Suppression of the CD8 T cell response by human papillomavirus type 16 E7 occurs in Langerhans cell-depleted mice.

Human papillomavirus (HPV) is an epitheliotropic virus that is the primary causal agent for cervical cancer. Langerhans cells (LC) are skin antigen presenting cells that are reduced in number in HPV-infected skin. The aim of this study was to understand the immune-modulatory effects of HPV16 E7 on LC and on the CD8 T cell response to a skin-expressed antigen. To test this, HPV16 E7 was expressed in mouse skin keratinocytes with the model antigen ovalbumin (Ova). Similar to what is observed in HPV-infected human skin, LC numbers were significantly reduced in E7-expressing mouse skin. This shows that expression of the E7 protein alone is sufficient to mediate LC depletion. Expression of E7 with Ova in keratinocytes strongly suppressed the Ova-specific CD8+ T cell response in the skin draining lymph node. When tested in LC-ablated mice, the CD8 T cell response to skin-expressed Ova in control mice was not affected, nor was the T cell response to Ova restored in E7-expressing skin. These data indicate a role for E7 in regulation of LC homeostasis in the skin and in suppression of antigen specific CD8 T cell expansion, but suggest that these two effects occur independent of each other.

cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.

Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner.

Altered expression of microRNA-451 in eutopic endometrium of baboons (Papio anubis) with endometriosis.

STUDY QUESTION: Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? SUMMARY ANSWER: Induction of endometriosis causes significant changes in the expression of eight miRs, including miR-451, in the baboon endometrium as early as 3 months following induction of the disease. WHAT IS KNOWN ALREADY: Endometriosis is one of the most common gynecological disorders and causes chronic pelvic pain and infertility in women of reproductive age. Altered expression of miRs has been reported in women and has been suggested to play an important role in the pathophysiology of several gynecological disorders including endometriosis. STUDY DESIGN, SIZE, DURATION: EuE was obtained from the same group of baboons before and 3 months after the induction of endometriosis. The altered expression of miR-451 was validated in the eutopic and ectopic endometrium of additional baboons between 3 and 15 months following disease induction. Timed endometrial biopsies from women with and without endometriosis were also used to validate the expression of miR-451. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was extracted from EuE samples before and after the induction of endometriosis, and miRNA expression was analyzed using a 8 × 15 K miR microarray. Microarray signal data were preprocessed by AgiMiRna software, and an empirical Bayes model was used to estimate the changes. The present study focused on quantitative RT-PCR validation of the microarray data, specifically on miR-451 and its target genes in both baboons (n = 3) and women [control (n = 7) and endometriosis (n = 19)]. Descriptive and correlative analysis of miR-451 and target gene expression was conducted using in situ hybridization and immunohistochemistry, while functional analysis utilized an in vitro 3' untranslated region (UTR) luciferase assay and overexpression of miR-451 in human endometrial and endometriotic cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of endometriosis results in the altered expression of miR-451, -141, -29c, -21, -424, -19b, -200a and -181a in the baboon endometrium. In the baboon, induction of endometriosis significantly decreased the expression of miR-451 at 3 months (P < 0.001), which was also associated with increased expression of its target gene YWHAZ (14.3.3ζ). A similar significant (P < 0.0001) decrease in miR-451 expression was observed in women with endometriosis. The 3' UTR luciferase assay confirmed the regulation of YWHAZ expression by miR-451. Furthermore, overexpression of miR-451 in 12Z cells (immortalized human endometriotic epithelial cell line) led to the decreased expression of its target YWHAZ and this was correlated with decreased cell proliferation. LIMITATIONS, REASONS FOR CAUTION: The study focused only on miR-451 and one of its targets, namely YWHAZ. A single miR could target number of genes and a single gene could also be regulated by number of miRs; hence, it is possible that other miRs and their regulated genes may contribute to the pathophysiology of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.

Characterization of GAB1 expression over the menstrual cycle in women with and without polycystic ovarian syndrome provides a new insight into its pathophysiology.

