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O-038 Stimulation of peritoneal macrophage (PM) mediated cytolysis of ectopic endometrial cells (EC) from women with endometriosis (endo pts)
Dmowski. Rush Medical College and Institute for the Study and Treatment of Endometriosis, Chicago, IL. Objective: Recent evidence suggests that apoptosis maintains cellular homeostasis during the menstrual cycle, eliminating senescent endometrial cells during the secretory and menstrual phases. The purpose of this study was to determine whether apoptosis is decreased in the endometrial tissue obtained from women with endometriosis. Design: Paired specimens of uterine and ectopic endometrial tissue from patients with endometriosis (n=13) and uterine endometrium from controls (n=6) were assessed for spontaneous apoptosis. These samples were obtained by curettage and laparoscopic biopsies. Materials and Methods: Single cell suspensions were prepared by enzymatic digestion of uterine and ectopic tissue with collagenase and DNAase and analyzed for spontaneous apoptosis using the cell death detection ELISA kit (Boehringer Mannheim Corporation, Indianapolis, IN). Results: The susceptibility of uterine endometrium to spontaneous apoptosis was significantly reduced (69%) in women with endometriosis as compared to healthy controls (p<0.001). In addition, among patients with endometriosis, the spontaneous apoptosis of ectopic endometrium was 67% less than the spontaneous apoptosis of eutopic endometrium (p<0.001). Decreased apoptosis of ectopic vs eutopic endometrium was independent of the cycle phase. Preliminary studies suggest that the stage of the disease contributes to the resistance/susceptibility of endometrial tissue to spontaneous apoptosis. Thus, when patients were segregated according to disease severity, eutopic and ectopic endometrium from patients with severe endometriosis displayed less susceptibility to spontaneous apoptosis than endometrial tissue obtained from patients with less advanced disease. Conclusions: Spontaneous apoptosis is significantly reduced in uterine endometrium obtained from women with endometriosis as compared to healthy controls. Furthermore, among patients with endometriosis, ectopic endometrium is even less susceptible than eutopic tissue to spontaneous apoptosis. Based on these data, we speculate that decreased susceptibility of endometrial cells to apoptosis contributes to the etiology/pathogenesis of endometriosis.
0-037 Increased Expression of Monocyte Chemotactic Protein-1 in the Endometrium of Women With Endometriosis. 1A. Akoum, 1Jolicoeur, 1M. Boutouil, 1I. Paradis, 2R. Drouin, ~R. Maheux and 1A. Lemay. 1Laboratoire d'Endocrinologie de la Reproduction, and 2Unit~ de Recherche en G~n~tique Humaine et Mol~culaire, Centre de Recherche, Pavillon Saint-Francois d'Assise, Centre Hospitalier Universitaire de Quebec, Universit~ Laval, Quebec, Canada. Objective: According to our previous data, cytokinestimulated uterine endometrial cells of women with endometriosis secrete higher levels of monocyte chemotactic protein-1 (MCP-1) than those of women without laparo-
scopical evidence of the disease. MCP-1 exerts a potent action on monocyte chemoattraction and activation, and may play an important role in the activation of peritoneal macrophages and peripheral blood monocytes observed in patients with endometriosis. In the present study, we examined the in situ expression of MCP-1 in the endometrium of women with and without endometriosis. Design: Immunohistochemical and in situ hybridization Studies. Material and Methods: Endometrial biopsies were obtained from women presenting for infertility or pelvic pain in whom endometriosis was diagnosed at laparoscopy (n=47) and women presenting for tubal ligation without laparoscopic evidence of the disease (n=22). Immunohistochemistry was carried out using a mouse monoclonal antiMCP-1 antibody, a Vectastain Elite ABC kit and diaminobenzidine as chromogen. In situ hybridization was performed using MCP-1 cDNA probes labelled with biotin, which was then detected by immunofluorescence. MCP-1 expression was evaluated in a blinded fashion using an arbitrary scale (0 = absent, 1 = light, 2 = moderate and 3 = intense), which takes into account the intensity and