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Background: Ovarian cancer cells are known to express myeloperoxidase (MPO), an oxidant-producing enzyme with a 150 kDa homodimer, consisting of two identical monomers connected by a disulfide bond. Here, we aim to validate monomeric MPO (mMPO) as a biomarker for the early detection of ovarian cancer. Methods: Human ovarian cancer cells, sera from patients at various stages, sera from non-cancer inflammatory gynecological diseases, and healthy volunteers were used. Monomeric and dimeric MPO were measured by ELISA. Receiver operating curves were used to compare the predictive powers of serum dimeric and monomeric MPO to discriminate between samples. Results: The expression of MPO was unique to ovarian cancer cells. Specifically, mMPO was found to be the only form of MPO in all ovarian cancer cell lines. Intriguingly, mMPO was detected in the sera from all patients with ovarian cancer at various stages, but not from healthy individuals. Serum mMPO discriminated between early-stage ovarian cancer, healthy controls, and benign inflammatory gynecologic disorders. In addition, mMPO discriminated between the early and late stages of the disease. Conclusion: This work highlights mMPO as a potential biomarker for early detection of ovarian cancer, which is critically needed. [ABSTRACT FROM AUTHOR]

Objective The objective of this study was to determine the expression, and effect of targeting CD11b with a monoclonal antibody in ovarian cancer cells. Methods CD11b expression was determined in epithelial ovarian cancer (EOC) cell lines and tissues by immunofluorescence and flow cytometry. Cytotoxicity of the CD11b antibody and synergism with chemothearapeutic drugs were determined by the MTT Cell Proliferation Assay in human macrophages, normal ovarian epithelial cells, and in both sensitive and chemoresistant EOC cell lines. Cell migration was assessed with a scratch assay and in vivo effects of the CD11b antibody was assessed with a nude mouse ovarian cancer xenograft model. Data was analyzed with either t -tests or one-way ANOVA. Results CD11b was unexpectedly expressed in several EOC lines and tissues, but not normal tissues. Targeting CD11b with its monoclonal antibody resulted in intriguing cytotoxic effects in sensitive and chemoresistant EOC lines, while surprisingly not affecting normal cells. More importantly, the cytotoxicity of the CD11b antibody when combined with chemotherapeutic drugs (cisplatin or docetaxel) was significantly synergistic, in both sensitive and chemoresistant EOC cells. The anti-tumorigenic effect of the CD11b antibody was confirmed in an ovarian cancer nude mouse xenograft model. Conclusion Here we identify CD11b as a novel target, which selectively induces cytotoxicity in ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Clinical and epidemiological investigations have provided evidence supporting the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS), collectively known as oxidative stress, in the etiology of cancer. Exogenous factors such as chronic inflammation, infection and hypoxia are major sources of cellular oxidative stress. Specifically, oxidative stress plays an important role in the pathogenesis, neoangiogenesis, and dissemination of local or distant ovarian cancer, as it is known to induce phenotypic modifications of tumor cells by cross talk between tumor cells and the surrounding stroma. Subsequently, the biological significance of the relationship between oxidative stress markers and various stages of epithelial ovarian cancer highlights potential therapeutic interventions as well as provides urgently needed early detection biomarkers. In the light of our scientific research and the most recent experimental and clinical observations, this review provides the reader with up to date most relevant findings on the role of oxidative stress in the pathogenesis of ovarian cancer and the possible therapeutic implications. [ABSTRACT FROM AUTHOR]

BACKGROUND: Adhesion development is the most common sequelae of intra-abdominal and pelvic surgery and represents a significant, yet poorly understood, cause of morbidity among post-operative patients. It remains unclear, for example, exactly why adhesions form more frequently in certain tissues and/or patients, or at specific locations within them, as opposed to others. This review contributes to the growing knowledge pool by elucidating factors that potentially predispose to the development of adhesions. Given the strong correlation between a hypofibrinolytic state and adhesion formation, this review article will examine not only those factors that have been shown to directly predispose to adhesion development, but also those that are likely do so indirectly by means of altering the coagulation/fibrinolytic profile. METHODS: A literature search was performed using the PubMed database for all relevant English language articles up to February 2014. All of the identified articles were reviewed with particular attention to predisposing factors to post-operative adhesion development. In addition, the reference lists of each article were reviewed to identify additional relevant articles. RESULTS: Various factors have been shown to directly increase the risk of post-operative adhesion development; namely, certain genetic polymorphisms in the interleukin-1 receptor antagonist, increased estrogen exposure, and endometriosis. In addition, numerous factors are knownto increase the risk of fibrosis, therefore likely increasing the risk of adhesion development indirectly. These factors include genetic polymorphisms in plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor, diabetes mellitus, metabolic syndrome, hyperglycemia, obesity, depression, binge alcohol consumption, anti-Parkinsonian medications, oral hormone therapy, pregnancy, and cancer. CONCLUSIONS: The literature reviewed in this paper will help to direct future research aimed at understanding the mechanisms that underlie the association of certain factors with adhesion development. This information will be crucial in the creation of adequate preventative and treatment strategies. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer (EOC) cells are under intrinsic oxidative stress, which alters metabolic activity and reduces apoptosis. Key oxidative stress enzymes, including myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS), are upregulated and colocalized in EOC cells. Oxidative stress is also regulated, in part, by superoxide dismutase (SOD) and hypoxia-inducible factor (HIF) 1a. Dichloroacetate (DCA) converts anaerobic to aerobic metabolism and thus was utilized to determine the effects on apoptosis, iNOS, MPO, extracellular SOD (SOD-3), and HIF-1a, in EOC cells. Protein and messenger RNA (mRNA) levels of iNOS, MPO, SOD-3, and HIF-1a were evaluated by immunoprecipitation/Western blot and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively, utilizing SKOV-3 and MDAH-2774 treated with DCA. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 assays. Dichloroacetate induced apoptosis, reduced MPO, iNOS, and HIF-1a, whereas increased SOD, in both EOC cell lines. In conclusion, reduction of enhanced oxidative stress-induced apoptosis of EOC cells, which may serve as future therapeutic intervention for ovarian cancer. [ABSTRACT FROM PUBLISHER]

Objective: To study the biochemical and morphologic implications of different hormonal levels on endometrial development during assisted reproductive technology (ART) cycles.Design: Prospective, controlled study.Setting: University center.Patient(s): Eleven women during a natural cycle (controls) and 11 oocyte donors during an ART cycle (treated).Intervention(s): At the time consistent with day-3 ET, a transvaginal ultrasound, an endometrial biopsy, and blood sampling were performed. Morphology and thickness of the endometrial stripe were recorded. Real-time reverse transcriptase-polymerase chain reaction was used to measure messenger RNA (mRNA) levels for estrogen receptor (ER)-α, ER-β, P receptor (PR)-A, and PR-B in the endometrial tissue.Main Outcome Measure(s): To evaluate morphologic and biochemical endometrial development.Result(s): Endometrium was mostly trilaminar (proliferative-like pattern) and thicker in the treated group, as opposed to homogeneous and thinner in the controls. The PR-B mRNA expression increased 41% in treated patients; PR-A mRNA expression, instead, was unchanged. Serum E(2) and P were higher in the treated group than in controls. In contrast, FSH and LH levels were lower in the treated group.Conclusion(s): When compared with natural cycles, exposure of the endometrium to high hormone levels during ovarian stimulation significantly increased PR-B receptor expression at the time of ET. Concurrently, a proliferative-like endometrial pattern persisted. These findings reflect a delayed endometrial development in ART. [ABSTRACT FROM AUTHOR]

