Expression of NS4 HCV in liver cells of HIV/HCV co-infected patients
Associated prof. N.V. Matsiyeuskaya, D.Sci (Med), associated prof. M.G. Zubritskiy, MD.PHDGrodno state medical university, BelarusGrodno regional pathological anatomical
bureau, Belarus
HIV/HCV co-infection in Belarus
• Incidence of HCV infectionamong HIV-infected patientsprevails the general populationindex in more than 10 times, in2013 it made up from 35.6% to40.8% among HIV-infectedpatients in different regions ofBelarus.
• The patients with HIV/HCV co-infection have a high incidenceliver cirrhosis (15.7%)compared to HIV-infectionwithout HCV co-infection (5%,p
Structure (%) of the leading causes of death among HIV-infected patients(Grodno, Belarus: 2000 – 2015, n=230)
22,417,1 15,3 13,7 12,4 10,5 8,6
Factors contributing to "liver" mortality include the absence of ART, high frequency of hepatotoxic factors: HCV co-infection (83.3%), drug use (75%), alcohol abuse (79.2%).
Диаграмма1
TuberculosisTuberculosis
"Liver" death"Liver" death
SuicidesSuicides
CNS pathologyCNS pathology
TumorsTumors
PneumoniaPneumonia
CVDCVD
22,4
17,1
15,3
13,7
12,4
10,5
8,6
Ряд 1
Ряд 2
47
0
36
0
32
0
29
0
26
0
22
0
0
18
Лист1
Ряд 1Ряд 2Ряд 3
Tuberculosis4702
"Liver" death3602
Suicides3203
CNS pathology2905
Tumors260
Pneumonia220
CVD018
NS4 HCV • NS4 HCV: NS4A and NS4B• NS4A protein is a 54 amino acid-long protein which acts as an activating
cofactor of NS3-4A serine protease for processing at the 3/4A, 4A/4B,4B/5A, and 5A/5B sites (Bartenschlager et al., 1994; Failla et al., 1994;Tanji et al., 1995).
• NS3-4A is tagged point for some direct anti-HCV agents (DAA) –boceprevir, telaprevir, simiprevir
• NS4B has been implicated in modulation of NS5B's RNA dependent RNApolymerase activity, has a possible role in HCV carcinogenesis, impairmentof ER function, and regulation of both viral and host translation.
• NS4B's apparent key role in the establishment of the replication (andpossible early assembly) platform represented by the membranous webplaces NS4B in a particularly prominent position in the viral life cycle(Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005)
2nd Central and Eastern European Meeting onViral Hepatitis and Co-infection with HIV6 - 7 October 2016, Bucharest, Romania
Aim of study: • to evaluate correlation between expression
of NS4 HCV and p24 HIV, HLA-DP, DQ, DR inliver tissue of HIV/HCV co-infected patients
Patients
• Liver samples of 18 dead anti HCV + HIV-infected patients were included in study
• Mean age of patients was 36,1±5,1, males - 7 (38,9%), females - 11 (61,1%);
• AIDS –15 (83,3), • ART – 12 (66,7%). • Liver cirrhosis - 6 (33,3%), • chronic hepatitis C (CHC) of mild activity – in 12
(66,7%) patients. • None of patients received antiviral therapy of CHC
Material and methods• Immunohistochemical studies were performed using streptavidin-biotin
method («Dako») in formalin-fixed, paraffin embedded specimens of the liver.
• Mouse monoclonal antibodies (anti-HIV p24, anti-human CD4, anti-human CD8, anti-human HLA-DP, DQ, DR antigen, anti-human CD56) and polyclonal rabbit antibodies (Anti-Herpes Simplex Virus types 1 and 2) were used in a standard dilution (DakoCytomation). To determine NS4 HCV in the cell cytoplasm murine anti-hepatitis C (AbDserotec) antibodies were used, to determine TNF-α in the liver cells monoclonal mouse anti-human TNF-α (AbDserotec) was used.
• Evaluation of IHC results was performed on a light microscope (magnification 100, 200, 400) in 6 random fields of view. Localization of staining in cells was evaluated as well as the percentage and intensity of positively stained cells. On this basis the degree of marker expression in points was determined. Scoring criteria: 0 points – negative staining, 1 –low intensity, 2 – moderate intensity, 3 – pronounced intensity.
Material and methods
• Assessment of structural changes of liver tissuewas performed by semiquantitative method usingKnodell RG and V. Serov scale, which helped tocalculate histological activity index (HAI), set thestage of fibrosis and the degree of steatosis byHornboll.
• Statistical analysis was performed on a personalcomputer using the package «Statistica», version10. Data are presented as Me and interquartileranges (IQRs).