CONTEXT: In a previous microarray analysis, GRB2-associated binding protein 1 (GAB1), a docking protein closely related to the insulin receptor substrate, was down-regulated in endometrium of women with polycystic ovary syndrome (PCOS). OBJECTIVE: The objective of the study was to characterize the cyclic expression of endometrial GAB1 in vivo in normal women and those with PCOS as well as investigate the possible mechanisms of endometrial regulation of GAB1 expression and action in vitro. DESIGN: This was an experimental and case-control study. SETTING: The study was conducted at a tertiary university hospital. PATIENTS: Normal proven fertile women (controls; n = 31) and women with PCOS (cases; n = 26) participated in the study. INTERVENTIONS: INTERVENTIONS included timed endometrial biopsies at different phases of the menstrual cycle. Ishikawa cells were cultured with ß-estradiol (E2), medroxyprogesterone acetate, and E2 + medroxyprogesterone acetate. Transfection of small interfering RNA for GAB1 in Ishikawa cells incubated with or without insulin. MAIN OUTCOME MEASURES: GAB1 mRNA expression in Ishikawa cells and in endometrium of cases and controls was measured. Protein expression of phosphorylated MAPK by Western blot was also measured. Immunohistochemical localization and expression of phosphorylated GAB1 in endometrium was also measured, using a digital histological score. RESULTS: In endometrial tissue, GAB1 mRNA was reduced in the proliferative phase of PCOS women, compared with controls (P = .003; ANOVA). When all the phases of the menstrual cycle were grouped, GAB1 protein expression was reduced in endometrium of PCOS women (P < .0001; Student t test). E2 increases GAB1 mRNA expression in Ishikawa cells (P = .001; ANOVA). Phosphorylated MAPK is reduced in cells transfected with small interfering RNA for GAB1 (P = .008; ANOVA) and incubated with insulin. CONCLUSIONS: GAB1 mRNA expression is positively modulated by E2. Endometrial GAB1 protein and mRNA expression are reduced in women with PCOS, suggesting that the endometrium of PCOS women have a defect in insulin signaling due to GAB1 down-regulation.

Expression of the transmembrane mucins, MUC1, MUC4 and MUC16, in normal endometrium and in endometriosis.

STUDY QUESTION: Are the transmembrane mucins, MUC1, MUC4 and MUC16, differentially expressed in endometriosis compared with normal endometrium? SUMMARY ANSWER: This study revealed that transmembrane mucin expression does not vary significantly in normal endometrium during the menstrual cycle and is not altered in endometriosis relative to the epithelial marker, cytokeratin-18 (KRT18). WHAT IS KNOWN ALREADY: Increased serum levels of the transmembrane mucin fragments MUC1, MUC4 and MUC16 that normally dominate the apical surface of simple epithelia are found in several pathological conditions, including endometriosis. Altered mucin expression in gynecologic diseases may promote infertility or endometrial pathologies. STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study of samples from 12 endometriosis patients as well as non-endometriosis control samples obtained from 31 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was isolated from endometrial biopsies of ectopic and eutopic endometrium from women with endometriosis and control patients from different stages of the menstrual cycle. Quantitative (q)-RT-PCR analyses were performed for the mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, cytokeratin-18 (KRT18), or ß-actin (ACTB). Frozen sections from endometrial biopsies of proliferative and mid-secretory stage women with endometriosis were immunostained for MUC1, MUC4 and MUC16. MAIN RESULTS AND THE ROLE OF CHANCE: qRT-PCR analyses of MUC1 and MUC16 mRNA revealed that these mucins do not vary significantly during the menstrual cycle nor are they altered in women with endometriosis relative to the epithelial marker, KRT18. MUC4 mRNA is expressed at very low levels relative to MUC1 and MUC16 under all conditions. There was little difference in MUC1 and MUC16 expression between eutopic endometrial and ectopic endometriotic tissues. MUC4 expression also was not significantly higher in the ectopic endometriotic tissues. Immunostaining for all three mucins reveals robust expression of MUC1 and MUC16 at the apical surfaces of endometrial epithelia, but little to no staining for MUC4. LIMITATIONS, REASONS FOR CAUTION: qRT-PCR analysis was the main method used for mucin detection. Additional studies with stage III-IV endometriotic tissue would be useful to determine if changes in MUC1 and MUC16 expression occur, or if MUC4 expression increases, at later stages of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: We report a comprehensive comparative profile of the major transmembrane mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, KRT18, in normal cycling endometrium and in endometriosis, and indicate constitutive expression. Previous studies have profiled the expression of individual mucins relative to ß-actin and indicate accumulation in the luteal phase. Thus, these differences in interpretation appear to reflect the increased epithelial content of endometrium during the luteal phase. STUDY FUNDING: This study was supported by: NIH R01HD29963 to D.D.C.; NIH U54HD007495 to S.M.H.; and NIH R01HD067721 to S.L.Y. and B.A.L. The authors have no competing interests to declare.

