VIP conditions human endometrial receptivity by privileging endoplasmic reticulum stress through ATF6α pathway.

Endometrial stromal cells undergo endoplasmic reticulum (ER) stress and unfolded protein response (UPR) during the decidualization linked with the inflammation and angiogenesis processes. Considering VIP (vasoactive intestinal peptide) induces the decidualization program, we studied whether modulates the ER/UPR pathways to condition both processes for embryo implantation. When Human Endometrial Stromal Cell line (HESC) were decidualized by VIP we observed an increased expression of ATF6α, an ER stress-sensor, and UPR markers, associated with an increase in IL-1ß production. Moreover, AEBSF (ATF6α -inhibitor pathway) prevented this effect and decreased the expansion index in the in vitro model of implantation. VIP-decidualized cells also favor angiogenesis accompanied by a strong downregulation in thrombospondin-1. Finally, ATF6α, VIP and VPAC2-receptor expression were reduced in endometrial biopsies from women with recurrent implantation failures in comparison with fertile. In conclusion, VIP privileged ATF6α-pathway associated with a sterile inflammatory response and angiogenesis that might condition endometrial receptivity.

Hospital management of colonic perforations complicating ambulatory outpatient colonoscopy via over-the-scope clips or surgery: a case series.

BACKGROUND: Colonoscopy is the standard of care for the diagnosis and treatment of many colonic disorders. Over the past few years, endoscopic closure of colonoscopy-related perforation has become more common. Endoscopic closure of perforation secondary to colonoscopy has been undertaken in patients in the hospital setting and often during the same colonoscopic procedure in which the perforation itself occurred. The aim of our study was to analyze our experience with emergency endoscopic closure of colonoscopy-related perforation with over-the-scope clip (OTSC) technique. METHODS: We report five cases of colonic perforation that occurred during colonoscopy in an outpatient facility remotely located from our hospital and then referred as an emergency to our institution for endoscopic closure. RESULTS: Bowel preparation was reported to be adequate in all cases. Prior to attempting endoscopic closure of colonic perforation, all patients were in stable clinical condition, early broad-spectrum antibiotic coverage was initiated, and a surgical consult was obtained. All patients had sigmoidoscopy and were found to have sigmoid colon perforations. In three cases, the perforations were closed successfully using an OTSC clip device 14 mm type t. Two patients were found to have greater than 4-cm sigmoid perforations with irregular margins, incompatible with OTSC closure, and were referred for emergency surgery. All patients had an uneventful course following either OTSC closure or surgery. CONCLUSIONS: Based on the characteristics of the five cases and a review of the literature, we suggest a practical approach for undertaking closure of colonic perforations occurring during colonoscopy in the outpatient setting, focusing on clinical criteria to determine eligibility of patients for attempted endoscopic closure and outlining required therapeutic and monitoring steps needed to optimize outcomes.

Adipokine levels in overweight women with early-onset gestational diabetes mellitus.

PURPOSE: The study of adipokines in overweight women with early-onset (diagnosed before 20 weeks) gestational diabetes mellitus (GDM) could help to understand the ethiopathological mechanisms of this disorder. Our aim was to assess adipokine levels in overweight pregnant women with early-onset GDM compared to patients with standard-onset (diagnosed at 24-28 weeks) GDM and to glucose-tolerant women at the same gestational ages. METHODS: This nested case-control study included 133 overweight pregnant women: 33 with early-onset (diagnosed < 20 weeks) GDM; 40 with standard-onset (diagnosed ≥ 24 weeks) GDM and 60 glucose-tolerant (normal oral glucose tolerance tests < 20 and ≥ 24 weeks). Adiponectin, leptin, resistin, visfatin and ghrelin serum levels were measured by ELISA. RESULTS: Adiponectin serum levels were significantly lower in early-onset GDM women than in standard-onset GDM patients or controls matched for gestational age. Leptin serum levels were significantly higher in women with early-onset GDM than in controls. Women with early-onset GDM had lower adiponectin/leptin ratio than those with standard-onset GDM. There were no significant differences in resistin, ghrelin and visfatin serum levels among the participants. CONCLUSIONS: Our results suggest that, compared to overweight glucose-tolerant women and patients with standard-onset GDM, overweight women with early-onset GDM have unbalanced adipokine levels, suggesting that they have a more inflammatory profile.

Impact of the Reticular Stress and Unfolded Protein Response on the inflammatory response in endometrial stromal cells.

During decidualization, endometrial stromal cells undergo reticular stress (RS) and unfolded protein response (UPR), allowing the endoplasmic reticulum-expansion and immunomodulators production. Physiological RS generates the activation of sensing proteins, inflammasome activation and mature-IL-1ß secretion, associated with pro-implantatory effects. We focus on the impact of RS and UPR on decidualized cells and whether they induce a physiological sterile inflammatory response through IL-1ß production. Human endometrial stromal cell line (HESC) after decidualization treatment with MPA + dibutyryl-cAMP (Dec) increased the expression of RS-sensors (ATF6, PERK and IRE1α) and UPR markers (sXBP1 and CHOP) in comparison with Non-dec cells. Then we found increased NLRP3 expression in Dec cells compared with Non-dec cells. In fact STF-083010 (an IRE1α inhibitor) prevented this increase. Downstream, increased levels of active caspase-1 on Dec cells were detected by FAM-Flica Caspase-1 associated with an increase in IL-1ß production. Moreover, the treatment with STF-083010 decreased the invasion index observed in Dec cells, evaluated by an in vitro model of implantation. In endometrial biopsies from recurrent spontaneous abortion patients an increased expression of IRE1α was found in comparison with fertile women; while recurrent implantation failure samples showed a lower expression of sXBP1, TXNIP and NLRP3 than fertile women, suggesting that RS/UPR tenors might condition endometrial receptivity.