Abstract: Nitric oxide, superoxide, and lipid peroxidation (LPO) produced under oxidative stress may contribute to the development of postoperative adhesions. The objective of this study was to determine the effects of polychlorinated biphenyls (PCBs) on LPO, superoxide dismutase, myeloperoxidase (MPO), and nitrite/nitrate in human normal peritoneal and adhesion fibroblasts. PCB treatment reduced inducible nitric oxide synthase (iNOS) expression as well as levels of nitrite/nitrate in both cell lines. Although there was no difference in iNOS expression between the two cell lines, adhesion fibroblasts manifested lower basal levels of MPO compared to normal peritoneal fibroblasts. There was a reduction in MPO expression and its activity in response to PCB treatment in normal peritoneal fibroblasts; however, this effect was minimal in adhesion fibroblasts. Moreover, adhesion fibroblasts manifested higher levels of LPO compared to normal peritoneal fibroblasts, whereas PCB treatment increased LPO levels in both cell types. We conclude that PCBs promote the development of the adhesion phenotype by generating an oxidative stress environment. This is evident by lower iNOS, MPO, and nitrite/nitrate and a simultaneous increase in LPO. Loss of MPO activity, possibly through a mechanism involving MPO heme depletion and free iron release, is yet another source of oxidative stress. [Copyright &y& Elsevier]

Abstract: Objectives: Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO+)-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO+. We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. Methods: MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT–PCR, ELISA, and TUNEL assays. Results: MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. Conclusion: MPO and iNOS are colocalized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions. [Copyright &y& Elsevier]

Background: Postoperative adhesions occur in the overwhelming majority of patients after laparotomy and laparoscopy. The clinical consequences of adhesions can be devastating. Adhesions are believed to contribute to infertility and are the most common cause of small bowel obstruction and abdominal pelvic pain. Our objective is to develop a model for ex vivo development of postoperative adhesions. Methods: Peritoneal tissue strips were obtained from Sprague Dawley rats and were abraded by scraping the tissue surface with a scalpel. Tissues were incubated in a special medium, and visible adhesion bands were assessed. Experiments were performed in a university setting. Results: Adhesion bands were observed as early as 24 hours after abrasion. No adhesion bands were observed in the control tissue strips. Conclusions: This is a model system for the creation of actual adhesive bands utilizing an ex vivo culture of peritoneal strips. [ABSTRACT FROM AUTHOR]

The article presents a study which evaluates the development of adhesion, prevention, and the use of adjunct therapies in gynecologic surgery that employs laparoscopy. It notes that laparoscopic surgery has proven benefits when employed in open surgery. However, it is unclear whether laparoscopy has its role in adhesion development and in the applicability to adhesion barrier.

Postoperative adhesions are a significant source of morbidity, including contributions to pelvic pain, bowel obstruction, and infertility. While the mechanisms of postoperative adhesion development are complex and incompletely understood, hypoxia appears to trigger a cascade of intracellular responses involving hypoxia-inducible factors, lactate, reactive oxygen species, reactive nitrogen species, and insulin-like growth factors that results in manifestation of the adhesion phenotype. Thus, substantial evidence exists to implicate the direct role of cellular metabolism in wound repair and adhesion development. [ABSTRACT FROM AUTHOR]

Hypoxia induces the adhesion phenotype, characterized by enhanced extracellular matrix molecule and cytokine expression. Additionally, hypoxia reduces myeloperoxidase (MPO) activity in normal peritoneal fibroblasts to basal levels of adhesion fibroblasts indicating the importance of this enzyme in the development of the adhesion phenotype and also in tissue fibrosis. Immunohistochemistry was used to detect and localize MPO and inducible nitric oxide synthase (iNOS) in fibroblasts. Silencing of these genes was performed using siRNA technology. Levels of iNOS, MPO, type I collagen, and transforming growth factor were detected using real-time reverse transcription-polymerase chain reaction (RT-PCR), while HPLC was used to measure nitrate/nitrite levels. Our results show a unique interaction between MPO and iNOS, which are colocalized in both cell lines. Silencing iNOS reduced MPO and nitric oxide levels while silencing MPO had similar results, but to a lesser extent in both cell types. Additionally, silencing iNOS reduced type I collagen and transforming growth factor-β in adhesion fibroblasts, but to a lesser extent in peritoneal fibroblasts. These studies identify MPO and iNOS as key enzymes in the cellular response to hypoxia and consequent development of tissue fibrosis. [ABSTRACT FROM AUTHOR]

Objective: To examine the effect of interferon (IFN)-γ treatment under normal and hypoxic conditions on the BCL-2/BAX ratio of fibroblasts obtained from normal peritoneal and adhesion tissues of the same patients. Design: Prospective experimental study. Setting: University medical center. Patient(s): Fibroblasts from peritoneum and adhesion tissues of 5 patients. Intervention(s): Hypoxia and IFN-γ treatments of fibroblasts. Main Outcome Measure(s): We used the multiplex polymerase chain reaction technique to measure expression of BCL-2 and BAX in normal peritoneal and adhesion fibroblasts exposed to hypoxia (2% O2), in the presence or absence of IFN-γ for different time points and dosages. Result(s): At baseline, adhesion fibroblasts manifested decreased basal levels of apoptosis compared with normal fibroblasts. Hypoxia treatment resulted in a time–response decrease in apoptosis in both cell lines. Interferon-γ treatment resulted in a dose–response increase in apoptosis in both cell lines. Hypoxia had a reduced or no effect on apoptosis in the presence of increasing doses of IFN-γ in both cell types. Conclusion(s): Interferon-γ can block the effects of hypoxia on apoptosis, supporting the antifibrogenic nature of this cytokine. This suggests that IFN-γ would be a good candidate for consideration for intervention in the development of peritoneal adhesions and fibrosis. [Copyright &y& Elsevier]

Purpose To assess the ability of fibroblasts isolated from normal peritoneum and adhesion tissues to express various hormone receptors when cultured with exogenous estradiol. Methods Primary cultures of fibroblasts from normal human peritoneum and adhesion tissue were treated with zero (control), 10-10, 10-8, and 10-6 M concentrations of 17β-estradiol. We performed real time reverse transcriptase polymerase chain reaction to determine mRNA levels of estradiol-a receptor (ER-α) and estradiol-β receptor (ER- β), progesterone receptor (P-R), androgen receptor (A-R), and prolactin receptor (PRL-R) in the two types of fibroblast cultures. Results In the control groups, P-R and A-R were higher in normal than in adhesion fibroblasts. In adhesion cells, ER- α were higher at 10-8 estradiol; ER-β were higher at 10-6 M estradiol; P-R remained constant; A-R showed a higher expression at 10-10 and 10-8 M estradiol; and PRLR showed an exponential increase at 10-10 M estradiol. Conclusions The inflammatory-like changes manifested by adhesion fibroblasts enhance the anabolic hormones receptor expression (ER-α, ER-β, PRL, and A-R), when exposed to estradiol. [ABSTRACT FROM AUTHOR]

