Cigarette smoke induces oxidative stress and apoptosis in normal term fetal membranes.

Cigarette smoking and bacterial infections are two major risk factors associated with preterm prelabor rupture of membranes (pPROM). We hypothesized that exposure of fetal membranes to cigarette smoke extracts might induce oxidative stress (OS) and fetal membrane apoptosis, culminating in an alternate pathway to that commonly activated by infection. To test this, we characterized the production of prostanoids and biomarkers of apoptosis in normal term human fetal membrane explant cultures. Fetal membrane explants collected at term (from cesarean deliveries, not in labor) were stimulated with cigarette smoke extract (CSE) for 24 h. Two classes of prostanoids, F2-Isoprostane (F2-IsoP), a marker of OS and PGF2α, a classical uterotonin, were measured by gas chromatography/mass spectrometry. Western blot analyses of tissue lysates were performed to quantify the anti-apoptotic protein Bcl2 and actin (as a control). Fetal membrane apoptosis was detected by immunohistochemistry for active caspase 3 and confirmed by TUNEL staining for nuclear fragmentation. CSE exposure resulted in significantly more F2-IsoP production from fetal membranes (242.8 ± 79.3 pg/ml/mg of total membrane protein) compared to unstimulated controls (131.5 ± 53.1 pg/ml/mg; p < 0.0001). By contrast, PGF2α was not different in CSE vs. controls (1083 ± 527 vs. 1136 ± 835 pg/ml/mg of protein; p = 0.80). CSE-exposed tissues demonstrated a dose-dependent decrease in Bcl2 expression and increases in active caspase 3 and nuclear fragmentation in both amnion and chorion cells compared to controls. In summary, fetal membranes exposed to CSE manifest evidence of OS and apoptosis. The differential pattern of prostanoid production observed in this study supports the hypothesis that an alternate non-inflammatory pathway mediated by OS and apoptosis in pPROM may promote proteolysis resulting in membrane weakening and rupture.

The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.

Although skeletal pain is a leading cause of chronic pain and disability, relatively little is known about the specific populations of nerve fibers that innervate the skeleton. Recent studies have reported that therapies blocking nerve growth factor (NGF) or its cognate receptor, tropomyosin receptor kinase A (TrkA) are efficacious in attenuating skeletal pain. A potential factor to consider when assessing the analgesic efficacy of targeting NGF-TrkA signaling in a pain state is the fraction of NGF-responsive TrkA+ nociceptors that innervate the tissue from which the pain is arising, as this innervation and the analgesic efficacy of targeting NGF-TrkA signaling may vary considerably from tissue to tissue. To explore this in the skeleton, tissue slices and whole mount preparations of the normal, adult mouse femur were analyzed using immunohistochemistry and confocal microscopy. Analysis of these preparations revealed that 80% of the unmyelinated/thinly myelinated sensory nerve fibers that express calcitonin gene-related peptide (CGRP) and innervate the periosteum, mineralized bone and bone marrow also express TrkA. Similarly, the majority of myelinated sensory nerve fibers that express neurofilament 200 kDa (NF200) which innervate the periosteum, mineralized bone and bone marrow also co-express TrkA. In the normal femur, the relative density of CGRP+, NF200+ and TrkA+ sensory nerve fibers per unit volume is: periosteum>bone marrow>mineralized bone>cartilage with the respective relative densities being 100:2:0.1:0. The observation that the majority of sensory nerve fibers innervating the skeleton express TrkA+, may in part explain why therapies that block NGF/TrkA pathway are highly efficacious in attenuating skeletal pain.

Blockade of nerve sprouting and neuroma formation markedly attenuates the development of late stage cancer pain.