Resveratrol and endometrium: a closer look at an active ingredient of red wine using in vivo and in vitro models.

Resveratrol is a natural phytoestrogen with antiproliferative properties present in red wine, grapes, and berries. Published reports on the effects of resveratrol in human endometrial function are limited. The objective of this study was to investigate the expression of estrogen receptor α (ESR1), Ki-67 (a proliferative marker), aryl hydrocarbon receptor (AhR), and members of the cytochrome P450 superfamily of enzymes (CYP1A1 and CYP1B1) in an in vitro and vivo assay. Alkaline phosphatase assay of estrogenicity was used to compare estrogen activity of different concentrations of resveratrol to estradiol (E2) and diethylstilbestrol (DES), using Ishikawa cell culture. Immunohistochemical expression of ESR1 and Ki67, and reverse transcriptase polymerase chain reaction of AhR, CYP1A1, and CYP1B1 were analyzed from xenograft implants of human endometrial tissue in ovariectomized immunodeficient RAG-2-γ(c) mice, after 30 days of treatment with subcutaneous pellets of E2, E2 plus progesterone (P4), or E2 plus resveratrol (6, 30, or 60 mg) for 30 days. Compared to E2, resveratrol acted as an agonist and antagonist of estrogen in low and high concentrations, respectively, when combined with E2. Xenografts of human endometrial tissues in RAG-2 mice exhibited reduced expression of ESR1 and proliferative activity (Ki67) with 60 mg of resveratrol. This study suggests that resveratrol, at high doses, has the potential benefit to reduce proliferation of human endometrium through ESR1.

Enhancing the immunogenicity of tumour lysate-loaded dendritic cell vaccines by conjugation to virus-like particles.

BACKGROUND: Tumour cell lysates are an excellent source of many defined and undefined tumour antigens and have been used clinically in immunotherapeutic regimes but with limited success. METHODS: We conjugated Mel888 melanoma lysates to rabbit haemorrhagic disease virus virus-like particles (VLP), which can act as vehicles to deliver multiple tumour epitopes to dendritic cells (DC) to effectively activate antitumour responses. RESULTS: Virus-like particles did not stimulate the phenotypic maturation of DC although, the conjugation of lysates to VLP (VLP-lysate) did overcome lysate-induced suppression of DC activation. Lysate-conjugated VLP enhanced delivery of antigenic proteins to DC, while the co-delivery of VLP-lysates with OK432 resulted in cross-priming of naïve T cells, with expansion of a MART1(+) population of CD8(+) T cells and generation of a specific cytotoxic response against Mel888 tumour cell targets. The responses generated with VLP-lysate and OK432 were superior to those stimulated by unconjugated lysate with OK432. CONCLUSION: Collectively, these results show that the combination of VLP-lysate with OK432 delivered to DC overcomes the suppressive effects of lysates, and enables priming of naïve T cells with superior ability to specifically kill their target tumour cells.

Identification, characterization, and evolution of a primate beta-defensin gene cluster.

