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Can Hypoxia Impact Lymphocyte-Mediated Elimination of Peritoneal Fibroblasts Leading to Development of Adhesion Phenotype?
achieved more than one positive test and of the patients using more than one donor, similar numbers of pregnancies occurred before and after switching donors. Overall, biochemical pregnancy rate was 13.7% per cycle and the live birth rate was 9.4% per cycle. CONCLUSION: The results of the study indicate that switching donors does not increase the likelihood of pregnancy for TDI. Supported by: None
REPRODUCTIVE SURGERY P-846 Can Hypoxia Impact Lymphocyte-Mediated Elimination of Peritoneal Fibroblasts Leading to Development of Adhesion Phenotype? Z. Alpay, M. S. Ozgonenel, S. Savasan, S. Buck, G. M. Saed, M. P. Diamond. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI; Children’s Hospital of Michigan, Division of Hematology/Oncology, Wayne State University, Detroit, MI. OBJECTIVE: Several characteristics of fibroblasts obtained from postsurgical intraperitoneal adhesion tissue (AT) could be induced by hypoxia treatment in normal peritoneal fibroblasts suggesting a central role for local hypoxic environment in the adhesion development. The natural immune response participates in the elimination of altered cells maintaining body homeostasis. The rates of this response and tissue regeneration ultimately determine the outcome of tissue healing. We have shown that lymphokineactivated killer (LAK) cells kill AT fibroblasts more efficiently than normal peritoneal (NP) fibroblasts. In this study, we investigated the effect of hypoxia on the expression of immune response-related surface molecules and LAK cell-mediated fibroblast elimination of NP and AT fibroblasts in vitro. DESIGN: Primary cell cultures of both NP and AT fibroblasts obtained from patients with post-surgical adhesions were expanded and used in the experiments. Immune response-related surface marker expression of untreated and hypoxia-treated NP and AT fibroblasts was studied at least three times. The effect of allogeneic LAK cells on those cells was simultaneously investigated following a 4-hour co-incubation of LAK cells. MATERIALS AND METHODS: Hypoxia treatment was achieved by incubating fibroblasts in an airtight Plexiglas chamber, which was deoxygenated by positive infusion of 2% CO2 /nitrogen gas mixture. Cultures in hypoxic chamber were then placed in a standard humidified tissue incubator for 24 hours prior to experimentation. The expression of intercellular adhesion molecule-1 (CD54), co-stimulatory molecule (CD40), tumor necrosis factor-alpha receptor type II (CD120b) and transferrin receptor (CD71) was studied by flow cytometry in hypoxia-treated and untreated NP and AT primary culture fibroblasts obtained from four different individuals. Allogeneic LAK cells were generated by incubating peripheral blood mononuclear cells from healthy donors with interleukin 2 and interleukin 15 for five days. The LAK cell-mediated fibroblast elimination was studied by our established flow cytometric cell mediated cytotoxicity assay. Paired t test of the mean values of individual cases was used for statistical evaluation. RESULTS: Hypoxia treatment of fibroblasts resulted in increased average LAK cell-mediated fibroblast killing compared to untreated cells. This effect was more prominent in NP than in AT fibroblasts (1.5 ⫾ 0.4 fold vs. 1.1 ⫾ 0.2 fold). The surface expression of CD54, CD40, CD120b and CD71 were not affected significantly by short term hypoxia exposure, however it resulted in a measurable decrease in CD71 expression in AT fibroblasts (0.95 ⫾ 0.08 in NP vs. 0.88 ⫾ 0.03 in AT). CONCLUSION: The finding of a more prominent increase in LAK cell-mediated fibroblast elimination following hypoxia exposure in NP than AT fibroblasts was supportive of a role of hypoxia in the pathogenesis of adhesion development. In other words, hypoxia treatment of NP could induce a phenotype similar to AT, but could not further affect AT fibroblasts. However, this increase in lymphocyte response was not correlated with enhanced immune response associated marker expressions following short term hypoxia exposure. The decrease in transferrin receptor expression might reflect an adaptive response to hypoxia resulting in decreased cellular iron uptake and this might be a transient phenomenon. Supported by: None.