For many patients, pain is the first sign of cancer and, while pain can be present at any time, the frequency and intensity of pain tend to increase with advancing stages of the disease. Thus, between 75 and 90% of patients with metastatic or advanced-stage cancer will experience significant cancer-induced pain. One major unanswered question is why cancer pain increases and frequently becomes more difficult to fully control with disease progression. To gain insight into this question we used a mouse model of bone cancer pain to demonstrate that as tumor growth progresses within bone, tropomyosin receptor kinase A (TrkA)-expressing sensory and sympathetic nerve fibers undergo profuse sprouting and form neuroma-like structures. To address what is driving the pathological nerve reorganization we administered an antibody to nerve growth factor (anti-NGF). Early sustained administration of anti-NGF, whose cognate receptor is TrkA, blocks the pathological sprouting of sensory and sympathetic nerve fibers, the formation of neuroma-like structures, and inhibits the development of cancer pain. These results suggest that cancer cells and their associated stromal cells release nerve growth factor (NGF), which induces a pathological remodeling of sensory and sympathetic nerve fibers. This pathological remodeling of the peripheral nervous system then participates in driving cancer pain. Similar to therapies that target the cancer itself, the data presented here suggest that, the earlier therapies blocking this pathological nerve remodeling are initiated, the more effective the control of cancer pain.

Chorioamnionitis--a complex pathophysiologic syndrome.

Chorioamnionitis, inflammation of the amniochorionic membrane (fetal membranes) is a very common disease but a complex syndrome associated with pregnancy. It presents a clinical impasse due to lack of knowledge of specific etiologies associated with this condition making confident clinical interventions difficult. Recent reports provide insight into genetic, epigenetic, behavioral, psychosocial, molecular and pathophysiological factors that are associated with chorioamnionitis. However, a coordinated approach in understanding causality and lack of early indicators (clinical and biomarkers) has hampered gaining knowledge about the disease status preventing proper intervention. Several reviews have provided in-depth analysis of the histologic and clinical evidence associated with chorioamnionitis. In this review, we provide a novel perspective on chorioamnionitis based on recent evidences from scientific literature on inflammation, apoptosis and genetics.

Identification, characterization, and regulation of the canonical Wnt signaling pathway in human endometrium.

Members of the Wnt family of signaling molecules are important in cell specification and epithelial-mesenchymal interactions, and targeted gene deletion of Wnt-7a in mice results in complete absence of uterine glands and infertility. To assess potential roles of the Wnt family in human endometrium, an endocrine-responsive tissue, we investigated in the proliferative and secretory phases of the menstrual cycle, endometrial expression of several Wnt ligands (Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, and Wnt-8b), receptors [Frizzled (Fz)-6 and low-density lipoprotein receptor-related protein (LRP)-6], inhibitors [FrpHE and Dickkopf (Dkk)-1], and downstream effectors (Dishevelled-1, glycogen synthase kinase-3beta, and beta-catenin) by RT-PCR, real-time PCR and in situ hybridization. No significant menstrual cycle dependence of the Wnt ligands (except Wnt-3), receptors, or downstream effectors, was observed. Wnt-3 increased 4.7-fold in proliferative compared with secretory endometrium (P < 0.05). However, both inhibitors showed dramatic changes during the cycle, with 22.2-fold down-regulation (P < 0.05) of FrpHE and 234.3-fold up-regulation (P < 0.001) of Dkk-1 in the secretory, compared with the proliferative phase. In situ hybridization revealed cell-specific expression of different Wnt family genes in human endometrium. Wnt-7a was exclusively expressed in the luminal epithelium, and Fz-6 and beta-catenin were expressed in both epithelium and stroma, without any apparent change during the cycle. Both FrpHE and Dkk-1 expression were restricted to the stroma, during the proliferative and secretory phase, respectively. These unique expression patterns of Wnt family genes in different cell types of endometrium and the differential regulation of the inhibitors during the proliferative and secretory phase of the menstrual cycle strongly suggest functions for a Wnt signaling dialog between epithelial and stromal components in human endometrium. Also, they underscore the likely importance of this family during endometrial development, differentiation and implantation.

Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility.

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants.

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.

Global gene profiling in human endometrium during the window of implantation.

Implantation in humans is a complex process that is temporally and spatially restricted. Over the past decade, using a one-by-one approach, several genes and gene products that may participate in this process have been identified in secretory phase endometrium. Herein, we have investigated global gene expression during the window of implantation (peak E2 and progesterone levels) in well characterized human endometrial biopsies timed to the LH surge, compared with the late proliferative phase (peak E2 level) of the menstrual cycle. Tissues were processed for poly(A(+)) RNA and hybridization of chemically fragmented, biotinylated cRNAs on high density oligonucleotide microarrays, screening for 12,686 genes and expressed sequence tags. After data normalization, mean values were obtained for gene readouts and fold ratios were derived comparing genes up- and down-regulated in the window of implantation vs. the late proliferative phase. Nonparametric testing revealed 156 significantly (P < 0.05) up-regulated genes and 377 significantly down-regulated genes in the implantation window. Up-regulated genes included those for cholesterol trafficking and transport [apolipoprotein (Apo)E being the most induced gene, 100-fold], prostaglandin (PG) biosynthesis (PLA2) and action (PGE2 receptor), proteoglycan synthesis (glucuronyltransferase), secretory proteins [glycodelin, mammaglobin, Dickkopf-1 (Dkk-1, a Wnt inhibitor)], IGF binding protein (IGFBP), and TGF-beta superfamilies, signal transduction, extracellular matrix components (osteopontin, laminin), neurotransmitter synthesis (monoamine oxidase) and receptors (gamma aminobutyric acid A receptor pi subunit), numerous immune modulators, detoxification genes (metallothioneins), and genes involved in water and ion transport [e.g. Clostridia Perfringens Enterotoxin (CPE) 1 receptor (CPE1-R) and K(+) ion channel], among others. Down-regulated genes included intestinal trefoil factor (ITF) [the most repressed gene (50-fold)], matrilysin, members of the G protein-coupled receptor signaling pathway, frizzled-related protein (FrpHE, a Wnt antagonist), transcription factors, TGF-beta signaling pathway members, immune modulators (major histocompatibility complex class II subunits), and other cellular functions. Validation of select genes was conducted by Northern analysis and RT-PCR using RNA from endometrial biopsies obtained in the proliferative phase and the implantation window and by RT-PCR using RNA from cultured endometrial epithelial and stromal cells. These approaches confirmed up-regulation of genes corresponding to IGFBP-1, glycodelin, CPE1-R, Dkk-1, mammaglobin, and ApoD and down-regulation for PR membrane component 1, FrpHE, matrilysin, and ITF, as with the microarray data. Cultured endometrial epithelial cells were found to express mRNAs for glycodelin, CPE-1R, Dkk-1, the gamma aminobutyric acid A receptor pi subunit, mammaglobin, matrilysin, ITF and PR membrane component 1. The expression of IGFBP-1, CPE1-R, Dkk-1, and ApoD mRNAs increased upon decidualization of stromal cells in vitro with progesterone after E2 priming, whereas FrpHE decreased, consistent with the microarray results. Overall, the data demonstrate numerous genes and gene families not heretofore recognized in human endometrium or associated with the implantation process. Reassuringly, several gene products, known to be differentially expressed in the implantation window or in secretory endometrium, were verified, and the striking regulation of select secretory proteins, water and ion channels, signaling molecules, and immune modulators underscores the important roles of these systems in endometrial development and endometrial-embryonic interactions. In addition, the current study validates using high density oligonucleotide microarray technology to investigate global changes in gene expression in human endometrium.

Endocrine and paracrine regulation of endometrial angiogenesis.