Defensins are members of a large diverse family of cationic antimicrobial peptides that share a signature pattern consisting of six conserved cysteine residues. Defensins have a wide variety of functions and their disruption has been implicated in various human diseases. Here we report the characterization of DEFB119-DEFB123, five genes in the human beta-defensin cluster locus on chromosome 20q11.1. The genomic structures of DEFB121 and DEFB122 were determined in silico. Sequences of the five macaque orthologs were obtained and expression patterns of the genes were analyzed in humans and macaque by semiquantitative reverse transcription polymerase chain reaction. Expression was restricted to the male reproductive tract. The genes in this cluster are differentially regulated by androgens. Evolutionary analyses suggest that this cluster originated by a series of duplication events and by positive selection. The evolutionary forces driving the proliferation and diversification of these defensins may be related to reproductive specialization and/or the host-parasite coevolutionary process.

Targeting early events in T cell activation to construct improved vaccines.

Live, attenuated vaccines currently offer the best protection against virulent pathogens. Recent advances in Immunology and Molecular Biology provide an opportunity to design vaccines that will be more effective and safer than existing ones. Immunologists are rapidly developing the capacity to identify and construct the minimal immunogenic units from pathogens. The molecular signals required to fully activate antigen presenting cells (APCs) and responder T cells are becoming apparent. Improved vaccine delivery systems are being designed which will mimic the actions of pathogens in vivo. These vaccines will incorporate protective epitopes fused to immunoregulatory cytokines in chimeric proteins. They will be encapsulated in formulations which allow for the slow release of these chimeric proteins thereby inducing the memory T cells required for long-lived immunity. These vaccine formulations will target receptors present on the most active APCs. Here we discuss how these advances will allow us to rationally construct "virtual pathogens" which will provide improved protection against new and old microbial foes.

Utilization of umbilical cords to assess in utero exposure to persistent pesticides and polychlorinated biphenyls.

In support of a study to relate developmental and cognitive effects with prenatal exposure to selected environmental toxicants, we developed and applied an analytical method to determine the concentration of two persistent pesticides, hexachlorobenzene (HCB) and p,p'-dichlorodiphenyldichloroethylene (DDE), and 32 specific polychlorinated biphenyl (PCB) congeners in 316 umbilical cords taken in 1986-1987 from women of the Faroe Islands. The analytical method consisted of homogenization of the cords, partitioning, microsilica gel column chromatography for clean-up, and dual-column capillary gas chromatography (DB-5 and DB-1701) with electron capture detection. Several quality control parameters were followed to monitor the performance of the method. Important criteria used before reporting unknown data were the recovery of in vitro-spiked analytes from a bovine umbilical cord (BUC) and the percentage lipid obtained for a Certified Reference Material (CRM)-350 of mackerel oil (MO). Recoveries of analytes that had been spiked at two concentration ranges (0.26-0.95 ng/g whole weight; 0.35-2.42 ng/g whole weight) into bovine cords ranged from 38.5% to 158% and from 50.4% to 145%, respectively, with a median recovery of 77.7%. Measurement of the percentage lipid for CRM-350 ranged from 73.8% to 107% with a median lipid value of 96.0%. The most prevalent analytes detected (%) in unknown umbilical cords were HCB (100), DDE (100), Ballschmiter/Zell PCBs 153 (100), 138 (98), 180 (98), 170 (93), 118 (88), 187 (86), and 146 (83), with corresponding median concentrations (ng/g whole weight) of 0.17, 1.19, 0.38, 0.30, 0.17, 0.11, 0.12, 0.09, and 0.07, respectively. Total PCB--sum of all measurable PCB congeners--had a median concentration of 1.37 ng/g whole weight. The analytes, which were very low in lipid content were also quantified on a lipid-adjusted basis, which provided an analytical challenge in these umbilical cord samples. The gravimetrically measured lipids in the human specimens ranged from 0.01% to 1.43% (median of 0.18%). In the pooled BUCs, our lipid measurements varied from 0.05% to 0.33% with a median value of 0.13%. The utility of using the umbilical cord as a matrix to assess in utero exposure to persistent environmental pollutants, compared with the use of umbilical cord blood or mother's blood, is worthy of debate.

Regulation of signal transducers and activators of transcription (STATs) by effectors of adipogenesis: coordinate regulation of STATs 1, 5A, and 5B with peroxisome proliferator-activated receptor-gamma and C/AAAT enhancer binding protein-alpha.

