NS 2.8 0.3 2.8 0.2 NS

The results of the current study are consistent with a limited effect of modest doses of LC n-3 PUFA on the outcomes studied here.Reasons for the lack of TAG lowering by fish oil may be too low an intake of LC n-3 PUFA, insufficient DHA or the inclusion of subjectswho were normotriacylglycerolaemic. The finding of the current study in relation to sICAM-1 is in general accordance with some previousstudies(2–5) and it can be concluded that some aspects of the inflammatory response are sensitive to modest-dose LC n-3 PUFA and thatEPA+DHA may exert similar modest anti-inflammatory effects in middle-aged men and in the elderly. In summary, findings from thepresent study indicate that a dose of 2.3 g/d EPA+DHA is not sufficient to demonstrate maximal effects on CVD risk factors, includinginflammatory markers, in fairly-healthy middle-aged men.

1. Harris WS (1996) Curr Opin Lipidol 7, 3–7.2. De Caterina R, Liao JK & Libby P (2000) Am J Clin Nutr 71, Suppl., 213S–223S.3. Abe Y, El Masri B, Kimball KT, Pownall H, Reilly CF, Osmundsen K, Smith, CW & Ballantyne CM (1998) Arterioscler Thromb Vasc Biol 18,

723–731.4. Berstad P, Seljeflot I, Veierod MB, Hjerkinn EM, Arnesen H & Pedersen JI (2003) Clin Sci (Lond) 105, 13–20.5. Hjerkinn EM, Seljeflot I, Ellingsen I, Berstad P, Hjermann I, Sandvik L & Arnesen H (2005) Am J Clin Nutr 81, 583–589.



A specific mixture of short-chain galacto-oligosaccharides andlong-chain fructo-oligosaccharides induced an anti-allergic Ig profile in

infants at risk for allergy

A. J. Nauta1, S. Arsnalognu2, G. Boehm3, G. Moro2, J. Faber1, E. Knol1, B. Ruiter4,E. van Hoffen4, L. M’Rabet5 and J. Garssen1,2

1Immunology, Numico-Research BV, Wageningen, The Netherlands, 2Macedonio Melloni Maternity Hospital, Center for

Infant Nutrition, Milano, Italy, 3Numico-Research, Friedrichsdorf, Germany, 4Academic Hospital Utrecht, Dermatology,

Utrecht, The Netherlands and 5Utrecht Institute for Pharmaceutical Sciences, Pharmacology and Pathophysiology,

Utrecht, The Netherlands

In a prospective study in infants with a family history of atopy a specific prebiotic oligosaccharide mixture (90% short-chain galacto-oligosaccharides and 10% long-chain fructo-oligosaccharides (GOS/FOS; IMMUNOFORTIS) reduced the cumulative incidence of atopicdermatitis at 6 months of age(1). In a subgroup of these infants (n 84) it was possible to obtain a blood sample at 6 months of age toanalyse the potential effect of these dietary oligosaccharides on the Ig profile.

In this prospective double-blind randomized placebo-controlled study the infants received a hypoallergenic formula with either 8 gGOS/FOS/l or 8 g/ maltodextrin (placebo)/l for 6 months. At 3 months of age children were vaccinated against diphtheria, tetanus andpolio (DTP). At 6 months of age total plasma levels of IgE, IgG1, IgG2, IgG3 and IgG4 as well as cow’s-milk protein (CMP)- and DTP-specific Ig were measured by ELISA.

Supplementation with GOS/FOS led to a significant reduction in plasma levels of total IgE (P=0.007), IgG2 (P=0.029) and IgG3(P=0.0343) whereas no significant effect on IgG4 was observed. The plasma levels of CMP-specific IgG1 was significantly decreased(P=0.015) in the GOS/FOS group. The levels of CMP-specific IgE were very low and no effect of GOS/FOS supplementation wasobserved. CMP-specific IgG4 was not detectable in the samples. No effect of GOS/FOS supplementation on any vaccine-specific antibodyisotype levels was found.

Evidently, GOS/FOS supplementation induced an anti-allergic Ig profile in infants at high risk for allergic diseases while thedesired specific immune responses were unaffected, indicating the potential role of oral GOS/FOS exposure for primary prevention ofallergies.

