Microchimerism following canine in utero hematopoietic stem cell transplantation: Development of...
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31 MICROCHIMERISM FOLLOWING CANINE IN UTERO HEMATOPOIETIC STEM CELL TRANSPLANTATION: DEVELOPMENT OF T-CELL DOSING REGIMENS SCOTT PETERSEN 1 , MARIYA GENDELMAN 1 , KATHLEEN MURPHY 2 , MICHAEL TORBENSON 2 , RICHARD JONES 3 , JANYNE ALTHAUS 1 , KARIN BLAKEMORE 1 , 1 Johns Hopkins University, Gynecology and Obstetrics, Baltimore, Maryland, 2 Johns Hopkins University, Pathology, Baltimore, Maryland, 3 Johns Hopkins University, Oncology Center, Baltimore, Maryland OBJECTIVE: In utero hematopoietic stem cell transplantation (IUHSCT) shows promise in treating immune disorders, storage disorders, and hemoglo- binopathies. There is data to suggest that T cell dosing in addition to HSC dosing may increase the degree of engraftment. In this study, we investigated the use of anti-canine anti-T cell antibodies for cell dosing of the donor graft in a canine model of IUHSCT. STUDY DESIGN: Canine IUHSCT was performed by ultrasound-guided intraperitoneal injection in day 37 (term 63), 4.5cm CRL, 7.1g fetal canines with CD34 + cells isolated from paternal bone marrow at doses of 4.5 ! 10 8 CD34 + cells/kg and T cells (CD3 + CD5 + ) isolated from paternal blood at 4.7 ! 10 8 cells/kg. Controls were marked intraperitoneally with ethiodol. Postnatal studies included tissue histology to assess GVHD and polymerase chain reaction-based chimerism analysis to assess degree of engraftment (TaqMan SRY for female recipients and unique microsatellite loci using capillary electrophoresis for both sexes). RESULTS: Term survival in this transplant cycle was 100%. Microchimerism (!1%) was detected in two of three recipients in multiple tissues including bone marrow, thymus, spleen, liver, kidney and large intestine. Levels of engraftment were highest in bone marrow and thymus at 0.5 % in one of the female offspring. Histopathological analysis did not reveal any signs of GVHD. CONCLUSION: Injection of relatively high doses of donor T cells in addition to haploidentical HSC yields microchimerism in multiple tissues without evidence of GVHD. The availability of canine CD34 + , CD3 + , and most recently CD5 + antibodies, permits accurate T-cell and HSC dosing experiments to determine the optimal conditions for engraftment without GVHD. Progress in the canine model will provide a framework for advancing IUHSCT toward human application. 32 URINARY ANGIOGENIC FACTORS CLUSTER HYPERTENSIVE DISORDERS AND IDENTIFY WOMEN WITH SEVERE PREECLAMPSIA CATALIN BUHIMSCHI 1 , ERROL NORWITZ 1 , SUSAN RICHMAN 1 , EDMUND FUNAI 1 , SETH GULLER 1 , CHARLES LOCKWOOD 1 , IRINA BUHIMSCHI 1 , 1 Yale University, Ob/Gyn & Reprod Sci, New Haven, Connecticut OBJECTIVE: The etiology and pathogenesis of preeclampsia remains unclear. Serum levels of soluble fms-like tyrosine kinase 1 (sFlt-1), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) are altered in women with clinical preeclampsia. We sought to identify whether similar alterations in urinary levels of these proteins occur in hypertensive disorders and identify women with severe preeclampsia (sPE). STUDY DESIGN: Free urinary levels of sFlt-1, VEGF and PlGF were measured by immunoassay in 68 women enrolled prospectively in the following groups: nonpregnant reproductive age (NP-CRL n = 14), healthy pregnant control (P-CRL n = 16), pregnant chronic hypertensive women (cr-HTN n = 21) and women with sPE (n = 17). Levels of angiogenic factors were normalized for creatinine and/or total proteins. RESULTS: There was no difference in median gestational age (sPE: 31, crHT: 33, P-CRL: 26 wks) among the groups. Urinary sFlt-1 was significantly increased in women with sPE relative to all other groups (P ! .001, Fig). PlGF was significantly decreased