the distribution of staining. Results: MCP-1 was found in the stroma and epithelial glands of the endometrium. An increased expression of MCP-1 in the endometrial glands of women having endometriosis compared to normal women was observed both at the level of the protein (P = 0.0001) and mRNA (P = 0.0001). The most significant elevation of the protein expression was found in the mild stage of endometriosis (stage II) (P = 0.0006), whereas the highest level of mRNA expression was observed in more severe disease. Conclusion: These findings indicate that MCP-1 expression is up-regulated in the eutopic endometrium of women with endometriosis and make plausible MCP-1 as a key effector cell mediator involved in the pathogenesis of the disease. 0-038
Stimulation of Peritoneal Macrophage (PM) Mediated Cytolysis of Ectopic Endometrial Cells (EC) From Women With Endometriosis (endo pts). D.P. Braun, H. Gebel, N. Rana and W. P. Dmowski. Institute for the Study and Treatment of Endometriosis and Rush Medical College, Chicago, IL. Objectives: We have previously reported that EC from peritoneal implants of endo pts are significantly less sensitive to PM-mediated cytolysis than eutopic EC from the same pts. We hypothesized that the resistence of ectopic EC to PM cytolysis was critical for the establishment of endo. The goal of this study was to assess the capacity of macrophage activators or monoclonal antibodies (mAB) against EC epitopes to enhance the cytolytic activity of PM against EC from peritoneal implants. Design: PM preincubated with medium, v-interferon + lipopolysaccharide (IFN/LPS) or Virulizin T M were tested for the capacity to lyse Chromium ~1 (Cr51)-labelled autologous implant EC in the presence or absence of the $2n8 mAB. Abstracts
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Materials and Methods: Pts with endo found to have implants suitable for biopsy (n= 10) were diagnosed and staged at the time of laparoscopy according to RAFS criteria. EC were obtained from implant tissues by digestion with collagenase/DNase and used in cytotoxicity assays following labelling with Cr 51. PM were isolated from peritoneal aspirate fluids by adherence to microtiter plates. PM were activated 18 hrs in 100 U/ml IFN+2 ng/ml LPS or with a 1:1000 dilution of Virulizin T M prior to washing, addition of Cr51-labelled EC _+ a 1:1000 dilution of $2n8 mAB, and an additional 24 hrs. incubation. Specific Cr 51 release from EC was determined with the Titertek system. Results: Cytolysis of EC by PM following incubation in medium was <2% and only slightly increased following incubation with IFN/LPS (mean_+SD=2.8_+4%, not significant compared to medium). In contrast, cytolysis of EC by PM activated with Virulizin T M was significantly increased (25.8_+11%, p=0.005) as was the cytolysis of EC pretreated with the $2n8 mAB (23.8_+14%, p=0.02). Conclusions: This study suggests that EC from peritoneal implants of endo pts that are resistent to PM-mediated cytolysis can be lysed by PM activated with Virulizi n T M o r following treatment with the $2n8 mAB. Therapy with macrophage activators _+ monoclonal antibodies against EC-associated epitopes should be considered.
0-039 Immunotolerance Induced by Intratesticular Antigen Priming: Expression of TGF-fl, Fas and Fas Ligand. 1H. K. Li, 2j. Ren, ~H. Shichi and 1C. B. Dhabuwala. 1Department of Urology and 2Kresge Eye Institute, Wayne State University, Detroit, MI. Objectives: A single injection of S-antigen (S-Ag) into rat testes prior to immunization induces systemic tolerance (orchidic tolerance) and protects the animals from Experimental autoimmune uveoretinitis (EAU). In order to understand the mechanism by which the immunosuppression signal or signal carrier is generated, we determined in this study changes in immunoreactivity for Transforming growth factor (TGF-~), Transducing molecule Fas (CD95) and Fas ligand in the testes following an injection of S-Ag. Design: The expression of TGF-~, Fas and Fas ligand were studied by immunohistochemistry. The effects of anti-TGF-~ and Fas peptide was evaluated by transfering of splenocytes into the recipient rats. Materials and Methods: S-Ag (100 #g/rat) or PBS (100 #1) was injected into the testes of Lewis rats and orchiectomy was performed at 1, 3, 6, 12, 24 and 48 hours. Testes were fixed, embedded in paraffin and sectioned for avidin/ biotin HRP immunohistochemistry. Immunization of rats was affected by a single footpad injection of S-Ag/CFA. Rat splenocytes were isolated and incubated overnight with rat testicular soluble extracts and S-Ag. Fas peptide or anti-TGF-fl antibody was included in the supernatant for incubation with splenocytes. Splenocytes were then injected intraperitoneally into recipient rats. Seventy two hours later, the animals were immunized with 100 #g SAg/CFA. S20