Placebo (median) GOS/FOS (median) P

Total IgE (kU/ml) 10.0 4.00 0.008Total IgG1 (g/L) 3.09 2.26 0.005Total IgG4 (mg/L) 187.7 427.2 0.728CMP IgE (ng/ml) 2.50 1.80 0.348CMP IgG1 (AU/ml) 3.40 1.00 0.015DTP IgE (AU/ml) 0.40 0.37 0.884DTP IgG1 (AU/ml) 441.3 329.6 0.748

1. Moro G, Arslanoglu S, Stahl B, Jelinek J, Wahn U & Boehm G (2006) Arch Dis Child 91, 814–819.



Immune-enhancing role of vitamin C and zinc and effect onclinical conditions

S. Beveridge1, E. S. Wintergerst2, S. Maggini1 and D. Hornig3

1Bayer Consumer Care, Basel, 2Bayer Diabetes Care, Basel, Switzerland and 3 Bayer Diabetes Care, Reinach, Switzerland

The present paper is intended to give an overview on the roles of vitamin C and Zn in immune functions. Vitamin C concentrations in theplasma and leucocytes rapidly decline during infections and stress. Supplementation of vitamin C improves components of the humanimmune system such as antimicrobial and natural killer (NK) cell activities, lymphocyte proliferation, chemotaxis and delayed-typehypersensitivity. Vitamin C contributes to maintaining the redox integrity of cells and thereby protects them against reactive oxygenspecies generated during the respiratory burst and in the inflammatory response. Similarly, Zn deficiency impairs cellular mediators ofinnate immunity such as phagocytosis, NK cell activity, and the generation of oxidative burst. Thus, both nutrients play important andcomplementary roles in immune function and the modulation of host resistance to infectious agents, reducing the risk, severity andduration of infectious diseases. A deficiency in one of these essential nutrients weakens immunity since vitamin C is crucial for cellularimmunity and Zn for the production of antibodies.A large number of randomized controlled intervention trials with intakes of £1 gvitamin C and £30 mg Zn are available. These trials show that adequate intakes of vitamin C and Zn ameliorate symptoms and shorten theduration of respiratory tract infections including the common cold. Natural defences can only provide full protection when the body hassufficient Zn, as well as high levels of vitamin C.The physiological effects of vitamin C provide clear evidence and rationale for a numberof ways in which it might help to protect against infection. This evidence is termed mechanistic evidence because it stems fromknowledge of the chemical reactions and biochemical processes in which vitamin C is known to play an important role (Table). Theactions of Zn not only complement the actions of vitamin C to provide ‘double protection’ (Table), but may even have a synergistic effect.Like vitamin C, in recent years research has proved that Zn is essential for effective immune defence at several different levels.

Table. Summary of the role of vitamin C and Zn in body defences(1,2,3)

Defence Vitamin C Zn

Skin and mucosal barriers Collagen synthesisImproved strength

Cellular proliferationMaintains thickness

Neutrophils and macrophages Improved motility and chemotaxisEnhanced killingOverall improvement in phagocytosis

Lymphocytes Proliferation of stem cellsB- and T-cell differentiationB- and T-cell interaction

B lymphocytes Antibody productionT lymphocytes Proliferation Proliferation and appropriate response

Destruction of infected tissue cells and tumoursInterferon Production enhanced

Adequate intakes of vitamin C and Zn are essential for health.This is of special importance in populations in which insufficient intake ofthese nutrients is prevalent. The current belief is that regular prophylactic intakes of vitamin C at doses of ‡ 200 mg daily have no effecton the incidence of the common cold, but may be beneficial in the reduction of the severity and duration of the symptoms, suggesting thatvitamin C plays some role in the respiratory defence mechanisms. However, the elderly, who have been shown to have a lowered vitaminC status and may therefore be more prone to infections, individuals exposed to continuous oxidative stress, such as chronic smokers, andindividuals exposed to heavy physical exercise and/or cold environment may benefit from a moderate continuous vitamin C intake. Othervulnerable population groups include children. As a result of the high prevalence of Zn deficiency, especially in children in developingcountries, and the impaired immune status, susceptibility to infectious diarrhoea, malaria and pneumonia is found to be substantiallyincreased. Large intervention trials with daily intakes of 10–30 mg Zn have shown that Zn supplementation could be an importantadjuvant therapy for treating these infectious diseases in children in developing countries. Given that both vitamin C and Zn havean important and synergistic effect on immune function and the modulation of host resistance to infectious agents it is hence appropriateand beneficial to combine the trace element Zn with a high dose of vitamin C in one supplement.