The human endometrium is a complex tissue comprised of different cell types, including epithelial, stromal, inflammatory, perivascular, and blood vessel cells. The hormonal receptivity and distribution of these cell populations change during the menstrual cycle. Cyclical endometrial growth is dependent on its ability to regenerate a vascular capillary network, which grows in parallel with the proliferation and differentiation of the endometrial lining. Natural hormonal effects on the endometrium and endocrine manipulation of this tissue, in response to the use of exogenous steroid therapies, can affect endometrial capillary proliferation and function, leading to clinical abnormalities of uterine bleeding. We propose that the regulation of endometrial angiogenesis is mediated indirectly via complex interactions among cell types. Our laboratory has focused on a prototypical member of the angiogenic proteins, vascular endothelial growth factor (VEGF)-A. In this paper we present data demonstrating that VEGF-A expression in normal endometrial epithelial and stromal cells and in Ishikawa adenocarcinoma cells is increased by an ovarian steroid, estradiol. Infiltrating immune cells, particularly polymorphonuclear granulocytes, also are sources of VEGF-A. In inflammatory conditions involving the endometrium (e.g., endometriosis), a proinflammatory cytokine, IL-1beta, can mediate neoangiogenesis by inducing VEGF-A gene transcription. Thus, endometrial vascularization is effected by both endocrine and paracrine pathways.

IL-1beta induction of RANTES (regulated upon activation, normal T cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-kappaB site in the proximal promoter.

A complex network of cytokines mediates immunoregulatory responses in the pathogenesis of endometriosis. RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemoattractant for monocytes and T cells. Endometriotic lesions express RANTES, and its concentration in peritoneal fluid correlates with the severity of endometriosis. We investigated the influence of IL-1beta, a potent macrophage cytokine, on RANTES production in endometriotic stromal cells and determined the region of the RANTES promoter responsible for IL-1beta action. RANTES mRNA was induced 5-fold in endometriotic stromal cells, and the conditioned medium RANTES protein concentrations were 12-fold higher in IL-1beta-treated endometriotic stromal cells vs. untreated controls (P < 0.05). IL-1beta activated the full-length (-940 bp) RANTES promoter as well as a truncated 456-bp 5'-flanking construct by 2-fold. Mutagenesis of a nuclear factor-kappaB response element at -30 bp abolished the IL-1beta effect, whereas mutation of a nearby TNF response element did not affect the IL-1beta induction. An IL-1beta time-course Western assay revealed a rapid diminution of IkappaB (endogenous inhibitor of nuclear factor-kappaB) in endometriotic stromal cells. Overexpression of IkappaB in endometriotic stromal cells inhibited the IL-1beta response of the RANTES gene promoter. Transcription of RANTES mRNA is up-regulated by IL-1beta via a nuclear factor-kappaB response element in the proximal RANTES gene promoter. These results demonstrate a feed-forward regulatory loop in the pathogenesis of endometriosis by which IL-1beta produced from activated macrophages can lead to further macrophage recruitment via RANTES production in endometriotic stromal cells.

Nuclear peroxisome proliferator-activated receptors alpha and gamma have opposing effects on monocyte chemotaxis in endometriosis.

The peroxisome proliferator-activated receptors (PPARs) alpha and gamma are nuclear receptors that play important roles in inflammatory diseases like ulcerative colitis and arthritis. In this study, we examined the possible role of PPARs in macrophage attraction into the peritoneal cavity of patients with endometriosis. We identified PPAR-alpha and -gamma messenger RNA by RT-PCR and protein by immunoblotting of lysates of peritoneal macrophages and monocytic U937 cells. Using immunocytochemistry, we localized PPAR-alpha and -gamma within the nuclei of both cell types. Monocyte chemotactic activity of peritoneal fluid from patients with endometriosis was quantified in Boyden chambers. Migration of U937 cells was increased by WY 14643 and reduced by rosiglitazone. Peritoneal fluid from patients with endometriosis activated U937 cells transiently transfected with a PPAR-alpha/GAL4 luciferase reporter. By contrast, peritoneal fluid did not cause significant activation of PPAR-gamma/GAL4 constructs. The U937 cells transiently transfected with a PPAR response element luciferase reporter showed disease stage-dependent up-regulation when treated with peritoneal fluid from patients with endometriosis. Treatment with peritoneal fluid from healthy controls down-regulated PPAR response element transactivation. We conclude that peritoneal fluid of endometriosis patients contains activators of PPAR-alpha that stimulate macrophage chemotaxis. Inhibitors of PPAR-alpha or activators of PPAR-gamma could be developed for the treatment of inflammation associated with endometriosis.

Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: assessment of autocrine/paracrine regulation by in situ hybridization.

Chemoattraction of macrophages and T cells into the normal endometrium and inflammatory sites within endometriotic foci is mediated by chemokine gene expression. mRNA transcripts encoding regulated on activation, normal T-cell-expressed and -secreted (RANTES), a monocyte and T-cell chemokine, were demonstrated in the stroma of normal endometrium and endometriotic implants using in situ mRNA hybridization. Epithelial glands failed to express RANTES mRNA. In histological serial sections, we observed CD68-positive macrophages in the stroma of endometriotic implants adjacent to regions with prominent RANTES mRNA hybridization. In adjacent sections, monoclonal antibodies against tumor necrosis factor (TNF)-alpha showed this cytokine to be localized to stromal and epithelial compartments of the endometriotic implant with weak staining in unaffected ovarian tissue. Subconfluent monolayers of endometriotic stromal cells were tested for RANTES gene expression in situ, but we could only detect RANTES mRNA in isolated stromal cells after treatment with TNF-alpha. No RANTES mRNA was observed in unstimulated stromal cells or TNF-alpha stimulated or unstimulated epithelial cells. The data are consistent with a model in which proinflammatory cytokines (eg, TNF-alpha) induce RANTES gene expression limited to specific cells within endometrial and endometriotic stroma. Production of this chemokine, in turn, stimulates recruitment of CD68-positive macrophages into these tissues.

Elevated levels of S-nitrosoalbumin in preeclampsia plasma.

The availability of nitric oxide (NO), which is required for the normal regulation of vascular tone, may be decreased in preeclampsia, thus contributing to the vascular pathogenesis of this pregnancy disorder. Because ascorbate is essential for the decomposition of S-nitrothiols and the release of NO, we speculated that the ascorbate deficiency typical of preeclampsia plasma might result in decreased rates of decomposition of S-nitrosothiols. We tested the hypothesis that total S-nitrosothiol and S-nitrosoalbumin concentrations are increased in preeclampsia plasma, reflecting a decreased release of NO from these major reservoirs of NO. Gestationally matched plasma samples were obtained (before labor or intravenous MgSO(4)) from 21 women with preeclampsia and 21 women with normal pregnancy, and plasma samples were also obtained from 12 nonpregnant women of similar age and body mass index during the follicular phase of the menstrual cycle. All were nonsmokers. The assay included ultraviolet-induced decomposition of S-nitrosothiols to liberate NO captured by a florigenic reagent, 4,5-diaminofluoresceine, to produce diaminofluoresceine-Triazole. Preeclampsia plasma contained significantly higher concentrations of total S-nitrosothiols (11.1+/-2.9 nmol/mL) than normal pregnancy samples (9.4+/-1.5 nmol/mL). Even greater differences were found between preeclampsia plasma and plasma samples from normal pregnancies and nonpregnant women (294+/-110, 186+/-25, and 151+/-25 pmol/mg protein, respectively) when S-nitrosothiol content was expressed per milligram protein. The albumin fraction contained 49.4% of total plasma S-nitrosothiols in the control samples and 53.7% and 56.8% of plasma S-nitrosothiols in normal pregnancy and preeclampsia, respectively. The level of S-nitrosoalbumin was significantly higher in preeclampsia than in normal pregnancy or nonpregnancy plasma (6.3+/-1.4, 5.1+/-0.7, and 4.2+/-1.0 nmol/mL, respectively). The increased concentration of S-nitrosoalbumin in preeclampsia almost completely accounted for the increased levels of S-nitrosothiols in total plasma. Due to combined increases in nitrosothiols and decreases in protein, the preeclampsia plasma concentration of S-nitrosoalbumin was greatly increased on a per milligram of protein basis (271% and 186% compared with normal nonpregnancy and normal pregnancy plasma, respectively). We conclude that S-nitrosoalbumin and total S-nitrosothiol concentrations are significantly increased in preeclampsia plasma and may reflect insufficient release of NO groups in this condition.