Objective: To determine the effects of blocking the translation of cyclooxygenase-2 (COX-2) mRNA on the mRNA levels of type I collagen, type III collagen, fibronectin, and transforming growth factor-beta (TGF-β1) in fibroblasts obtained from normal peritoneal and adhesion tissues. Design: Prospective experimental study. Setting: University medical center. Patient(s): Fibroblasts established from peritoneal and adhesion tissue of the same patients. Intervention(s): Adenovirus with COX-2 treatment of the primary cultured fibroblasts. Main Outcome Measure(s): Fibroblasts of normal peritoneal and adhesion tissues were isolated from the same patients. Adhesion and normal peritoneal fibroblasts were transfected with an adenovirus COX-2 mRNA in sense or antisense orientation. RNA was extracted from each treatment and subjected to real time reverse transcriptase–polymerase chain reaction to quantify change in mRNA levels of type I collagen, type III collagen, fibronectin, and TGF-β1. Result(s): Type I collagen, type III collagen, fibronectin, and TGF-β1 mRNAs were present in both normal peritoneal and adhesion fibroblasts with significantly higher levels in adhesion fibroblasts. Normal fibroblasts transfected with the COX-2 sense virus exhibited an elevated mRNA level in those molecules that reached the mRNA levels seen for adhesion fibroblasts. There were no effects seen on the mRNA levels in those molecules when adhesion fibroblasts were transfected with COX-2 sense virus. Normal fibroblasts transfected with COX-2 antisense virus exhibited no difference in mRNA levels in those molecules as compared with untransfected controls. In contrast, adhesion fibroblasts transfected with COX-2 antisense exhibited markedly reduced mRNA levels in those molecules to levels that were seen in normal peritoneal fibroblasts. Conclusion(s): Our data suggest that inhibition of COX-2 may reduce the development of postoperative adhesions by preventing the formation of the adhesion phenotype. [Copyright &y& Elsevier]

Objective: To determine whether docosahexaenoic acid (DHA) reduces adhesion marker mRNA levels in normal peritoneal and adhesion fibroblasts. Design: Prospective experimental study. Setting: University Medical Center. Patient(s): Three patients undergoing laparotomy with excision of adhesions and normal peritoneum. Intervention(s): DHA treatment (100 μM) of cell cultures for 24 hours. Main Outcome Measure(s): Real-time reverse transcriptase polymerase chain reaction (RT-PCR) quantification of relative changes (mRNA copies/μg mRNA) in mRNA levels of type I collagen, vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1). Result(s): The DHA treatment significantly reduced type I collagen and VEGF, but not TGF-β1 mRNA levels in normal peritoneal fibroblasts compared to normal controls. The DHA treatment of adhesion fibroblasts reduced type I collagen mRNAs to those of normal peritoneal fibroblasts, decreasing mRNAs by 35% compared to untreated adhesion fibroblasts. The VEGF mRNA levels were 50% lower in DHA-treated adhesion fibroblasts versus untreated adhesion fibroblasts. Docasahexaenoic acid reduced TGF- β1 mRNA to normal levels in treated adhesion fibroblasts compared to untreated normal peritoneal fibroblasts. Conclusion(s): Docasahexaenoic acid substantially reduces levels of adhesion-related markers in normal peritoneal and adhesion fibroblasts. This study provides the molecular basis for an easily administered and potentially, highly efficacious, antiadhesion adjuvant. [Copyright &y& Elsevier]

Objective: To determine whether the insulin receptor (IR), insulin-like growth factor-I receptor (IGF-IR), and IGF-I are expressed differentially in fibroblasts isolated from normal peritoneal and adhesion tissue before and after 24-hour treatment with increasing glucose concentrations. Design: Prospective experimental study. Setting: University medical center. Patient(s): Primary cultures of fibroblasts established from peritoneal and adhesion tissues of the same patients. Intervention(s): Glucose treatment of the primary cultured fibroblasts for 24 hours with increasing concentrations of glucose (100–850 mg/dL). Main Outcome Measure(s): Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) levels for IR, IGF-IR, and IGF-I. Enzyme-linked immunosorbent assay was used to determine protein levels. Result(s): At the normal glycemic level (100 mg/dL), adhesion fibroblasts have significantly higher mRNA levels of the IR (7.96 ± 0.15 vs. 6.97 ± 0.16; P<.05), IGF-IR (7.72 ± 0.22 vs. 6.88 ± 0.06; P<.05), and IGF-I (7.04 ± 0.10 vs. 5.92 ± 0.10; P<.05) when compared with normal fibroblasts, respectively. Data are expressed as log(mRNA/μg RNA). Normal fibroblasts respond to increasing glucose concentrations by increasing the expression levels of the IR, IGF-IR, and IGF-I, whereas adhesion fibroblasts respond by decreasing the expression of the IR, IGF-IR, and IGF-I. Conclusion(s): The differential expression of the IR, IGF-IR, and IGF-I in adhesion fibroblasts may contribute to the pathogenesis of fibrosis observed in diabetic patients. [Copyright &y& Elsevier]

Free radicals are highly reactive molecules produced in the cell either as part of, or as end-products of, biochemical reactions that have crucial roles in the homeostasis of the organism. Thus, excess production or impaired elimination of free radicals leads to increased oxidative stress, which has been implicated in the development of several different disease states, including hypoxia–reperfusion injury, cancer, and aging. Peritoneal adhesions and endometriosis are relatively commonly identified in women, and are known to be associated with infertility without clearly understood pathophysiology. The prevention and treatment strategies of these conditions, both of which have tremendous propensity to recur, have not been completely established. The development of both disorders has been shown to be closely related to the presence of increased oxidative stress in the tissues. In this article, we review this relationship with reference to the mechanistic steps involved and their regulation. As our knowledge of both conditions expands, we believe there will be opportunities for specific steps to intervention in free radical metabolism to reduce and/or prevent further development of endometriosis and adhesions. [Copyright &y& Elsevier]

Objective: To determine the response of adhesion and normal peritoneal fibroblasts to interferon-γ (IFN-γ) under normal and hypoxic conditions. Design: Prospective experimental study. Setting: University medical center. Patient(s): Primary cultures of fibroblasts established from peritoneal and adhesion tissue of the same patients. Intervention(s): Hypoxia and IFN-γ treatment of the primary cultured fibroblasts. Main Outcome Measure(s): Primary cultures of fibroblasts were established from peritoneal and adhesion tissues of the same patients (n = 5). The expression of extracellular matrix components (type I collagen and fibronectin) in adhesion and peritoneal fibroblasts under normal (20% O2) and hypoxic (2% O2) conditions was evaluated by multiplex reverse-transcription polymerase chain reaction analysis. Result(s): Adhesion fibroblasts (ADF) have increased basal levels of type I collagen as compared with normal peritoneal fibroblasts (NF). Interferon-γ treatment resulted in a dose-response decrease in type I collagen and fibronectin mRNA levels in both ADF and NF. Hypoxia treatment resulted in a time-response increase in type I collagen and fibronectin mRNA levels in NF and ADF. Hypoxia had no effect on type I collagen and fibronectin mRNA levels in the presence of increasing dose of IFN-γ in both NF and ADF. Interferon-γ can block the stimulating effects of hypoxia on type I collagen expression, supporting the antifibrogenic nature of this cytokine. Conclusion(s): Understanding the mechanism by which IFN-γ exerts its effect will be important in the utilization of this cytokine as a therapy for postoperative adhesion and tissue fibrosis. [Copyright &y& Elsevier]

We examined the potential physiological relevance of myeloperoxidase (MPO)–nitric oxide (NO) interactions as they may relate to the cosubstrate, pseudo halide thiocyanate (SCN-), and substrate switching. Direct spectroscopic and rapid kinetics studies revealed that SCN- interaction with MPO facilitates formation of the MPO catalytic intermediate Compound II, limiting overall activity. However, a physiological NO concentration (2 μM or less) dramatically influences the build-up, duration, and decay of the steady-state level of MPO Compound II during the metabolism of SCN-, allowing the enzyme to function at full capacity. At higher NO concentrations, we observed significant increases in the rate of MPO Compound II formation, along with proportional increases in its duration as determined by the time elapsed during catalysis. Surprisingly, the decay rate of MPO Compound II remained unaltered as NO concentrations were increased. Computer simulations were carried out to model the kinetics of MPO Compound II formation, duration, and decay during the metabolism of SCN- as a function of NO concentration. These simulation traces closely approximate what was observed experimentally and support the involvement of a conformational intermediate of MPO Compound II complex decay, altering the overall capacity of MPO to promote two electrons versus one-electron oxidation reactions during steady- state catalysis. Collectively, the present studies reveal that (patho)physiologically relevant levels of NO have significant effects on MPO Compound II accumulation. Thus, NO affects the overall rate of peroxidation of substrates and the overall ability of the peroxidase to execute one- versus two-electron oxidation reactions. [ABSTRACT FROM AUTHOR]