1. Wintergerst ES, Maggini S & Hornig DH (2006) Ann Nutr Met 50, 85–94.2. Wintergerst ES, Maggini S & Hornig DH (2007) Ann Nutr Met 51, 301–323.3. Maggini S, Wintergerst ES, Beveridge S & Hornig DH (2007) Br J Nutr 98, Suppl. 1, S29–S35.



Contribution of selected vitamins and trace elements to immune function

S. Maggini1, E. S. Wintergerst2, S. Beveridge1 and D. Hornig3

1Bayer Consumer Care, Basel, Switzerland, 2Bayer Diabetes Care, Basel, Switzerland and 3Bayer Diabetes Care,

Reinach, Switzerland

The immune system requires essential nutrients to function efficiently. Inadequate intake and status of vitamins and trace elements maylead to suppressed immunity, which predisposes to infections and aggravates undernutrition. Available data indicate a role for vitamins A,B6, B12, C, D and E and folate and for the trace elements Se, Zn, Cu and Fe in the immune response. Evidence has accumulated that inhuman subjects these nutrients selectively influence the immune response, induce dysregulation of a coordinated host response to infec-tions in the case of deficiency and oversupply, and that deficiency may impact on the virulence of otherwise harmless pathogens. Thus,micronutrients are required at appropriate intakes for the immune system to function optimally and contribute to the body’s naturaldefences on three levels by supporting the physical barriers (skin and mucosa), cellular and humoral immunity. Vitamins A, C and E andthe trace element Zn assist in enhancing the skin barrier function. The vitamins A, B6, B12, C, D and E and folic acid and the traceelements Fe, Zn, Cu and Se work in synergy to support the protective activities of immune cells. Finally, all these micronutrients, exceptvitamin C and Fe, are essential for antibody production.

The table summarises the most important roles of selected vitamins and trace elements in immune function(1–3).

Micronutrient Main roles in the immune system

Vitamin A Normal differentiation of epithelial tissue and gene expressionImportant role both in humoral antibody response and cell-mediated immunityDeficiency impairs innate immunity, induces inflammation, potentiates existing inflammatory conditions and impairs ability

to defend against extracellular pathogensSupplementation is of benefit in reducing the morbidity and mortality from infectious diseases (especially children)

Vitamin D Potent immune system modulator when metabolized to 1,25-dihydroxycholecalciferol, is involved in cell proliferation and enhancesinnate immunity by increasing the differentiation of monocytes to macrophages

Deficiency correlates with higher susceptibility to infections as a result of impaired localized innate immunity and defects in antigenspecific cellular immune response

Supplementation together with a high-Ca diet inhibits progression of autoimmune disorders

Vitamin E Optimizes and enhances immune response (T-helper (Th) 1 response)Supplementation in healthy adults increases T-cell proliferation, improves the CD4 + :CD8 + , and decreases indices of oxidative stressSupplementation of elderly individuals improves overall immune function

Vitamins B6 and B12

and folic acidInterfere with immune function through involvement in nucleic acid and protein biosynthesisMaintain Th1 immune response (vitamin B6), innate immunity (natural killer (NK) cell activity; folate) and act as

immunomodulatory for cellular immunity, especially with effects on cytotoxic cells (NK; CD8 + T lymphocytes; vitamin B12)

Vitamin C Maintains redox integrity of cells and protection against reactive oxygen species (ROS) generated during respiratory burst and inflammatory responseStimulates leucocyte functions (neutrophil, monocytes movement)Role in antimicrobial and NK cell activities, lymphocyte proliferation, chemotaxis and delayed-type hypersensitivity responseDecreases duration and severity of common cold

Se Antioxidant essential for optimal immune response, influencing both the innate and the acquired immunityDeficiency impairs antibody production and causes viruses to undergo mutations to more virulent formsSupplementation normalizes age-related decline in immune response

Zn Influences both innate and acquired immunity, supports Th1 response, helps to maintain skin and mucosal integrityDeficiency leads to thymic atrophy, decreased cell-mediated cytotoxicity, helper T-cell and NK cell activities

Cu Part of Cu/Zn-superoxide dismutase, a key enzyme in the defence against ROSMaintains intracellular antioxidant balance, suggesting an important role in inflammatory responseAdequate intake supports a Th1 response and both deficiency and oversupply modulate the immune response

Fe Essential for cell differentiation and growth, component of enzymes critical for functioning of immune cellsInvolved in the regulation of cytokine production and action

Insufficient intake of micronutrients occurs in individuals with eating disorders, in smokers (both active and passive), in individualswith chronic alcohol abuse, in patients with certain diseases, during pregnancy and lactation, and in the elderly. With aging a variety ofchanges are observed in the immune system, which translate to less-effective innate and adaptive immune responses and increasesusceptibility to infections. Overall, inadequate intake and status of vitamins A, B6, B12, C, D and E and folate and of the trace elementsSe, Zn, Cu and Fe may lead to suppressed immunity, which predisposes to infections and aggravates malnutrition. Thus, supplementationwith a combination of these selected micronutrients can support the body’s natural defence system by enhancing all three levels ofimmunity: epithelial barriers; cellular immunity; antibody production.