in all hypertensive women versus normotensive controls (P ! .001). Urinary excretion of VEGF was significantly increased in sPE compared to NP-CRL (P = .023). A cut-off > 1.15 in the ratio sFlt-1/PlGF had 88.2% sensitivity and 100% specificity in differentiating women with sPE from normotensive controls, significantly better than proteinuria alone (ROC analysis P = .03). CONCLUSION: sPE is associated with increased urinary output of sFlt at the time of clinical manifestation. Our findings support the idea of a rapid noninvasive screening of hypertensive women based on urinary sFlt/PlGF ratio, which may be used as a marker of disease severity and which appears to be superior to urinary protein measurements. 33 PROSPECTIVE RANDOMIZED CLINICAL TRIAL OF INPATIENT CERVICAL RIPENING WITH STEPWISE ORAL MISOPROSTOL VERSUS VAGINAL MISOPROSTOL IRIS COLON 1 , KAYTHA CLAWSON 1 , MARK TASLIMI 1 , MAURICE DRUZIN 1 , 1 Stanford University, Obstetrics and Gynecology, Stanford, California OBJECTIVE: To compare the efficacy and safety of stepwise oral misoprostol versus vaginal misoprostol for cervical ripening prior to induction of labor. STUDY DESIGN: Two hundred and four women between 32 to 42 weeks of gestation with an unfavorable cervix (Bishop score % 6) and an indication for labor induction were randomized to receive oral or vaginal misoprostol every 4 hours up to 4 doses. The oral misoprostol group received 50 mg initially followed by 100 mg in each subsequent dose. The vaginal group received 25 mg in each dose. The primary outcome was the interval from first misoprostol dose to delivery. The sample size calculation (n = 194) was based on an alpha of .05 and a beta of .20 to detect a 4-hour difference. Patient satisfaction and side effects were assessed by surveys completed after delivery. Statistical analyses were performed using Student t test, c 2 test, Fisher exact test, and ANOVA where appropriate. RESULTS: Ninety three ( 45.6%) women received oral misoprostol; 111 (54.4%) received vaginal misoprostol. There was no difference in the average interval from first dose to vaginal delivery between the oral (19.3 G 6.7 hrs ) and vaginal (18.0 G 8.3 hrs, P = NS) groups. There was a trend towards a lower incidence of hyperstimulation in the oral group 2.2% vs. vaginal group 5.4%, P = NS. Eighteen patients in the oral group (19.4%) and 36 (32.4%) in the vaginal group underwent cesarean section (P ! .05). This difference remained significant after controlling for confounders of cesarean delivery. There was no difference in side effects (nausea, vomiting, diarrhea, shivering) between groups but more patients in the vaginal group were dissatisfied with the use of misoprostol (14% vs. 7.5%, P = NS). CONCLUSION: Stepwise oral misoprostol (50 mg followed by 100 mg) is as effective as vaginal misoprostol (25 mg) for cervical ripening with a low incidence of hyperstimulation, lower rate of cesarean section, no increase in side effects and a trend towards improved patient satisfaction. 34 EP4 RECEPTOR ACTIVATION PRODUCES QUANTIFIABLE DECREASE IN CERVICAL COLLAGEN CONTENT HELEN FELTOVICH 1 , HUILING JI 1 , COLLEEN CARROLL 1 , JESSIE JANOWSKI 1 , ELIZABETH BONNEY 1 , EDWARD CHIEN 2 , 1 University of Vermont, Obstetrics and Gynecology, Burlington, Vermont, 2 Brown University, Women and Infants Hospital of Rhode Island, Providence, Rhode Island OBJECTIVE: Prostaglandin E analogs are used for cervical ripening because they simulate the physiologic rise in PGE2 production prior to normal parturition. Ripening involves disruption of collagen, which has been previously noted but not quantified. Effects of PGE2 are mediated through its receptors (Ptger-EP1, 2, 3, and 4). Based on their functions and expression patterns through gestation, we hypothesized that PGE2-induced cervical ripening is mediated through