This article demonstrates the ability to measure the actual oxygen and nitric oxide levels in cultured fibroblasts established from normal peritoneum and adhesion tissues with and without hypoxia treatment and to measure levels of oxygen and nitric oxide in vivo in various tissues before and after abrasion. [Copyright &y& Elsevier]

Objective: To determine the levels of COX-1, COX-2, and prostaglandin (PG) E(2) in human fibroblasts isolated from normal peritoneal and adhesion tissues. Design: Prospective experimental study. Setting: University medical center. Patient(s): Fibroblast cultures from both peritoneum and adhesion tissues of five patients. Intervention(s): Treatment of the primary cultured fibroblasts with NS398. Main Outcome Measure(s): We used Western blot to determine the effects of hypoxia on COX-1 and COX-2 levels from lysates of normal peritoneal and adhesion fibroblasts before and after hypoxia. We also used the ELISA techniques to determine PGE(2) levels in media collected from these fibroblasts before and after hypoxia and with and without NS398, a COX-2-specific inhibitor. Result(s): There was no difference in COX-1 levels between normal and adhesion fibroblasts with and without hypoxia. Basal COX-2 and PGE(2) levels were significantly higher in adhesion than normal fibroblasts. Hypoxia gradually increased COX-2 and PGE(2) levels in normal peritoneal fibroblasts over time, reaching a peak at 24 hours but had no effect on adhesion fibroblasts. Inhibition of COX-2 by NS398 significantly reduced PGE(2) levels in both normal and adhesion fibroblasts. Conclusion(s): The presence of higher levels of COX-2 in adhesion fibroblasts and the induction of COX-2 in normal peritoneal fibroblasts in response to hypoxia indicate a possible inflammatory response. Regulation of COX-2 may alter peritoneal healing and may provide the opportunity to reduce postoperative adhesion development. [ABSTRACT FROM AUTHOR]

Background: Injury to the peritoneum during surgery is followed by a healing process that frequently results in the attachment of adjacent organs by a fibrous mass, referred commonly as adhesions. Because injuries to the peritoneum during surgery are inevitable, it is imperative that we understand the mechanisms of adhesion formation to prevent its occurrence. This requires thorough understanding of the molecular sequence that results in the attachment of injured peritoneum and the development of fibrous tissue. Recent data show that fibroblasts from the injured peritoneum may play a critical role in the formation of adhesion tissues. Therefore, identifying changes in gene expression pattern in the peritoneal fibroblasts during the process may provide clues to the mechanisms by which adhesion develop. Methods: In this study, we compared expression patterns of larger number of genes in the fibroblasts isolated from adhesion and normal human peritoneum using gene filters. Contributions of TGF-beta1 and hypoxia in the altered expression of specific genes were also examined using a semiquantitative RT-PCR technique. Results: Results show that several genes are differentially expressed between fibroblasts of normal and adhesion peritoneum and that the peritoneal fibroblast may acquire a different phenotype during adhesion formation. Genes that are differentially expressed between normal and adhesion fibroblasts encode molecules involved in cell adhesion, proliferation, differentiation, migration and factors regulating cytokines, transcription, translation and protein/vesicle trafficking. Conclusions: Our data substantiate that adhesion formation is a multigenic phenomenon and not all changes in gene expression pattern between normal and adhesion fibroblasts are the function of TGF-beta1 and hypoxia that are known to influence adhesion formation. Analysis of the gene expression data in the perspective of known functions of genes connote to additional targets that may be manipulated to inhibit adhesion development. [ABSTRACT FROM AUTHOR]

To determine whether α smooth muscle cell actin (αSMCA) is expressed in human fibroblasts isolated from normal peritoneal and adhesion tissues.Prospective, experimental study.University medical center.Five patients undergoing laparotomy for adhesiolysis for pelvic pain.Primary culture of fibroblasts from both peritoneum and adhesion tissues.Immunohistochemistry techniques were used to determine whether αSMCA was present in normal peritoneal and adhesion fibroblasts from the same patients. Cultured fibroblasts from all tissues were also fixed on slides and stained with αSMCA monoclonal antibody labeled with immunofluorescence.Alpha SMCA protein was present in very low levels in the cytoplasm of normal peritoneal fibroblasts from all five subjects, as compared with high levels present in adhesion fibroblasts from the same patients.Adhesion fibroblasts develop a specific phenotype, an adhesion phenotype, which in part is characterized by the over-expression of αSMCA. The over-expression of αSMCA in adhesion fibroblasts indicates a possible response to peritoneal injury. Regulation of αSMCA might alter peritoneal healing and might provide the opportunity to reduce postoperative adhesion development. [Copyright &y& Elsevier]

Objective: To study the modulation of the inducible nitric oxide synthase/nitric oxide (iNOS/NO) expression system in fibroblasts isolated from human peritoneum and adhesion tissues by hypoxia. Design: Prospective experimental study. Setting: University medical center. Patient(s): Cultures of fibroblasts from both peritoneum and adhesion tissues of five patients. Intervention(s): Hypoxia treatment of the primary cultured fibroblasts. Main Outcome Measure(s): We used Western and Northern blots to determine whether iNOS mRNA and its protein were present in peritoneal and adhesion fibroblasts and whether this expression is modulated by hypoxia. Multiplex reverse transcription polymerase chain reaction (RT-PCR) technique was used to quantify type I collagen mRNA in response to NG-nitro-L-arginine methyl ester (L-NAME). A terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL) assay was used to quantify apoptosis in response to NO donor S-nitro-N-acetyl-penicillamine (SNAP) treatment. A Griess assay was used to measure NO levels. Result(s): Peritoneal fibroblasts have significantly higher NO levels than adhesion fibroblasts. Hypoxia decreased NO in peritoneal fibroblasts to levels observed for adhesion fibroblasts. In addition, hypoxia increased both mRNA and protein levels of the iNOS gene in peritoneal and adhesion fibroblasts. Augmentation of NO by SNAP treatment increased apoptosis in adhesion fibroblasts. In contrast, SNAP had no effect on apoptosis of peritoneal fibroblasts. Inhibition of NO by L-NAME treatment increased type I collagen mRNA levels in peritoneal fibroblasts. Conclusion(s): Our findings confirm that adhesion fibroblasts produce less NO than normal peritoneal fibroblasts; NO may be the mechanism responsible for the creation and persistence of the adhesion phenotype. [ABSTRACT FROM AUTHOR]