1. Wintergerst ES, Maggini S & Hornig DH (2006) Ann Nutr Met 50, 85–94.2. Wintergerst ES, Maggini S & Hornig DH (2007) Ann Nutr Met 51, 301–323.3. Maggini S, Wintergerst ES, Beveridge S & Hornig DH (2007) Br J Nutr 98, Suppl. 1, S29–S35.



Differential effect of Bifidobacterium species characteristic of thegut microbiota of breast-fed and formula-fed infants on

in vitro cytokine production

E. Puertollano1, T. Pozo1, I. Nadal2, A. Marcos1, Y. Sanz2 and E. Nova1

1Immunonutrition Group, Metabolism and Nutrition Department, Instituto del Frıo (CSIC), Madrid, Spain and2Instituto de Agroquımica y Tecnologıa de Alimentos (CSIC), Valencia, Spain

The importance of the bacterial colonization process of the newborn intestine in the maturation of the immune system has beenrecognized. The presence of a healthy microflora (adequate composition of species and their frequency) in the gut is thought to play a rolein the balance of T-helper (Th) 1–Th2 immune responses. Some differences have been observed in the composition of bifidobacterialspecies in relation to the type of milk fed. While B. breve is one of the predominant species of the gut microbiota of breast-fed (BF)babies, B. catenulatum and B. adolescentis are characteristic of that of formula-fed (FF) infants(1). The aim of the current study was toassess the differential effect of the bifidobacterial species identified in the intestinal microbiota of BF and FF infants on cytokineproduction by peripheral blood mononuclear cells (PBMC). The effects of different bifidobacterial species were assessed individually andin combinations representing their proportions in infants under both feeding types. Live bacterial cell suspensions (107 colony-formingunits/ml) were incubated with PBMC in the proportion 10:1 for 48 h (5% CO2, 37�C) and cytokine concentrations were measured in thesupernatant fraction by flow cytometry (CBA; BD Biosciences, Madrid, Spain). The experiments were performed with blood from fourvolunteers and duplicates were done of each experimental condition and analysed separately. Statistical analyses were carried out usingANOVA and Mann-Whitney tests. Results for representative cytokines of Th1 and Th2 responses (interferon-g (IFN-g) and IL-4respectively) and the anti-inflammatory cytokine IL-10 are presented. Significant differences were found among different bifidobacterialspecies for the induction of IFN-g and IL-10 production (P<0.001 and P=0.003). B. catenulatum was the strongest enhancer of IFN-gproduction, followed by B. breve. B. catenulatum is more frequent in FF infants and B. breve is more frequent in BF infants(1), but nodifferences were found in the induction of IFN-g production by BF and FF mixtures of bifidobacteria used. B. catenulatum also inducedsignificantly higher levels of IL-4 than B. adolescentis and B. infantis (P=0.029). B. infantis was a mild cytokine inducer, showing thelowest effect among the assayed species for all three cytokines. B. longum, which is the third most frequent bifidobacterial species ininfant flora of both BF and FF infants, showed the strongest IL-10-inducing capacity. The effects of BF and FF bifidobacterial speciescombinations on cytokine production were not significantly different.

Mean values and standard deviations represented by vertical bars.Bifidobacterium adolescentis Bifidobacterium angulatum Bifidobacterium breve Bifidobacterium catenulatum

Bifidobacterium infantis Bifidobacterium longum Breast-fed Formula-fed

Phytohaemaglutinin Lipopolysaccharide Control, non-stimulated

IFN-gamma (pg/ml)

0

2000

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IL4 (pg/ml)

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IL10 (pg/ml)

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These results suggest that the presence or absence of particular bifidobacterial species as well as the overall composition ofthe bifidobacterial population in the infant gut could be key factors defining the immunomodulatory effect of the gut microbiota inearly life.

This work received financial support from CSIC; project references 200570F0091 and 200570F0093.

1. Haarman M & Knol J (2005) Appl Environ Microbiol 71, 2318–2324.


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