Abstracts
Results: 1. TGF-~ immunoreactivity reached a maximum at 6 to 12 hours and decreased thereafter to the basal level at 48 hours. TGF-fl immunoreactivity was found predominantly in the interstitial tissue and not in the germ cells. 2. Both in control and experimental animals, immunoreactivity for Fas ligand was high in the interstitial tissue, while Fas immunoreactivity was associated with the germ cells in the seminiferous tubule. 3. Inclusion of anti-TGF-p or a Fas peptide in the testis extract reduced the potency of incubated splenocytes to induce systemic tolerance in recipient rats. Conclusions: Our studies suggest that TGF-fl and Fas ligand expressed in MHC-positive interstitial cells may have an important role in the generation of orchidic tolerance induction signals. Generation of the orchidic tolerance signal does not require the anatomical structure of the testes but is mediated by molecular entities such as TGF-fl and Fas ligand. 0-040
The Effect of Leukocyte Immunotherapy on the Development of Maternal Sperm Antibodies. R. Pyrzak, D. N. Curole, P. H. Rye, R. P. Dickey, S. N. Taylor, P.Y. Lu, A. S. Ports and C. C. Grady. The Fertility Institute, New Orleans, LA. Objective: Paternal Leukocyte Immunization therapy (PLIT) is used to prevent further abortions in women with a history of recurrent pregnancy loss (RPL). This study evaluates the effect of PLIT on the appearance of sperm antibodies (SAb) in maternal serum. Design: A prospective study of women undergoing immunological evaluation resulting from RPL. If the leukocyte antibody detection (LAD) was negative, then PLIT was recommended. Sperm antibodies in the maternal serum, before and after treatment, was measured. Materials and Methods: Twelve women, who had been diagnosed with negative LAD, were included in this study. Prior to PLIT, maternal peripheral blood was collected. Patients were innoculated intradermally with a paternal leukocyte (WBC). The concentrate of WBC for immunotherapy was prepared from 80 ml of paternal blood. WBC were prepared in a manner to remove the erythrocytes and reduce the amount of platelets. Patients were innoculated with 80-100 × l0 GWBC, once, twice or three times, 4 - 6 weeks apart. Between one and six months after PLIT, the maternal blood was collected again. Patients' sera were tested for levels of IgG and IgA using sperm antibodies test. Indirect SAb test using immunobeads was performed. The level of SAb in maternal sera was compared, before and after the PLIT. SAb levels less than 20% (percent of sperm with antibodies) were considered as not significant, levels higher than 20% were positive and levels higher than 50% were clinically significant. Results: Before the treatment, 10 of 12 wemen had negative SAb with ranges between 0-9%, and two women had positive level of SAb ranging between 32-41%. After PLIT, 5 of the 10 initially negative patients developed clinically significant level of SAb, ranging between 5 0 95%. Two women developed a positive level of SAb with
0-037 Increased Expression of Monocyte Chemotactic Protein-1 in the Endometrium of Women With Endometriosis. 1A. Akoum, 1Jolicoeur, 1M. Boutouil, 1I. Paradis, 2R. Drouin, ~R. Maheux and 1A. Lemay. 1Laboratoire d'Endocrinologie de la Reproduction, and 2Unit~ de Recherche en G~n~tique Humaine et Mol~culaire, Centre de Recherche, Pavillon Saint-Francois d'Assise, Centre Hospitalier Universitaire de Quebec, Universit~ Laval, Quebec, Canada. Objective: According to our previous data, cytokinestimulated uterine endometrial cells of women with endometriosis secrete higher levels of monocyte chemotactic protein-1 (MCP-1) than those of women without laparo-