FERTILITY & STERILITY威
P-847 If This Is No Pollution the Solution Is Not Dilution: The Consequences of Irrigation on Peritoneal Fluid and Cell Homeostasis. D. E. Ott. Mercer University, Macon, GA. OBJECTIVE: Since peritoneal fluid and its contents are intimately involved with peritoneal homeostasis and repair it is necessary under normal circumstances to maintain as normal a physiologic condition and environment as possible. Removal of peritoneal fluid cells may effect tissue repair and immunologic response to peritoneal damage and have significant local and peritoneal effects. Thus the objective of this work is to evaluate the effect of peritoneal irrigation on peritoneal cell count. DESIGN: Prospective analysis of forty-eight (48), four groups of twelve (12) patients, having sequential pelvic irrigation sampling to determine the effect of irrigation on peritoneal cell count done in University research laboratory and a private research facility. MATERIALS AND METHODS: Forty-eight female patients undergoing benign, non-endometriosis, non-infectious laparoscopy had all visible peritoneal fluid evacuated from the cul-de-sac after establishing a pneumoperitoneum. Irrigation was done using lactated ringer’s solution as 500 milliliter (cc) aliquots for three (3) liters by using ten (10) 500 cc lavages, or 250 cc aliquots (12 lavages) or 100 cc aliquots (thirty (30) lavages). Samples were measured for cell type and count after each lavage. Data compared the number of cells retrieved in each lavage cycle and was analyzed using a Mann-Whitney U test and variance between groups was analyzed using analysis of variance. Probability values of ⬍ 0.05 were deemed significant. RESULTS: Initial cell type and number of groups did not significantly differ. A similar proportion of cells were removed from each group in each successive irrigation suction cycle. A significantly greater number of cells were removed from the first to third lavage irrigations than from subsequent irrigations. A distinctive washout curve was found showing progressive cell count reduction with the greatest change occurring within the first lavage irrigation retrieval progressing to 99.9% removal by the third cycle and no significant changes after further irrigations. Peritoneal cell depletion as a result of lavage was not statistically different regardless of the volume of irrigation used. CONCLUSION: Cells within the peritoneal fluid, adherent mesothelial cells and free floating cells are rapidly removed from the peritoneal cavity by three lavage irrigation suction cycles. Three irrigations of any volume or irrigation from 100 to 500 cc at a time removes virtually all peritoneal cells. Three or more peritoneal lavages regardless of volume used significantly reduces peritoneal resident cell population. Elimination of peritoneal fluid and peritoneal resident cell population by irrigation severely affects at least the immediate homeostatic balance of the peritoneal cavity. Supported by: Georgia BioMedical, Inc. P-848 Cross Talk Between Inducible Nitric Oxide Syntheses (iNOS) and Myeloperoxidase (MPO) in Fibroblasts Isolated From Normal Peritoneal and Adhesion Tissues. G. M. Saed, H. Lu, Z. Jiang, S. Abuolba, H. Abu-Soud, M. P. Diamond. Wayne State University/Detroit Medical Center, Detroit, MI. OBJECTIVE: Fibroblasts beneath the mesothelium in the human peritoneum play an important role in the healing process of the peritoneum. Adhesion fibroblasts are characterized by lower levels of nitric oxide (NO) although there were no differences in iNOS, the enzyme which generates NO, between normal and adhesion fibroblasts. Additionally, adhesion fibroblasts exhibited significantly lower levels of MPO. We have previously shown that there is a cross talk between MPO and iNOS utilizing purified enzymes. MPO upregulate the catalytic activity of iNOS by preventing the feed back inhibition attributed to the formation of iNOS nitrosyl complex. Our objective is to determine whether there is a cross talk between MPO and iNOS in vivo utilizing fibroblasts isolated from normal peritoneum and from adhesions. DESIGN: Laboratory Study. MATERIALS AND METHODS: We have obtained fibroblast primary cultures from normal peritoneum and adhesion tissues of the same patients. We have utilized the small interfering RNA (SiRNA) technology to specifically knock-out the expression of MPO alone, iNOS alone, and combination of both and then quantify the expression levels of MPO and iNOS using the real-time RTR/PCR developed in our laboratory.