Heme reduction of ferric lactoperoxidase (LPO) into its ferrous form initially leads to the accumulation of the unstable form of LPO-Fe(II), which spontaneously converts to a more stable species, the two of which can be identified by Soret peaks at 440 and 434 nm, respectively. Our data demonstrate that both LPO-Fe(II) species are capable of binding O2 at a similar rate to generate the ferrous-dioxy complex. Its formation with respect to O2 was first order and monophasic and with rate constants of kon = 3.8 × 104 M-1 S-1 and koff = 11.2 s-1. The dissociation rate constant for the formation of LPO-Fe(II)-O2 is relatively high, in contrast to hemoprotein model compounds. This high dissociation rate can be attributed to a combination of effects that include the positive trans effect of the proximal ligand, the heme pocket environment, and the geometry of the Fe-O2 linkage. Our results have also shown that the decay of the LPO-Fe(II)-O2 complex occurs by two sequential O2-independent steps. The first step involves formation of a short-lived intermediate that can be characterized by its Soret absorption peak at 416 nm and may be attributed to the weakening of the Fe(II)-O2 linkage with a rate constant of 0.5 s-1. The second step is spontaneous conversion of this intermediate to generate the native enzyme and presumably superoxide as end products with a rate constant of 0.03 s-1. A comprehensive kinetic model that links LPO-Fe(II)-O2 complex formation to the LPO catalase-like activity, combined with the classic catalytic cycle, is presented here. [ABSTRACT FROM AUTHOR]

We have previously shown that fibroblasts obtained from adhesions produce greater amounts of transforming growth factor-beta1 (TGF-Β1) and extracellular matrix (ECM) molecules than normal fibroblasts isolated from normal peritoneum. The purpose of the current studies was to examine the effect of Tisseel (Baxter Healthcare Corporation, Glendale, CA), a fibrin sealant containing fibrinogen, aprotinin (a protease inhibitor), thrombin, and CaCl2, on TGF-Β1 and ECM production by human peritoneal mesothelial cells, normal peritoneal fibroblasts, and adhesion fibroblasts. Multiplex reverse transcription-polymerase chain reaction using Β-actin as a housekeeping gene was used to determine mRNA levels of TGF-Β1 and ECM in these cells at 6, 12, 24, and 48 hours under normoxic conditions in the following treatment groups: fibrin sealant (Tisseel) along; fibrin sealant with the two components alluted 1:2: fibrin sealant with the sealer protein component reconstituted without aprotinin (a protease inhibitor); fibrin sealant with the sealer protein component reconstituted without aprotinin (and both components diluted 1:2); fibrin sealant components diluted to physiologic concentrations; and control (culture media). The test compositions had little effect on TGF-Β1 mRNA expression in mesothelial cells and normal peritoneal fibroblasts, but resulted in a marked reduction of TGF-Β1 from adhesion fibroblasts. Expression on type I collagen by human peritoneal mesothelial cells was not detected; the compositions reduced type I collagen mRNA expression by both types of fibroblasts. Type III collagen was detected at six hours, and increased approximately 50 percent by culturing for 48 hours. Tisseel at full strength and with both components diluted 1:2 initially increased type III collagen mRNA levels; in contract, type III collagen mRNA levels were reduced in mesothelial cells by the fibrin sealant without aprotinin at both concentrations and at physiologic concentrations. In both types of fibroblasts, the Tisseel compositions reduced type III collagen mRNA expression. Fibronectin mRNA were transiently reduced at six hours by approximately 50 percent in the presence of the Tisseel components, but then returned to control levels. Fibronectin mRNA levels were not altered in normal peritoneal fibroblasts, but were reduced by all but the physiologic concentration in adhesion fibroblasts. Tisseel may modulate human peritoneal mesothelial cell, normal peritoneal fibroblast, and adhesion fibroblast function. These results suggest that fibrin sealant prepared from the Tisseel kit without aprotinin has the ability to reduce ECM and TGF-Β1 mRNA levels, especially from adhesion fibroblasts, which may indicate a role in reduction of postoperative adhesion development. [ABSTRACT FROM AUTHOR]

Objective. Emerging evidence supports a role for prostaglandins (PG) and their synthesizing enzyme, cyclooxygenase (COX), in tumor angiogenesis. The objective of this study was to investigate the effects of prostaglandin E2 (PGE2) on the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible transcription factor-1 alpha (HIF-1α) genes in epithelial ovarian cancer (EOC) cells.Methods. Two human EOC cell lines, MDAH-2774 and SKOV-3, were treated with exogenous dimethyl prostaglandin E2 (dmPGE2) at two doses of 10 and 50 μg/ml and cultured for 24 h under both normoxic and hypoxic conditions. Total RNA was extracted from EOC cells with the use of a monophasic solution of phenol and GITC/Trizol method. The levels of COX-2, VEGF, and HIF-1α mRNA were measured by quantitative reverse transcription PCR (RT-PCR).Results. Under normoxic conditions, treatment of both ovarian cancer cell lines with dmPGE2 resulted in a significant increase in VEGF expression but had no effect on HIF-1α. Culturing the cells under hypoxic conditions resulted in an increase in HIF-1α and VEGF mRNAs. The combination of hypoxia and dmPGE2 treatment resulted in the highest levels of VEGF and HIF-1α when compared to either individual treatment.Conclusion. PGE2 is a potent stimulator of VEGF expression in ovarian cancer cells. This effect of PG is further potentiated under hypoxic conditions where it is also associated with a significant increase in HIF-1α expression. [Copyright &y& Elsevier]

Kinetic and structure analysis of inducible nitric oxide synthase (iNOS) revealed that, in addition to the increase of iNOS expression in inflamed areas, the major pathway causing overproduction of NO is destabilization of the iNOS-nitrosyl complex(es) that form during steady-state catalysis. Formation of such a complex allows iNOS to operate at only a fraction (20-30%) of its maximum activity. Thus, bioavailability of NO scavengers at sites of inflammation may play an essential role in up-regulation of the catalytic activity of iNOS, by preventing the catalytic activity inhibition that is attributed to nitrosyl complex formation. Myeloperoxidase (MPC)), a major NO scavenger, is a pivotal enzyme involved in leukocyte-mediated host defenses. It is thought to play a pathogenic role under circumstances such as acute inflammatory tissue injury and chronic inflammatory conditions. However, a detailed understanding of the interrelationship between iNOS and MPO at sites of inflammation is lacking. We used direct spectroscopic, HPLC, and selective NO-electrode measurements to determine the interdependent relationship that exists between iNOS and MPO and the role of the MPO/H[sub2]O[sub2] system in up-regulating the catalytic activity of iNOS that occurs at sites of inflammation. Scavenging free NO from the iNOS milieu by the MPO/H[sub2]O[sub2] system subsequently restores the full capacity of iNOS to convert L-aginine to product (NO), as judged by the increase in the rates of citrulline and nitrite/nitrate production. Studies of iNOS catalytic mechanisms and function are essential to a more fundamental understanding of these factors, which govern iNOS-dependent processes in human health and disease. [ABSTRACT FROM AUTHOR]

Objective To compare the mRNA levels of transforming growth factor beta (TGF-β) isoforms 1, 2, and 3 among surgically injured peritoneum that healed with adhesions, surgically injured peritoneum that healed without adhesions, and uninjured peritoneum. Design Prospective experimental study. Setting University vivarium. Animal(s) Fourteen sexually mature female Sprague-Dawley rats, 226–250 grams. Interventions(s) Standardized cecal abrasion was performed on 120 Sprague-Dawley rats. The areas of abrasion and any resultant adhesions were harvested at necropsy 7 days later. Total RNA was then extracted from the serosal adhesions of four rats, from the serosa of five rats that healed without adhesion formation, and from analogous areas of cecum from five rats that did not undergo abrasion and served as controls. Main outcome measure(s) Quantification of the expression of the TGF-β1, TGF-β2, and TGF-β3 mRNA transcripts in peritoneal adhesions, normally healed peritoneum, and fresh, uninjured peritoneal tissue through the use of multiplex reverse transcriptase–polymerase chain reaction analysis. Results(s) Peritoneal adhesion TGF-β1 and TGF-β3 mRNA expression was 3.7-fold and 8.6-fold higher, respectively, than in abraded tissues that healed normally; and 5.6-fold and 4.6-fold higher, respectively, than in nonabraded tissues. While TGF-β2 mRNA levels were also higher in serosal adhesions compared with normally healed and uninjured peritoneum, this rise was not statically significant. Conclusion(s) Peritoneal adhesions resulting from surgical abrasion of a serosal surface have statistically significant increased levels of TGF-β1 and -β3 mRNA transcripts compared with both uninjured and normally healed peritoneum. [ABSTRACT FROM AUTHOR]