activation of the EP4 receptor, thus a decrease in collagen concentration should be quantifiable microscopically with EP4 activation. Using selective and non-selective PGE2 receptor agonists, we explored whether ripening is mediated by EP4 as evidenced by changes in cervical collagen concentration. STUDY DESIGN: Pregnant Sprague-Dawley rats (n = 16) were placed into groups: PGE2 (non-selective EP1-4 agonist), Butaprost (EP2 agonist), Sulpro- stone (EP1, EP3 agonist) and Control. Indomethacin was administered to prevent effects of endogenous prostaglandin. Treatments were given intra- vaginally on day 20 of gestation (typical rat gestation is 22-23 days). Cervical tissue was obtained 24 hours post-treatment. Sections were stained with picosirius red then analyzed for polarized light birefringence. Images were captured with Olympus BX50 Light Microscope and Optronics MagnaFire digital camera at 40! magnification and analyzed with Metamorph software. RESULTS: Selective agonists for EP1, 2, and 3 did not produce differences in collagen concentration compared to Controls. The non-selective agonist, PGE2, did produce a significant 30% decrease in collagen content [Controls 27.5 + 5.22 %, PGE2 17.6 + 6.33 % ROI] (t test P ! .05). CONCLUSION: The decrease in cervical collagen content after PGE2 administration indicates that changes are quantifiable microscopically and that they are mediated through the EP4 receptor. Although PGE2 is non-selective, the absence of change in collagen concentration with EP1, 2, and 3-specific agonists suggests that the decrease observed with PGE2 was due exclusively to EP4 activation. SMFM Abstracts S15
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Transcript of Microchimerism following canine in utero hematopoietic stem cell transplantation: Development of...
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33 PROSPECTIVE RANDOMIZED CLINICAL TRIAL OF INPATIENT CERVICAL RIPENINGWITH STEPWISE ORAL MISOPROSTOL VERSUS VAGINAL MISOPROSTOL IRIS COLON1,KAYTHA CLAWSON1, MARK TASLIMI1, MAURICE DRUZIN1, 1Stanford University,Obstetrics and Gynecology, Stanford, California
OBJECTIVE: To compare the efficacy and safety of stepwise oral misoprostolversus vaginal misoprostol for cervical ripening prior to induction of labor.
STUDY DESIGN: Two hundred and four women between 32 to 42 weeks ofgestation with an unfavorable cervix (Bishop score % 6) and an indication forlabor induction were randomized to receive oral or vaginal misoprostol every 4hours up to 4 doses. The oral misoprostol group received 50 mg initially followedby 100 mg in each subsequent dose. The vaginal group received 25 mg in eachdose. The primary outcome was the interval from first misoprostol dose todelivery. The sample size calculation (n = 194) was based on an alpha of .05 anda beta of .20 to detect a 4-hour difference. Patient satisfaction and side effectswere assessed by surveys completed after delivery. Statistical analyses wereperformed using Student t test, c2 test, Fisher exact test, and ANOVA whereappropriate.
RESULTS: Ninety three ( 45.6%) women received oral misoprostol; 111(54.4%) received vaginal misoprostol. There was no difference in the averageinterval from first dose to vaginal delivery between the oral (19.3 G 6.7 hrs ) andvaginal (18.0 G 8.3 hrs, P = NS) groups. There was a trend towards a lowerincidence of hyperstimulation in the oral group 2.2% vs. vaginal group 5.4%,P = NS. Eighteen patients in the oral group (19.4%) and 36 (32.4%) in thevaginal group underwent cesarean section (P ! .05). This difference remainedsignificant after controlling for confounders of cesarean delivery. There was nodifference in side effects (nausea, vomiting, diarrhea, shivering) between groupsbut more patients in the vaginal group were dissatisfied with the use ofmisoprostol (14% vs. 7.5%, P= NS).