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REPRODUCTIVE SURGERY P-846 Can Hypoxia Impact Lymphocyte-Mediated Elimination of Peritoneal Fibroblasts Leading to Development of Adhesion Phenotype? Z. Alpay, M. S. Ozgonenel, S. Savasan, S. Buck, G. M. Saed, M. P. Diamond. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI; Children’s Hospital of Michigan, Division of Hematology/Oncology, Wayne State University, Detroit, MI. OBJECTIVE: Several characteristics of fibroblasts obtained from postsurgical intraperitoneal adhesion tissue (AT) could be induced by hypoxia treatment in normal peritoneal fibroblasts suggesting a central role for local hypoxic environment in the adhesion development. The natural immune response participates in the elimination of altered cells maintaining body homeostasis. The rates of this response and tissue regeneration ultimately determine the outcome of tissue healing. We have shown that lymphokineactivated killer (LAK) cells kill AT fibroblasts more efficiently than normal peritoneal (NP) fibroblasts. In this study, we investigated the effect of hypoxia on the expression of immune response-related surface molecules and LAK cell-mediated fibroblast elimination of NP and AT fibroblasts in vitro. DESIGN: Primary cell cultures of both NP and AT fibroblasts obtained from patients with post-surgical adhesions were expanded and used in the experiments. Immune response-related surface marker expression of untreated and hypoxia-treated NP and AT fibroblasts was studied at least three times. The effect of allogeneic LAK cells on those cells was simultaneously investigated following a 4-hour co-incubation of LAK cells. MATERIALS AND METHODS: Hypoxia treatment was achieved by incubating fibroblasts in an airtight Plexiglas chamber, which was deoxygenated by positive infusion of 2% CO2 /nitrogen gas mixture. Cultures in hypoxic chamber were then placed in a standard humidified tissue incubator for 24 hours prior to experimentation. The expression of intercellular adhesion molecule-1 (CD54), co-stimulatory molecule (CD40), tumor necrosis factor-alpha receptor type II (CD120b) and transferrin receptor (CD71) was studied by flow cytometry in hypoxia-treated and untreated NP and AT primary culture fibroblasts obtained from four different individuals. Allogeneic LAK cells were generated by incubating peripheral blood mononuclear cells from healthy donors with interleukin 2 and interleukin 15 for five days. The LAK cell-mediated fibroblast elimination was studied by our established flow cytometric cell mediated cytotoxicity assay. Paired t test of the mean values of individual cases was used for statistical evaluation. RESULTS: Hypoxia treatment of fibroblasts resulted in increased average LAK cell-mediated fibroblast killing compared to untreated cells. This effect was more prominent in NP than in AT fibroblasts (1.5 ⫾ 0.4 fold vs. 1.1 ⫾ 0.2 fold). The surface expression of CD54, CD40, CD120b and CD71 were not affected significantly by short term hypoxia exposure, however it resulted in a measurable decrease in CD71 expression in AT fibroblasts (0.95 ⫾ 0.08 in NP vs. 0.88 ⫾ 0.03 in AT). CONCLUSION: The finding of a more prominent increase in LAK cell-mediated fibroblast elimination following hypoxia exposure in NP than AT fibroblasts was supportive of a role of hypoxia in the pathogenesis of adhesion development. In other words, hypoxia treatment of NP could induce a phenotype similar to AT, but could not further affect AT fibroblasts. However, this increase in lymphocyte response was not correlated with enhanced immune response associated marker expressions following short term hypoxia exposure. The decrease in transferrin receptor expression might reflect an adaptive response to hypoxia resulting in decreased cellular iron uptake and this might be a transient phenomenon. Supported by: None.