: ObjectiveTo characterize the molecular changes that occur in normal fibroblasts, adhesion fibroblasts, and mesothelial cells as a result of exposure to modified hyaluronic acid and carboxymethylcellulose (Seprafilm).: SettingUniversity research laboratory.: DesignHuman mesothelial and fibroblast cell culture.: Main outcome measure(s)Multiplex reverse transcriptase polymerase chain reaction was used to examine control and Seprafilm-treated normal peritoneal fibroblasts, adhesion fibroblasts, and mesothelial cells in culture for levels of messenger RNA from molecules known to be associated with adhesion development (transforming growth factor-β1, type I collagen, matrix metalloproteinase-1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, and tissue plasminogen activator).: Result(s)Seprafilm treatment of normal peritoneal fibroblasts, adhesion fibroblasts and mesothelial cells did not alter the expression of markers examined.: Conclusion(s)In the absence of a biological effect of Seprafilm on markers known to be involved in postoperative adhesion development, the ability of Seprafilm to reduce postoperative adhesions is most likely caused by its effect as a physical barrier. [Copyright &y& Elsevier]

: ObjectiveTo determine whether the COX-2 gene is expressed in human fibroblasts isolated from normal peritoneal and adhesion tissues.: DesignProspective experimental study.: SettingUniversity medical center.: Patient(s)Five patients undergoing laparotomy for pelvic pain. Primary cultures of fibroblasts were taken from both peritoneum and adhesion tissues.: Intervention(s)Hypoxia treatment of the primary cultured fibroblasts.: Main outcome measure(s)We used the multiplex reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry techniques to determine whether COX-2 mRNA and its protein were present in normal peritoneal and adhesion fibroblasts from the same patients. Total RNA was extracted from cultured fibroblasts and subjected to multiplex RT-PCR to detect the presence of COX-2 mRNA in these cells. Cultured fibroblasts from all tissues were also fixed on slides and stained with COX-2 monoclonal antibody labeled with immunofluorescence.: Result(s)COX-2 mRNA and its protein were absent in normal peritoneal fibroblasts from all five subjects but were present in adhesion fibroblasts from the same patients, as indicated by the multiplex RT-PCR and immunohistochemistry techniques. Hypoxia treatment significantly induced the mRNA and COX-2 protein levels in normal peritoneal fibroblasts to levels seen in adhesion fibroblasts under normoxic conditions. However, hypoxia had no effects on COX-2 expression by adhesion fibroblasts.: Conclusion(s)Adhesion fibroblasts develop a specific phenotype, an adhesion phenotype, which is in part characterized by the expression of COX-2. The expression of COX-2 mRNA in adhesion fibroblasts and the induction of COX-2 in peritoneal fibroblasts in response to hypoxia indicate a possible inflammatory response. Regulation of COX-2 may alter peritoneal healing and may provide the opportunity to reduce postoperative adhesion development. [Copyright &y& Elsevier]

: ObjectiveTo determine whether normal peritoneal and adhesion fibroblasts express tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-I) and whether their expression is regulated by oxygen.: DesignProspective experimental study.: SettingUniversity medical center.: Patient(s)Cultures of human fibroblasts established from peritoneal and adhesion tissues.: Intervention(s)Hypoxia treatment of the primary cultured fibroblasts.: Main outcome measure(s)We have used the multiplex reverse transcription polymerase chain reaction (RT-PCR) technique to determine the effect of hypoxia on the expression of tPA and PAI-I in normal peritoneal (NPF) and adhesion (ADF) fibroblasts. Cultures of NPF and ADF were exposed to hypoxia (2% O2) for 24 hours. RNA was extracted from cells and subjected to multiplex RT-PCR to quantitate relative changes in mRNA levels of tPA and PAI-I in response to hypoxia treatment.: Result(s)Basal tPA mRNA levels are present in both NPF and ADF and were 45% higher in NPF than ADF. Hypoxia decreased tPA in both NPF and ADF by 74% and 95%, respectively. Basal PAI-I mRNA levels were 64% higher in ADF than in NPF. Hypoxia increased PAI-I mRNA levels by 67% and 53% in NPF and ADF, respectively.: Conclusion(s)Plasminogen activator activity (PAA) of the peritoneum does not solely reside in the mesothelial cells, as previously identified, but also exists within fibroblasts, thus providing the potential to resolve postoperative fibrinous collections even at sites at which the mesothelial cells have been injured, removed, or destroyed. Furthermore, PAA in fibroblasts is regulated by oxygen; creation of a hypoxic state markedly attenuates PAA, thereby leading to adhesion development. [Copyright &y& Elsevier]

: ObjectiveTo determine the effect of high glucose levels on the expression of type I collagen, and whether this effect is regulated by transforming growth factor (TGF)-β1 in human peritoneal fibroblasts in culture.: DesignProspective experimental study.: SettingUniversity medical center.: Patient(s)Primary cultures of fibroblasts established from peritoneal tissues of five patients.: Intervention(s)High glucose treatment of the primary cultured fibroblasts.: Main outcome measure(s)Primary cultures of human peritoneal fibroblasts were incubated with varying amounts of glucose (1–5 g/L) for 24 hours. Total RNA was extracted from human peritoneal fibroblasts and converted to cDNA by reverse transcriptase. Multiplex reverse transcriptase/polymerase chain reaction (PCR) simultaneously co-amplifying β-actin with TGF-β1 or type I collagen mRNAs was used to quantitate type I collagen and TGF-β1 mRNA levels in response to increasing glucose concentrations with and without TGF-β1 antibody treatment.: Result(s)There was a significant increase in the mRNA for type I collagen and TGF-β1 in response to increasing glucose concentrations in a dose response-dependent manner. The TGF-β1 antibody treatment resulted in an 83% and 68% decrease in type I collagen and TGF-β1 mRNA levels, respectively.: Conclusion(s)Increasing glucose concentrations stimulated type I collagen expression in human peritoneal fibroblasts in culture. A potential mediator for this effect is TGF-β1. These results have implications not only for individuals with diabetes mellitus who may be predisposed to greater postoperative adhesion development, but also for individuals with surgical stress responses after surgery. [Copyright &y& Elsevier]

Presents a study that determined the effect of hypoxia and transforming growth factor (TGF)-β1 treatment on the expression of TGF-β1-3 and TGF-βI and βII receptors in human peritoneal fibroblasts. Factors that are central to the development of adhesions; Effects of TGF-β on aspects of growth and development; Description of the membrane proteins.