Histocompatibility leukocyte antigen-G is not expressed by endometriosis or endometrial tissue.

OBJECTIVE: The immunological mechanisms that support persistence and proliferation of ectopic endometrial implants within the peritoneal cavity of women with endometriosis are unknown. Inhibition of natural killer (NK) and cytotoxic T-cell function has been proposed as a mechanism. We tested the hypothesis that expression of a nonclassical major histocompatibility antigen, HLA-G, might explain the local immunosuppression associated with ectopic endometrium. DESIGN: Nested case-control study of women with and without laparoscopic evidence of endometriosis. SETTING: Reproductive endocrinology clinic at a university hospital. PATIENT(S): Peritoneal fluid specimens from 10 women with revised AFS stage I-IV endometriosis and from 10 age-matched normal controls without laparoscopic evidence of endometriosis were tested for the presence of HLA-G protein. Endometriosis and normal endometrial biopsies from four patients were used to prepare stromal cell cultures directly evaluated for HLA-G protein. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression of HLA-G in peritoneal fluid, tissue, and cell cultures was determined by immunoblotting with a specific monoclonal antibody. RESULT(S): HLA-G protein was not detectable in peritoneal fluid specimens of endometriosis patients or controls. Moreover, ectopic and normal endometrial tissues and stromal cells did not express HLA-G. CONCLUSION(S): Immune cell inhibition in endometriosis must be mediated by factors other than HLA-G.

Purification and characterization of an immunomodulatory endometrial protein, glycodelin.

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.

Acute myocardial infarction associated with amphetamine use.

Myocardial infarction is a rarely reported complication of amphetamine use. We report the case of a healthy 31-year-old man who presented to our emergency department with no clinical evidence of an acute coronary event after intravenous injection of amphetamines. However, he subsequently experienced a non-Q-wave anterior wall myocardial infarction associated with the use of amphetamines.

Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid.

Endometriotic lesions secrete chemokines that recruit immune cells into the peritoneal cavity. The accumulation of these immune cells, especially activated macrophages and T lymphocytes, is thought to mediate inflammatory symptoms associated with endometriosis. Previous studies have demonstrated that RANTES (regulated on activation, normal T cell expressed and secreted) is synthesized by endometriotic stromal cells and circulates in peritoneal fluid, commensurate with the stage of endometriosis. In the current studies, we used the human monocytic cell line, U937, to assay chemotactic activity in cell culture conditioned media and peritoneal fluid from patients with endometriosis and normal controls. We demonstrated expression of the human RANTES receptors CCR-1 and CCR-5 in U937 cells and peritoneal macrophages. Over a range of 0-1000 pg/ml recombinant human RANTES had a direct, linear effect on monocyte migration. Conditioned media and peritoneal fluid induced dose-dependent effects on monocyte migration that were correlated with concentrations of immunoreactive RANTES (as measured by enzyme-linked immunosorbent assay) and the severity of endometriosis. Heat denaturation of the RANTES protein or addition of anti-human RANTES antibodies neutralized the chemoattractant effects of conditioned media and peritoneal fluid. RANTES stimulation of monocyte recruitment may be an important pathogenetic target for the treatment of infertility and pain associated with endometriosis.