We have recently demonstrated that three signal transducers and activators of transcription (STAT) family members are induced during adipocyte differentiation (Stephens et al., J. Biol. Chem. 271 (1996) 10441-10444). Since STATs 1, 5A, and 5B are induced during adipocyte differentiation, we have examined the ability of these proteins to be regulated by components of the differentiation cocktail. In addition, we have examined the effects of potent effectors of differentiation on STAT protein expression during adipogenesis. A negative effector, tumor necrosis factor-alpha (TNFalpha), and a positive effector, a thiazolidinedione, were used in these experiments. Our results demonstrate that the expression of STATs 1, 5A, and 5B is not dramatically influenced by individual components of the differentiation cocktail. However, the expression of these three STAT family members tightly correlates with lipid accumulation. Moreover, the expression of STATs 1, 5A, and 5B, but not STATs 3 and 6, are regulated in an identical fashion to both C/AAAT enhancer binding proteins alpha and peroxisome proliferator-activated receptor-gamma by TNFalpha and a thiazolidinedione. Furthermore, the expression of adipocyte-expressed JAK kinases are unaffected by effectors of differentiation. These findings suggest that three STAT family members may play a role in the regulation of adipocyte gene expression.

Dietary NaC1 during pregnancy and lactation: effect on brain angiotensin II receptors and behavior.

Female rats were fed diets containing either a basal (0.12%), mid- (1%) or high (3%) level of NaCl during pregnancy and lactation. Plasma aldosterone was elevated approximately 5- and 15-fold in dams fed basal compared with either the mid- or high-NaCl diets at the end of both pregnancy and lactation (Postnatal Day 21), respectively. Dams fed basal diet and killed at the end of lactation had a higher density of angiotensin II receptors in the organum vasculosum laminae terminalis, paraventricular hypothalamus, and median preoptic nucleus than did rats fed either mid- or high-NaCl diets. Other dams, treated identically, were returned to rodent chow (approximately 0.2% NaCl) at the end of lactation for intake tests during the next week. Dams that had received basal diet did not differ from mid-NaCl and high-NaCl groups in sodium appetite induced by either acute sodium depletion or mineralocorticoid administration but showed the lowest spontaneous intake of NaCl solution.

Immediate-early gene expression in the amygdala following footshock stress and contextual fear conditioning.

This study investigated the increase in expression in the amygdala of 2 immediate-early genes, c-fos and NGFI-A, following contextual fear conditioning. The immediate-shock freezing deficit paradigm was used to compare rats that received footshock after exploring a context to rats that received footshock immediately after placement in the chamber. The former procedure produces contextual fear conditioning while the latter does not. Rats were either handled (handled group), placed in a test chamber without receiving footshock (context-no-footshock group), received footshock immediately upon being placed in the chamber (immediate-footshock group), or received footshock after a 1 min delay (delayed-footshock group). Only the delayed-footshock group displayed a fear response (freezing behavior). Rats were sacrificed either 15 min after the experience or after a retention test 24 h later. The c-fos mRNA was increased in the medial nucleus of the amygdala in all of the groups that were placed in the test chamber. However, rats that received footshock (immediate- and delayed-footshock groups) had greater levels of c-fos mRNA expression than rats of the context-no-footshock group. The c-fos mRNA expression in the immediate- and delayed-footshock groups did not differ. However, after the retention test, the expression of c-fos mRNA in the medial nucleus of the amygdala did not differ between groups. In contrast to c-fos, NGFI-A mRNA expression in the lateral nucleus of the amygdala was greater in the delayed-footshock group than the handled and context-no-footshock groups 15 min after the footshock. This elevation in NGFI-A mRNA was not seen in the immediate-footshock group. This suggests that NGFI-A mRNA in the lateral nucleus of the amygdala may play a role in contextual fear conditioning.

Induction of tenascin in rat lungs undergoing bleomycin-induced pulmonary fibrosis.