Objective: To determine which ovarian sex steroid(s), when removed from an intact organism, reduce(s) postoperative adhesion development.Design: Randomized, prospective, blinded study.Setting: University vivarium.Patient(s): One hundred twenty sexually mature female Sprague-Dawley rats, 226–250 g.Intervention(s): Day 0, sham ovariectomy or bilateral ovariectomy, accompanied by continuous-release sex steroid replacement of either no steroids (control), 17β-E2, natural P (P4), or combined E2/P4. Day 7, standardized cecal abrasion; day 14, necropsy with assessment of adhesion presence or absence.Main Outcome Measure(s): Adhesion formation.Result(s): Three rats died because of anesthesia or surgical complications, and 117 rats reached necropsy. The ovary-intact (sham) rats adhesion incidence was 60.9%; ovariectomized control rats, 20.8%; E2, 28.6%; P4, 33.3%; and combined E2/P4, 24.0%. Despite differing sex steroid replacement, two-tailed χ2 testing with correction for multiple comparisons showed no statistical difference in adhesion incidence among the four ovariectomy groups. A statistically significant lower adhesion incidence was noted between the ovary-intact sham cohort and the collective ovariectomy groups and between the sham and ovariectomized control cohorts.Conclusion(s): Ovarian presence or absence at the time of surgical wounding, and not the 17β-E2 or P milieu, modulates adhesion development. This implicates other ovarian factor(s) in postoperative adhesion development. [Copyright &y& Elsevier]

Objective: To determine the effects of TGF-beta1 on the expression of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and on vinculin, and F-actin in human peritoneal fibroblasts.Design: Descriptive study using cell culture, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescent and confocal microscopy.Setting: Academic medical center.Patient(s): Gynecological surgery patients.Intervention(s): None.Main Outcome Measure(s): Effects of TGF-beta1 on the steady state levels of alpha5, alpha(v), and alpha6 integrin transcripts were examined in the normal peritoneal fibroblasts using RT-PCR. Expression levels of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and of F-actin were measured by immunofluorescent microscopy. The distribution pattern of the integrin subunits, vinculin, and F-actin were examined using confocal microscopy.Result(s): TGF-beta1 significantly up-regulated the expression levels of the alpha5, alpha(v), and alpha6 integrin subunits and modulated their expression pattern, resulting in relatively higher levels of these subunits in the focal contacts of peritoneal fibroblasts. It allocated vinculin expression primarily to the focal contacts of cells and caused distortion of F-actin structure. The transcript levels of the alpha5, alpha(v), and alpha6 integrin subunits were not altered by TGF-beta1 treatment.Conclusion(s): TGF-beta1 may promote postoperative adhesion formation by inducing the migration of peritoneal fibroblasts by altering the expression levels and patterns of specific integrin subunits, vinculin, and F-actin. [ABSTRACT FROM AUTHOR]

Objective: To determine the effects of TGF-β1 on the expression of the α1, α2, α5, αv, and α6 integrin subunits and on vinculin, and F-actin in human peritoneal fibroblasts.Design: Descriptive study using cell culture, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescent and confocal microscopy.Setting: Academic medical center.Patient(s): Gynecological surgery patients.Intervention(s): None.Main Outcome Measure(s): Effects of TGF-β1 on the steady state levels of α5, αv, and α6 integrin transcripts were examined in the normal peritoneal fibroblasts using RT-PCR. Expression levels of the α1, α2, α5, αv, and α6 integrin subunits and of F-actin were measured by immunofluorescent microscopy. The distribution pattern of the integrin subunits, vinculin, and F-actin were examined using confocal microscopy.Result(s): TGF-β1 significantly up-regulated the expression levels of the α5, αv, and α6 integrin subunits and modulated their expression pattern, resulting in relatively higher levels of these subunits in the focal contacts of peritoneal fibroblasts. It allocated vinculin expression primarily to the focal contacts of cells and caused distortion of F-actin structure. The transcript levels of the α5, αv, and α6 integrin subunits were not altered by TGF-β1 treatment.Conclusion(s): TGF-β1 may promote postoperative adhesion formation by inducing the migration of peritoneal fibroblasts by altering the expression levels and patterns of specific integrin subunits, vinculin, and F-actin. [Copyright &y& Elsevier]

Objective: To determine whether restoration of normoxia after a hypoxic insult returns the molecular expression of type I collagen and TGF-beta1 to baseline levels.Design: Prospective experimental study.Setting: University medical center.Patient(s): Primary cultures of fibroblasts established from peritoneal tissues of five patients.Intervention(s): Hypoxia treatment of the primary cultured fibroblasts.Main Outcome Measure(s): Cultured human peritoneal fibroblasts (HPF) were maintained under hypoxic conditions (2% oxygen) for 24 hours and then transferred into normal culture conditions (normoxia) for another 24 hours. Total cellular RNA from cells was collected and subjected to multiplex reverse transcription polymerase chain reaction to quantitate type I collagen and transforming growth factor (TGF)-beta1 mRNA levels in response to these treatments.Result(s): Hypoxia treatment resulted in 30% and 50% increases in type I collagen and TGF-beta1 expression, respectively. Restoration of normoxia after hypoxia treatment failed to restore type I collagen and TGF-beta1 expression to their baseline levels.Conclusion(s): These data support the hypothesis that hypoxia induces irreversible molecular changes in peritoneal fibroblasts that produce a phenotype that increases extracellular matrix expression and thereby would promote adhesion development. Thus once a phenotype consistent with increased adhesion development is manifested, restoration of oxygen supply does not reverse the stimulation of HPF type I collagen and TGF-beta1 expression. This observation may in part explain the clinical observation that adhesion reformation is more difficult to prevent than de novo adhesion formation. [ABSTRACT FROM AUTHOR]

Objective: To determine the effect of hypoxia on apoptosis and proliferation of fibroblasts cultured from normal peritoneum or adhesions.Design: Prospective experimental study.Setting: University medical center.Patient(s): Primary cultures of fibroblasts established from peritoneal and adhesion tissues of the same patients.Intervention(s): Hypoxia treatment of the primary cultured fibroblasts.Main Outcome Measure(s): Apoptosis was assessed by the TUNEL assay and by the BCL-2-BAX expression ratio, as determined using multiplex reverse transcription polymerase chain reaction. The proliferation rate of these cells was determined by measuring the proliferative fraction using the flow cytometry in adhesion and peritoneal fibroblasts under normal and hypoxic conditions.Result(s): Compared with fibroblasts from normal peritoneum, fibroblasts from adhesions had significant complementary decreases in apoptosis and an increase in proliferation. In response to hypoxia, the Bcl-2-bax mRNA ratio was significantly higher in fibroblasts from adhesions as compared with fibroblasts from normal peritoneum. The degree of apoptosis induced by hypoxia correlated with the increase in the bcl-2-bax ratio in fibroblasts derived from both normal peritoneum and adhesions.Conclusion(s): Hypoxia induces proliferation while inhibiting apoptosis in fibroblasts from adhesion, thereby creating a phenotype that would favor adhesion development. [ABSTRACT FROM AUTHOR]

Overexpression and accumulation of extracellular matrix is central to peritoneal adhesion formation following surgically induced tissue trauma. Transforming growth factor‐β1 and hypoxia have been implicated in tissue fibrosis and postoperative adhesion formation. To extend this observation we examined whether transforming growth factor‐β1 and/or hypoxia regulate the expression of type I and III collagen in human peritoneal mesothelial cells. Cultured human mesothelial cells were maintained under hypoxia (2% oxygen), or treated with transforming growth factor‐β1 (1 ng/ml) or a combination of hypoxia and transforming growth factor‐β1. Total cellular RNA from treated and untreated cells were collected and subjected to multiplex reverse transcription/polymerase chain reaction to quantitate collagen I and III mRNA levels in response to these treatments. The results indicate that 6 hours of hypoxia increased collagen III mRNA by 7.2 fold which was further increased to 9.4 fold following transforming growth factor‐β1 treatment; in contrast collagen I mRNA decreased by 0.42 fold which was further decreased by 0.3 fold following transforming growth factor‐β1 treatment. Transforming growth factor‐β1 treatment under normal conditions resulted in an 8.4‐fold increase and a 0.3‐fold decrease in collagen III and I mRNA levels, respectively. Hypoxia treatment also resulted in a 1.9‐fold increase in transforming growth factor‐β1 mRNA level compared with control. The ratio of type III/I collagen was increased in response to transforming growth factor‐β1 treatment under hypoxic condition. In conclusion, the data suggest that hypoxia may modulate extracellular matrix production by human mesothelial cells via a transforming growth factor‐β1 dependent mechanism. (WOUND REP REG 1999;7:504–510) --> [ABSTRACT FROM AUTHOR]