CONCLUSION: Stepwise oral misoprostol (50 mg followed by 100 mg) is aseffective as vaginal misoprostol (25 mg) for cervical ripening with a low incidenceof hyperstimulation, lower rate of cesarean section, no increase in side effects anda trend towards improved patient satisfaction.
SMFM Abstracts S15
MICROCHIMERISM FOLLOWING CANINE IN UTERO HEMATOPOIETIC STEM CELLTRANSPLANTATION: DEVELOPMENT OF T-CELL DOSING REGIMENS SCOTT PETERSEN1,MARIYA GENDELMAN1, KATHLEEN MURPHY2, MICHAEL TORBENSON2, RICHARD JONES3,JANYNE ALTHAUS1, KARIN BLAKEMORE1, 1Johns Hopkins University, Gynecologyand Obstetrics, Baltimore, Maryland, 2Johns Hopkins University, Pathology,Baltimore, Maryland, 3Johns Hopkins University, Oncology Center, Baltimore,Maryland
OBJECTIVE: In utero hematopoietic stem cell transplantation (IUHSCT)shows promise in treating immune disorders, storage disorders, and hemoglo-binopathies. There is data to suggest that T cell dosing in addition to HSCdosing may increase the degree of engraftment. In this study, we investigated theuse of anti-canine anti-T cell antibodies for cell dosing of the donor graft ina canine model of IUHSCT.
STUDY DESIGN: Canine IUHSCT was performed by ultrasound-guidedintraperitoneal injection in day 37 (term 63), 4.5cm CRL, 7.1g fetal canineswith CD34+ cells isolated from paternal bone marrow at doses of 4.5 ! 108
CD34+ cells/kg and T cells (CD3+ CD5+) isolated from paternal blood at 4.7! 108 cells/kg. Controls were marked intraperitoneally with ethiodol. Postnatalstudies included tissue histology to assess GVHD and polymerase chainreaction-based chimerism analysis to assess degree of engraftment (TaqManSRY for female recipients and unique microsatellite loci using capillaryelectrophoresis for both sexes).
RESULTS: Term survival in this transplant cycle was 100%. Microchimerism(!1%) was detected in two of three recipients in multiple tissues including bonemarrow, thymus, spleen, liver, kidney and large intestine. Levels of engraftmentwere highest in bone marrow and thymus at 0.5 % in one of the female offspring.Histopathological analysis did not reveal any signs of GVHD.
CONCLUSION: Injection of relatively high doses of donor T cells in addition tohaploidentical HSC yields microchimerism in multiple tissues without evidenceof GVHD. The availability of canine CD34+, CD3+, and most recently CD5+
antibodies, permits accurate T-cell and HSC dosing experiments to determinethe optimal conditions for engraftment without GVHD. Progress in the caninemodel will provide a framework for advancing IUHSCT toward humanapplication.
URINARY ANGIOGENIC FACTORS CLUSTER HYPERTENSIVE DISORDERS ANDIDENTIFY WOMEN WITH SEVERE PREECLAMPSIA CATALIN BUHIMSCHI1,ERROL NORWITZ1, SUSAN RICHMAN1, EDMUND FUNAI1, SETH GULLER1,CHARLES LOCKWOOD1, IRINA BUHIMSCHI1, 1Yale University, Ob/Gyn & ReprodSci, New Haven, Connecticut
OBJECTIVE: The etiology and pathogenesis of preeclampsia remains unclear.Serum levels of soluble fms-like tyrosine kinase 1 (sFlt-1), vascular endothelialgrowth factor (VEGF), and placental growth factor (PlGF) are altered in womenwith clinical preeclampsia. We sought to identify whether similar alterations inurinary levels of these proteins occur in hypertensive disorders and identifywomen with severe preeclampsia (sPE).