scopical evidence of the disease. MCP-1 exerts a potent action on monocyte chemoattraction and activation, and may play an important role in the activation of peritoneal macrophages and peripheral blood monocytes observed in patients with endometriosis. In the present study, we examined the in situ expression of MCP-1 in the endometrium of women with and without endometriosis. Design: Immunohistochemical and in situ hybridization Studies. Material and Methods: Endometrial biopsies were obtained from women presenting for infertility or pelvic pain in whom endometriosis was diagnosed at laparoscopy (n=47) and women presenting for tubal ligation without laparoscopic evidence of the disease (n=22). Immunohistochemistry was carried out using a mouse monoclonal antiMCP-1 antibody, a Vectastain Elite ABC kit and diaminobenzidine as chromogen. In situ hybridization was performed using MCP-1 cDNA probes labelled with biotin, which was then detected by immunofluorescence. MCP-1 expression was evaluated in a blinded fashion using an arbitrary scale (0 = absent, 1 = light, 2 = moderate and 3 = intense), which takes into account the intensity and the distribution of staining. Results: MCP-1 was found in the stroma and epithelial glands of the endometrium. An increased expression of MCP-1 in the endometrial glands of women having endometriosis compared to normal women was observed both at the level of the protein (P = 0.0001) and mRNA (P = 0.0001). The most significant elevation of the protein expression was found in the mild stage of endometriosis (stage II) (P = 0.0006), whereas the highest level of mRNA expression was observed in more severe disease. Conclusion: These findings indicate that MCP-1 expression is up-regulated in the eutopic endometrium of women with endometriosis and make plausible MCP-1 as a key effector cell mediator involved in the pathogenesis of the disease. 0-038
Stimulation of Peritoneal Macrophage (PM) Mediated Cytolysis of Ectopic Endometrial Cells (EC) From Women With Endometriosis (endo pts). D.P. Braun, H. Gebel, N. Rana and W. P. Dmowski. Institute for the Study and Treatment of Endometriosis and Rush Medical College, Chicago, IL. Objectives: We have previously reported that EC from peritoneal implants of endo pts are significantly less sensitive to PM-mediated cytolysis than eutopic EC from the same pts. We hypothesized that the resistence of ectopic EC to PM cytolysis was critical for the establishment of endo. The goal of this study was to assess the capacity of macrophage activators or monoclonal antibodies (mAB) against EC epitopes to enhance the cytolytic activity of PM against EC from peritoneal implants. Design: PM preincubated with medium, v-interferon + lipopolysaccharide (IFN/LPS) or Virulizin T M were tested for the capacity to lyse Chromium ~1 (Cr51)-labelled autologous implant EC in the presence or absence of the $2n8 mAB. Abstracts
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Materials and Methods: Pts with endo found to have implants suitable for biopsy (n= 10) were diagnosed and staged at the time of laparoscopy according to RAFS criteria. EC were obtained from implant tissues by digestion with collagenase/DNase and used in cytotoxicity assays following labelling with Cr 51. PM were isolated from peritoneal aspirate fluids by adherence to microtiter plates. PM were activated 18 hrs in 100 U/ml IFN+2 ng/ml LPS or with a 1:1000 dilution of Virulizin T M prior to washing, addition of Cr51-labelled EC _+ a 1:1000 dilution of $2n8 mAB, and an additional 24 hrs. incubation. Specific Cr 51 release from EC was determined with the Titertek system. Results: Cytolysis of EC by PM following incubation in medium was <2% and only slightly increased following incubation with IFN/LPS (mean_+SD=2.8_+4%, not significant compared to medium). In contrast, cytolysis of EC by PM activated with Virulizin T M was significantly increased (25.8_+11%, p=0.005) as was the cytolysis of EC pretreated with the $2n8 mAB (23.8_+14%, p=0.02). Conclusions: This study suggests that EC from peritoneal implants of endo pts that are resistent to PM-mediated cytolysis can be lysed by PM activated with Virulizi n T M o r following treatment with the $2n8 mAB. Therapy with macrophage activators _+ monoclonal antibodies against EC-associated epitopes should be considered.