Lung injury induced by bleomycin is associated with early inflammation and subsequent excessive deposition of extracellular matrix. In the present study, we investigated the expression of extracellular matrix glycoprotein tenascin (TN) during pulmonary injury induced by bleomycin. After the initial lung injury induced by intratracheal bleomycin instillation, TN and collagen type III (COL III) mRNAs were greatly induced. The pattern of induction of TN was distinct from that of COL III. TN was primarily induced during the early inflammatory phase, whereas the increase in COL III synthesis continued during the reparative phase. The induction and localization of TN mRNA during bleomycin-induced pulmonary injury were also examined by in situ hybridization. TN mRNA was focally induced in rat lungs 3 days after bleomycin administration. Induction of TN mRNA was spatially restricted in the areas of tissue inflammation. The interstitial cells in alveolar septal walls and secondary septal tips in the areas of tissue damage were the major source of TN mRNA production. Expression of TN mRNA was decreased as the inflammation attenuated and development of fibrosis proceeded. Immunocytochemical analyses of TN protein distribution in the lung yielded corroborative results. Immunoreactive TN protein was found in a patchy distribution in alveolar septal walls and secondary septal tips in the areas of damaged tissues. This study demonstrated that TN is a unique early-response extracellular matrix component to bleomycin-induced pulmonary injury and is induced at the sites of the inflammation, suggesting a potential role of TN as a modulator of pulmonary inflammation and repair.

Changes in the lung after prolonged positive pressure ventilation in normal baboons.

PURPOSE: The effects of prolonged positive pressure ventilation on lung ultrastructure are not well defined in primates. This study was designed to measure cardiopulmonary and morphological responses to 4 days of positive pressure ventilation in normal baboons. MATERIALS AND METHODS: Six adult male baboons were mechanically ventilated on air for 96 hours with 2.5 cm positive end-expiratory ventilation and a tidal volume of 12 to 15 mL/kg. Physiological measurements were obtained every 12 hours and serial measurements of ventilation-perfusion (VA/Q) were performed using the multiple inert gas elimination technique. Quantitative morphotometry, lung dry-to-wet ratio, and surfactant analysis were performed at the end of the experiment. RESULTS: Cardiovascular variables, except for a small increase in mean pulmonary artery pressure at 84 and 96 hours, were not significantly affected by positive pressure ventilation. Arterial Po2 decreased, and shunt fraction increased from 0.7% of cardiac output to 5.4% (P < .01). Dispersion of perfusion increased threefold (P < .01), and dispersion of ventilation doubled (P < .01) indicating increased VA/Q mismatch mismatch. Respiratory system compliance decreased by 30% (P < .01). There was no lung edema or change in surfactant composition. Lung morphometry showed increases in polymorphonuclear cells and type II cell volume. Vacuolated endothelial cells and bare basement membrane were observed consistently. CONCLUSION: Four days of positive pressure ventilation decreases lung compliance and worsens gas exchange by increasing shunt and VA/Q mismatch in healthy baboons. These effects are accompanied by only minor ultrastructural changes and mild inflammatory responses in the lung.

Integrins and other cell adhesion molecules in endometrium and endometriosis.

Endometriosis is a disease that affects about 5% of women of reproductive age, but is found much more frequently in those with pelvic pain and/or infertility. In affected individuals, shed endometrium is thought to attach and spread within the pelvis through specific cell adhesion receptors. To understand why some women develop endometriosis while others do not, researchers have begun to examine these receptors and their extracellular matrix ligands. Cell adhesion molecules fall into 4 major groups including cadherins, selectins, members of the immunoglobulin superfamily, and integrins. Based on our current understanding, each may potentially play a role in the development or progression of this disease. In addition, the use of integrins as markers of the normal endometrial phenotype may be useful for the diagnosis of endometriosis and may identify women with defects in endometrial function leading to infertility or recurrent pregnancy loss. As more information becomes available, it may be possible to develop better treatments for endometriosis based on these concepts and to identify those women at risk for development of this common yet serious disorder.

Composition of lung lavage in pulmonary alveolar microlithiasis.