FERTILITY & STERILITY威
P-847 If This Is No Pollution the Solution Is Not Dilution: The Consequences of Irrigation on Peritoneal Fluid and Cell Homeostasis. D. E. Ott. Mercer University, Macon, GA. OBJECTIVE: Since peritoneal fluid and its contents are intimately involved with peritoneal homeostasis and repair it is necessary under normal circumstances to maintain as normal a physiologic condition and environment as possible. Removal of peritoneal fluid cells may effect tissue repair and immunologic response to peritoneal damage and have significant local and peritoneal effects. Thus the objective of this work is to evaluate the effect of peritoneal irrigation on peritoneal cell count. DESIGN: Prospective analysis of forty-eight (48), four groups of twelve (12) patients, having sequential pelvic irrigation sampling to determine the effect of irrigation on peritoneal cell count done in University research laboratory and a private research facility. MATERIALS AND METHODS: Forty-eight female patients undergoing benign, non-endometriosis, non-infectious laparoscopy had all visible peritoneal fluid evacuated from the cul-de-sac after establishing a pneumoperitoneum. Irrigation was done using lactated ringer’s solution as 500 milliliter (cc) aliquots for three (3) liters by using ten (10) 500 cc lavages, or 250 cc aliquots (12 lavages) or 100 cc aliquots (thirty (30) lavages). Samples were measured for cell type and count after each lavage. Data compared the number of cells retrieved in each lavage cycle and was analyzed using a Mann-Whitney U test and variance between groups was analyzed using analysis of variance. Probability values of ⬍ 0.05 were deemed significant. RESULTS: Initial cell type and number of groups did not significantly differ. A similar proportion of cells were removed from each group in each successive irrigation suction cycle. A significantly greater number of cells were removed from the first to third lavage irrigations than from subsequent irrigations. A distinctive washout curve was found showing progressive cell count reduction with the greatest change occurring within the first lavage irrigation retrieval progressing to 99.9% removal by the third cycle and no significant changes after further irrigations. Peritoneal cell depletion as a result of lavage was not statistically different regardless of the volume of irrigation used. CONCLUSION: Cells within the peritoneal fluid, adherent mesothelial cells and free floating cells are rapidly removed from the peritoneal cavity by three lavage irrigation suction cycles. Three irrigations of any volume or irrigation from 100 to 500 cc at a time removes virtually all peritoneal cells. Three or more peritoneal lavages regardless of volume used significantly reduces peritoneal resident cell population. Elimination of peritoneal fluid and peritoneal resident cell population by irrigation severely affects at least the immediate homeostatic balance of the peritoneal cavity. Supported by: Georgia BioMedical, Inc. P-848 Cross Talk Between Inducible Nitric Oxide Syntheses (iNOS) and Myeloperoxidase (MPO) in Fibroblasts Isolated From Normal Peritoneal and Adhesion Tissues. G. M. Saed, H. Lu, Z. Jiang, S. Abuolba, H. Abu-Soud, M. P. Diamond. Wayne State University/Detroit Medical Center, Detroit, MI. OBJECTIVE: Fibroblasts beneath the mesothelium in the human peritoneum play an important role in the healing process of the peritoneum. Adhesion fibroblasts are characterized by lower levels of nitric oxide (NO) although there were no differences in iNOS, the enzyme which generates NO, between normal and adhesion fibroblasts. Additionally, adhesion fibroblasts exhibited significantly lower levels of MPO. We have previously shown that there is a cross talk between MPO and iNOS utilizing purified enzymes. MPO upregulate the catalytic activity of iNOS by preventing the feed back inhibition attributed to the formation of iNOS nitrosyl complex. Our objective is to determine whether there is a cross talk between MPO and iNOS in vivo utilizing fibroblasts isolated from normal peritoneum and from adhesions. DESIGN: Laboratory Study. MATERIALS AND METHODS: We have obtained fibroblast primary cultures from normal peritoneum and adhesion tissues of the same patients. We have utilized the small interfering RNA (SiRNA) technology to specifically knock-out the expression of MPO alone, iNOS alone, and combination of both and then quantify the expression levels of MPO and iNOS using the real-time RTR/PCR developed in our laboratory.
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