0-039 Immunotolerance Induced by Intratesticular Antigen Priming: Expression of TGF-fl, Fas and Fas Ligand. 1H. K. Li, 2j. Ren, ~H. Shichi and 1C. B. Dhabuwala. 1Department of Urology and 2Kresge Eye Institute, Wayne State University, Detroit, MI. Objectives: A single injection of S-antigen (S-Ag) into rat testes prior to immunization induces systemic tolerance (orchidic tolerance) and protects the animals from Experimental autoimmune uveoretinitis (EAU). In order to understand the mechanism by which the immunosuppression signal or signal carrier is generated, we determined in this study changes in immunoreactivity for Transforming growth factor (TGF-~), Transducing molecule Fas (CD95) and Fas ligand in the testes following an injection of S-Ag. Design: The expression of TGF-~, Fas and Fas ligand were studied by immunohistochemistry. The effects of anti-TGF-~ and Fas peptide was evaluated by transfering of splenocytes into the recipient rats. Materials and Methods: S-Ag (100 #g/rat) or PBS (100 #1) was injected into the testes of Lewis rats and orchiectomy was performed at 1, 3, 6, 12, 24 and 48 hours. Testes were fixed, embedded in paraffin and sectioned for avidin/ biotin HRP immunohistochemistry. Immunization of rats was affected by a single footpad injection of S-Ag/CFA. Rat splenocytes were isolated and incubated overnight with rat testicular soluble extracts and S-Ag. Fas peptide or anti-TGF-fl antibody was included in the supernatant for incubation with splenocytes. Splenocytes were then injected intraperitoneally into recipient rats. Seventy two hours later, the animals were immunized with 100 #g SAg/CFA. S20
Abstracts
Results: 1. TGF-~ immunoreactivity reached a maximum at 6 to 12 hours and decreased thereafter to the basal level at 48 hours. TGF-fl immunoreactivity was found predominantly in the interstitial tissue and not in the germ cells. 2. Both in control and experimental animals, immunoreactivity for Fas ligand was high in the interstitial tissue, while Fas immunoreactivity was associated with the germ cells in the seminiferous tubule. 3. Inclusion of anti-TGF-p or a Fas peptide in the testis extract reduced the potency of incubated splenocytes to induce systemic tolerance in recipient rats. Conclusions: Our studies suggest that TGF-fl and Fas ligand expressed in MHC-positive interstitial cells may have an important role in the generation of orchidic tolerance induction signals. Generation of the orchidic tolerance signal does not require the anatomical structure of the testes but is mediated by molecular entities such as TGF-fl and Fas ligand. 0-040
The Effect of Leukocyte Immunotherapy on the Development of Maternal Sperm Antibodies. R. Pyrzak, D. N. Curole, P. H. Rye, R. P. Dickey, S. N. Taylor, P.Y. Lu, A. S. Ports and C. C. Grady. The Fertility Institute, New Orleans, LA. Objective: Paternal Leukocyte Immunization therapy (PLIT) is used to prevent further abortions in women with a history of recurrent pregnancy loss (RPL). This study evaluates the effect of PLIT on the appearance of sperm antibodies (SAb) in maternal serum. Design: A prospective study of women undergoing immunological evaluation resulting from RPL. If the leukocyte antibody detection (LAD) was negative, then PLIT was recommended. Sperm antibodies in the maternal serum, before and after treatment, was measured. Materials and Methods: Twelve women, who had been diagnosed with negative LAD, were included in this study. Prior to PLIT, maternal peripheral blood was collected. Patients were innoculated intradermally with a paternal leukocyte (WBC). The concentrate of WBC for immunotherapy was prepared from 80 ml of paternal blood. WBC were prepared in a manner to remove the erythrocytes and reduce the amount of platelets. Patients were innoculated with 80-100 × l0 GWBC, once, twice or three times, 4 - 6 weeks apart. Between one and six months after PLIT, the maternal blood was collected again. Patients' sera were tested for levels of IgG and IgA using sperm antibodies test. Indirect SAb test using immunobeads was performed. The level of SAb in maternal sera was compared, before and after the PLIT. SAb levels less than 20% (percent of sperm with antibodies) were considered as not significant, levels higher than 20% were positive and levels higher than 50% were clinically significant. Results: Before the treatment, 10 of 12 wemen had negative SAb with ranges between 0-9%, and two women had positive level of SAb ranging between 32-41%. After PLIT, 5 of the 10 initially negative patients developed clinically significant level of SAb, ranging between 5 0 95%. Two women developed a positive level of SAb with