A case of pulmonary alveolar microlithiasis (PAM) is reported wherein total lung lavage was performed for relief of dyspnea. Characterization of the lavage material and examination of the microliths isolated from the lavage fluid confirmed previous reports of their spherical-ovoid shape and a 2:1 calcium to phosphate composition. The microliths contained considerable amounts of ionizable iron and generated oxidants in an in vitro system. A detailed biochemical analysis of the lavage fluid reflected elevations in total protein, phosphatidylserine, phosphatidylglycerol and the ratio of phosphatidylglycerol to phosphatidylinositol. Surfactant apoprotein-A levels approximated that of normal patients. The potential roles of oxidant generation and alterations in surfactant metabolism are discussed in the context of the pathogenesis of PAM.

An age-related difference in hyperoxia lethality: role of lung antioxidant defense mechanisms.

The role of animal age in the lethal response to > 98% oxygen has been extensively studied, with the observation that neonatal rats were resistant while mature animals were sensitive. Antioxidant enzymes increased during the oxygen exposure in neonatal but not in mature rats, suggesting they were important in the age-related toxicity difference. Because no studies had compared the response of mature and old rats to hyperoxia, we exposed Fischer 344 rats, aged 2 and 27 mo, to > 98% oxygen. Unexpectedly, the old rats lived significantly longer than young, 114 and 65 h, respectively. No histopathological differences were found to explain the results. Of the antioxidants, only glutathione peroxidase (GPx) activity was higher in the lungs of nonexposed old rats. Superoxide dismutase (SOD) was higher in the young, results opposite those expected if SOD was important in the lethality difference. No antioxidant induction occurred in the old oxygen-exposed rats. These results suggest that although there may be a role for GPx, mechanisms in addition to antioxidant protection and inflammation are likely responsible for the age-related difference in hyperoxia lethality.

TGF-beta regulates expression of tenascin alternative-splicing isoforms in fetal rat lung.

Two distinct mRNA-splice isoforms of tenascin (TN) are expressed differentially during rat lung development. The unique temporal expression pattern of two TN isoforms suggests the expression of tenascin is strictly developmentally regulated in rat lung tissue. We investigated molecular mechanisms which modulate alternative-splicing expression of TN in lung development. The effect of transforming growth factor-beta 1 (TGF-beta 1) on regulation of expression of TN isoforms was examined by in vitro lung explant culture. Immunoblotting with anti-TN antibody detected two TN polypeptides in rat lung explant culture, the larger [relative molecular weight (M(r)) 230, TN230] polypeptide and the smaller (M(r) 180, TN180). TGF-beta 1 markedly induced the TN180 isoform and caused only a moderate increase of the TN230 isoform. The effects of TGF-beta 1 were shown to be dose dependent over a physiological range of TGF-beta 1 protein concentration. The induction of TN isoform biosynthesis by TGF-beta 1 was detected 12 h after addition of the growth factor, and the effects endured for up to 48 h at a dose of 5 ng/ml. By reverse transcriptase-polymerase chain reaction through amplification of the entire fibronectin type III (FN-III) splicing domain, two distinct TN isoforms were detected in total RNA isolated from gestational day 21 rat lung explant culture treated with TGF-beta 1 and from postnatal day 8 rat lung. The larger isoform contained five FN-III alternative splicing repeats [1,420 base pairs (bp)], but the shorter splicing isoform lacked four FN-III alternative splicing repeats (340 bp).(ABSTRACT TRUNCATED AT 250 WORDS)

NMDA processes mediate anterograde amnesia of contextual fear conditioning induced by hippocampal damage: immunization against amnesia by context preexposure.

Hippocampal lesions in rats produce both a retrograde and an anterograde amnesia of contextual fear conditioning. The present experiments examined the anterograde deficit in context conditioning. The deficit produced by electrolytic hippocampal lesions was apparent when training occurred on 7, 14, or 28 days following surgery, confirming the durability of the amnesia. The role of the hippocampus in context conditioning may be related to an NMDA (N-methyl-D-aspartate) receptor-mediated process. Both NMDA hippocampal lesions and intrahippocampal administration of an NMDA antagonist produced anterograde amnesia. Animals preexposed to the conditioning context 28 days prior to hippocampal lesioning were protected from the deficit normally produced by the lesions. Thus, the hippocampus must form a contextual representation during preexposure that is subsequently stored elsewhere. Once formed this representation of the context can be associated with an unconditional stimulus.