EXPRESSION OF NS4 HCV IN HEPATOCYTES (HC) OF HIV/HCV CO-INFECTED PATIENTS
Distribution of HIV/HCV + patients according to clinical data in dependence of NS4 expression
NS4 HCV+N=9
NS4HCV –N=9
p
Age 37,4+5,6 34,8+5,6 >0,05
women 5 6 >0,05
men 4 3 >0,05
IDU 5 2 >0,05
AIDS 6 9 >0,05
ART 8 4 >0,05 NS4 HCV moderate expression in HC
0
2
4
6
8
10
NS4 HCV+ NS4 HCV-
9 9
Distribution of the patients according to NS4 expression
Expression of p24 HIV, HLA-DP at Kupffer cells in patients with productive HCV infection (NS4+) of HC vs
non productive HCV infection (NS4-)
Маркер HIV/HCV, n=9 HIV/HCV, n=9
productive HCV infection (NS4+) of HC non productive HCV infection (NS4-) of HC
1, abs/% >1, abs/% Me, IQR 1, abs/% >1, abs/% Me, IQR
p24 HIV in KC
4/44,4 3/33,3 1 (1 – 2)* 1/5,6 0 0 (0 – 0)
HLA-DP, DQ, DR in KC
4/44,4 2/11,1 1 (0 –1)* 0 0 0 (0 – 0)
HLA-DP, DQ, DR in liver tissue
Positive correlation (Spearman): betweenexpression of NS4 HCV in HC and LiverCirrhosis presence: R=0,37 (p=0,03)
KC – Kupffer cells, HC – hepatocytes, *- p
Productive HIV infection (p24 HIV+) in hepatocytes and Kupffercells of HIV/HCV+patient
p24 HIV+ in HC
p24 HIV+ in KCPolychromatic staining with azure II
electron microscopy
KC
A
B
C
D
Expression of immunological markers in hepatocytes and Kupffer cells in patients with productive HIV infection (p24+) of
HC and KC vs. non productive HIV (p24-)
МаркерHIV/HCV, n=8 HIV/HCV, n=10
productive HIV infection (p24+) of HC and KC
non productive HIV infection (p24-) of HC and KC
1 points, abs/%
>1points,abs/%
Me,IQRs
1points, abs/%
>1 points,abs/%
Me,IQRs
NS4 HCVin HC
4/50,0 3/37,5 1,0 (1,0 -2,0) *
2/20 0 0 (0,0 - 0,0)
HSV1 inHC
5/62,0 2/25 1,0 (1,0 -1,0) *
3/30 1/10 0 (0,0 - 0,1)
HLA-DP,DQ, DR+ inKC
3/37,5 2/25 0,5 (0,0 -1,0)*
1/10 0 0 (0,0 -0,0)
TNF-α inKC
6/75,0 0 1 (0,5 -1,0)*
1/10 0 0 (0,0 -0,0)
KC – Kupffer cells, HC – hepatocytes; *- p
Expression of HSV 1 (3 points) in hepatocytes of HIV and HCV-infected patient, 34 yrs., AIDS
Conclusions
• Synergism in expression of replicative markersof HCV (NS4) and HIV (p24) in liver cells ofHIV/HCV co-infected patients has beenestablished which associated with activationof Kupffer cells, and higher expression of HSV1in liver cells.
• We can propose that effect of DAA in HIV/HCVco-infected patients may be enhanced byachievement of strong HIV suppression.
Acknowledgments
Grodno State medical university, Belarus• Prof. V.A. Snezhitskiy, D.Sci (Med) corresponding
member of NAS of Belarus • Prof. V.M. Tsyrcunov, D.Sci (Med)• Prof. V.P. Andreev, MD. PHD• Senior researcher R.I. Kravchuk, MD. PHD
Grodno regional pathological anatomical bureau• Associated prof. N.I. Prokopchik, MD. PHD
Gomel regional pathological anatomical bureau• V.N. Tischenko - pathomorphologist
Expression of NS4 HCV in liver cells of HIV/HCV co-infected patients�HIV/HCV co-infection in Belarus Structure (%) of the leading causes of death �among HIV-infected patients�(Grodno, Belarus: 2000 – 2015, n=230)NS4 HCV ���Aim of study: PatientsMaterial and methodsMaterial and methodsEXPRESSION OF NS4 HCV IN HEPATOCYTES (HC) OF HIV/HCV CO-INFECTED PATIENTSExpression of p24 HIV, HLA-DP at Kupffer cells in patients with productive HCV infection (NS4+) of HC vs non productive HCV infection (NS4-)Productive HIV infection (p24 HIV+) in hepatocytes and Kupffer cells of HIV/HCV+patientExpression of immunological markers in hepatocytes and Kupffer cells in patients with productive HIV infection (p24+) of HC and KC vs. non productive HIV (p24-)Expression of HSV 1 (3 points) in hepatocytes of HIV and HCV-infected patient, 34 yrs., AIDSConclusionsAcknowledgments
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