Canine cutaneous T-cell lymphoma (CTCL) is a morphologic and immunophenotypic simulant of human mycosis fungoides (MF) characterized by an infiltrate of atypical, hyperconvoluted, epidermotropic T cells. To further support our hypothesis that canine MF is a useful model for the study of human CTCL, we have used Southern blotting to search for clonal T-cell proliferations in canine MF. Cellular DNA was extracted from normal dog buffy coat cells (n = 8), lesional canine MF skin (n = 8), canine MF buffy coat cells (n = 7), normal dog skin (n = 3), and normal human buffy coat cells (n = 5), digested with a panel of restriction enzymes and Southern blotted onto nylon membranes. All cases of canine MF were also immunophenotyped with anti-canine monoclonal antibodies to CD4, CD8, CD18, CD45RA, canine class II, T-cell activation antigens, and pan-B-cell antigens. Normal dogs gave reproducible digestion patterns in blood and skin, which differed from the human germline patterns when probed with a human T-cell receptor (TCR), beta chain constant region (Cβ) cDNA. Common germline bands between the species included the 3.5-kb Eco RI, 3.4-kb Bam HI, 5.4-kb Sac I. These results confirmed that the TCR-β gene is evolutionarily conserved between dog and man. Immunostaining revealed that 3/7 cases were CD4+ canine CTCL and 4/7 were CD8+ canine CTCL. Rearranged bands, deletion of germline bands, as well as minor alterations in electrophoretic mobility were observed in lesional DNA from seven of eight cases of canine MF, with at least two restriction digests in each case. Dog rearrangements were best detected with Bgl II, Eco RI, Eco RV, and Sac I, whereas deletions were detected with Bgl II, Sac I, Eco RV, and Bam HI. These studies demonstrate the presence of clonal TCR rearrangement in canine MF, further supporting the similarity of this tumor to human MF and its role as an animal model of CTCL. [ABSTRACT FROM AUTHOR]

To test whether recombinant anti-Müllerian hormone (rAMH) could exert an inhibitory function on BRCA1/2 expression in human ovarian cortex. Pilot study on ovariectomized nude mice xenotransplanted with human vitrified/warmed ovarian cortex and treated with rAMH via infusion pump. Twelve nude mice were ovariectomized and Alzet pumps delivering 1.23 mcg rAMH/day to reach a serum concentration of 17.5 ng/mL, or placebo (controls), were inserted intraabdominally. Previously vitrified/warmed 2x2 mm ovarian cortex fragments were transplanted on day 7 and then harvested on day 14 after pump placement. PCR analyses determined mRNA levels for BRCA1 and BRCA2 in the human ovarian cortex. In mice treated with rAMH, BRCA1 expression was significantly lower (0.196 fg/µg RNA, IQR 0.158, 0.236) than in controls (0.544 fg/µg RNA, IQR 0.458, 0.554; p =.030), while BRCA2 expression remained similar in rAMH mice (5.355 fg/µg RNA, IQR 4.479, 6.230) and in controls (4.011 fg/µg RNA, IQR 3.650, 4.182; p =.327). Administration of rAMH in the peri-transplant period caused downregulation of BRCA1, but not of BRCA2 expression, in human ovarian cortex. These results help our understanding of DNA repair mechanism in the ovarian cortex and identify AMH's possible protective effect on ovarian reserve in BRCA1 mutation carriers. [ABSTRACT FROM AUTHOR]

Objective: To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG.Materials and methods: This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7 days and then harvested after 14 days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count.Results: Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group.Conclusions: Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers’ tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion. [ABSTRACT FROM AUTHOR]

Objective: To test whether recombinant anti-Müllerian hormone (AMH) can inhibit ovarian cortex function by modulating the expression of other hormone receptors. Materials and Methods: Pilot experimental study with ovarian cortex obtained from 5 patients. Immediately after explant, the ovarian cortex specimens were divided into 5 equal fragments. One fragment was flash-frozen (uncultured) and 4 were incubated for 48 hours at 37°C in a pH-adjusted gamete buffer medium with increasing AMH concentrations of 0, 5, 25, and 50 ng/mL. After incubation, all specimens were rinsed and flash-frozen for polymerase chain reaction (PCR) executed in triplicates. We utilized real-time reverse transcription–polymerase chain reaction (RT-PCR) to determine messenger RNA (mRNA) levels of AMH and its receptor Anti-Müllerian Hormone-Receptor 2 (AMH-R2), follicle stimulating hormone receptor (FSH-R), luteinizing hormone receptor (LH-R), inhibin B, and insulin-like growth factor 1 receptor 1 (IGF1-R1) in ovarian cortex tissue. In addition, we performed Ki-67 immunostaining to evaluate cell proliferation in the treatment groups. Results: Absence of recombinant human AMH (rAMH) caused upregulation of all markers. Exposure to increasing rAMH concentrations caused tissue AMH expression downregulation (P = .024), while AMH-R2 (P = .005), FSH-R (P = .009), LH-R (P = .003), and inhibin B (P = .001) mRNA expression followed a bell-shaped response with an increased expression at low dose, followed by a decreased expression at higher doses. Expression of IGF1-R1 was independent (P = .039) of rAMH exposure. The Ki-67 immunostaining showed an increased cell proliferation in the media control compared to the uncultured and the tissue cultured with rAMH. Conclusions: Culture with increasing rAMH concentrations caused downregulation of its own, as well as other hormone receptors, and a decreased ovarian cortex cell proliferation. These results help understanding the inhibitory effects of AMH on follicular development. [ABSTRACT FROM AUTHOR]

We were the first to report that epithelial ovarian cancer (EOC) cells and tissues express myeloperoxidase (MPO) that is known to play a role in immune surveillance and inflammation by myeloid cells. Additionally, we reported that MPO is colocalized with inducible nitric oxide synthase (iNOS), a key pro-oxidant enzyme, and plays a key role in regulating apoptosis in EOC cells. Whereas myeloid cells express MPO in a dimeric form, intriguingly, here we report the unique expression of only the monomeric form of MPO in EOC cells, tissues, and blood of an ovarian cancer patient. Additionally, we have identified a cell membrane receptor, αV/β1 integrin, that is uniquely expressed by both chemosensitive and chemoresistant EOC cells with significantly higher expression in chemoresistant EOC cells. More importantly, we have demonstrated that monoclonal antibodies against αV/β1 integrin induced cytotoxicity in EOC cells, but not in normal cells, that is also synergistic with conventional chemotherapies. Cytotoxicity of αV/β1 antibodies is due to conformational changes in αV/β1 integrin which prevents monomeric MPO binding to αV/β1 integrin inhibiting the activation of MPO, leading to increased apoptosis. Since normal epithelial cells and macrophages lack monomeric MPO and αV/β1 integrin system, targeting this unique MPO-dependent survival mechanism will selectively eliminate EOC cells and will be the target for developing specific ovarian cancer therapies. [ABSTRACT FROM AUTHOR]