STUDY DESIGN: Free urinary levels of sFlt-1, VEGF and PlGF weremeasured by immunoassay in 68 women enrolled prospectively in the followinggroups: nonpregnant reproductive age (NP-CRL n = 14), healthy pregnantcontrol (P-CRL n = 16), pregnant chronic hypertensive women (cr-HTNn= 21) and women with sPE (n = 17). Levels of angiogenic factors werenormalized for creatinine and/or total proteins.
RESULTS: There was no difference in median gestational age (sPE: 31, crHT:33, P-CRL: 26 wks) among the groups. Urinary sFlt-1 was significantly increasedin women with sPE relative to all other groups (P ! .001, Fig). PlGF wassignificantly decreased in all hypertensive women versus normotensive controls(P ! .001). Urinary excretion of VEGF was significantly increased in sPEcompared to NP-CRL (P= .023). A cut-off > 1.15 in the ratio sFlt-1/PlGF had88.2% sensitivity and 100% specificity in differentiating women with sPE fromnormotensive controls, significantly better than proteinuria alone (ROC analysisP = .03).
CONCLUSION: sPE is associated with increased urinary output of sFlt at thetime of clinical manifestation. Our findings support the idea of a rapidnoninvasive screening of hypertensive women based on urinary sFlt/PlGF ratio,which may be used as a marker of disease severity and which appears to besuperior to urinary protein measurements.
34 EP4 RECEPTOR ACTIVATION PRODUCES QUANTIFIABLE DECREASE IN CERVICALCOLLAGEN CONTENT HELEN FELTOVICH1, HUILING JI1, COLLEEN CARROLL1,JESSIE JANOWSKI1, ELIZABETH BONNEY1, EDWARD CHIEN2, 1University ofVermont, Obstetrics and Gynecology, Burlington, Vermont, 2BrownUniversity, Women and Infants Hospital of Rhode Island, Providence, RhodeIsland
OBJECTIVE: Prostaglandin E analogs are used for cervical ripening becausethey simulate the physiologic rise in PGE2 production prior to normalparturition. Ripening involves disruption of collagen, which has been previouslynoted but not quantified. Effects of PGE2 are mediated through its receptors(Ptger-EP1, 2, 3, and 4). Based on their functions and expression patternsthrough gestation, we hypothesized that PGE2-induced cervical ripening ismediated through activation of the EP4 receptor, thus a decrease in collagenconcentration should be quantifiable microscopically with EP4 activation. Usingselective and non-selective PGE2 receptor agonists, we explored whetherripening is mediated by EP4 as evidenced by changes in cervical collagenconcentration.
STUDY DESIGN: Pregnant Sprague-Dawley rats (n = 16) were placed intogroups: PGE2 (non-selective EP1-4 agonist), Butaprost (EP2 agonist), Sulpro-stone (EP1, EP3 agonist) and Control. Indomethacin was administered toprevent effects of endogenous prostaglandin. Treatments were given intra-vaginally on day 20 of gestation (typical rat gestation is 22-23 days). Cervicaltissue was obtained 24 hours post-treatment. Sections were stained withpicosirius red then analyzed for polarized light birefringence. Images werecaptured with Olympus BX50 Light Microscope and Optronics MagnaFiredigital camera at 40! magnification and analyzed with Metamorph software.
RESULTS: Selective agonists for EP1, 2, and 3 did not produce differences incollagen concentration compared to Controls. The non-selective agonist, PGE2,did produce a significant 30% decrease in collagen content [Controls27.5 + 5.22 %, PGE2 17.6 + 6.33 % ROI] (t test P ! .05).
CONCLUSION: The decrease in cervical collagen content after PGE2administration indicates that changes are quantifiable microscopically and thatthey are mediated through the EP4 receptor. Although PGE2 is non-selective,the absence of change in collagen concentration with EP1, 2, and 3-specificagonists suggests that the decrease observed with PGE2 was due exclusively toEP4 activation.