Association of cerebral palsy with consanguineous parents and other risk factors in a Palestinian population.

This case-control study investigated risk factors for cerebral palsy in a Palestinian population. Cases were 107 children aged 1-15 years at a cerebral palsy referral centre in Jerusalem; controls were 233 children without cerebral palsy from West Bank outpatient clinics. Data were collected from medical records and a structured questionnaire to parents. In stepwise logistical regression, consanguinity and birth deficits in other family members were positively associated with cerebral palsy (OR = 4.62; 95% CI: 2.07-10.3 and OR = 12.7; 95% CI: 3.13-51.7 respectively), suggesting a possible genetic link. Other risk factors were: perinatal hypoxia (OR = 92.5; 95% CI: 24.5-350), low birth weight (OR = 4.98; 95% CI: 2.01-12.3), twin births (OR = 9.25; 95% CI: 1.29-66.8) and no prenatal medical care (OR = 5.22; 95% CI: 1.18-23.1). This first stepwise model of significant and modifiable risk factors in our population provides useful evidence for policy-makers.

No added value of performing Ziehl-Neelsen on auramine-positive samples for tuberculosis diagnostics.

SETTING: Regional Laboratory for Tuberculosis, Amsterdam, The Netherlands. BACKGROUND: There is a push to switch from Ziehl-Neelsen (ZN) to auramine microscopy. Despite World Health Organization guidelines that one staining method is sufficient, in some countries national guidelines prescribe that auramine-positive samples should be confirmed by ZN. OBJECTIVE: To investigate the added value of confirming auramine-positive samples using ZN. DESIGN: Using diagnostic data from 10 276 respiratory samples collected from 5525 patients tested for tuberculosis (TB) at the Municipal Health Service of Amsterdam between May 2006 and October 2011, we determined the diagnostic accuracy of auramine alone and of confirmation of auramine-positive samples using ZN. RESULTS: Of 141 M. tuberculosis complex-positive samples detected using auramine on which ZN was performed, 32 (22.7%) were ZN-negative. A similar percentage (6/25, 24.0%) of negatives was found for samples containing non-tuberculous mycobacteria (NTM) species, thus making it impossible to distinguish between TB and NTM on the basis of ZN results. CONCLUSIONS: A positive auramine result followed by a negative ZN result could not be used to exclude TB or to indicate the presence of NTM species. Confirming auramine-positive samples using ZN in this setting thus provided no clinically informative information and was a waste of resources.

Development of innovative paclitaxel-loaded small PLGA nanoparticles: study of their antiproliferative activity and their molecular interactions on prostatic cancer cells.

Taxanes, including paclitaxel, are anti-cancer drugs approved for the treatment of prostate cancer but which have limited clinical application due to their hydrophobicity, their low therapeutic index and the emergence of chemoresistance. These side effects may be avoided through the use of new drug delivery systems such as nanoparticles, and paclitaxel-loaded PLGA nanoparticles up to 200 nm in size have shown encouraging results. As it is known that size affects the tissular penetration and distribution of tumors via the enhanced permeability and retention effect, so nanoparticles smaller than 100 nm are potentially interesting vehicles for improving paclitaxel delivery and efficacy. In this work, new paclitaxel-loaded small PLGA nanoparticles, between 49 nm and 95 nm in size and with positive or negative surface charges, were prepared without detergent. They were stable in the presence of serum, and HPLC showed that high paclitaxel loading and stability were achieved. Intracellular uptake of these nanoparticles was studied in PC3 cells by flow cytometry. Confocal studies confirmed a high tubulin destructuration at very low dose with these nanoparticles. This study suggests that both positively and negatively charged paclitaxel-loaded small PLGA nanoparticles deliver this drug into PC3 cells, and that this nanoparticle mode of delivery highly improves paclitaxel efficiency by up to two log-increase. These results also highlight the importance of small nanoparticles for drug delivery in cancer applications and are extremely promising for in vivo studies.

Association of serum levels of vascular endothelial growth factor and early ectopic pregnancy.

BACKGROUND: This study evaluated serum vascular endothelial growth factor (VEGF) concentrations in women with ectopic pregnancy (EP), miscarriage, and normal pregnancy (NP). MATERIALS AND METHODS: This was a case-control study comparing serum VEGF concentrations among 72 women with ectopic pregnancy (n = 35), miscarriage (n = 15), and normal pregnancy (n = 22) matched for gestational age. For the determination of serum VEGF concentration a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) was used. Patients were stratified according to serum VEGF above or below 200 pg/ml. RESULTS: The serum level of VEGF was significantly higher in women with EP (median 211.1 pg/ml; range 5-1,017.0 pg/ml) than in women with normal pregnancy (median 5 pg/ml; range 5-310.6 pg/ml) p < 0.0001. Serum VEGF concentrations did not show any statistically significant difference between women with miscarriage (median 231.9 pg/ml; range 5-813.7 pg/ml) and EP (median 211.1 pg/ml; range 5-1,017.0 pg/ml). When threshold concentrations of serum VEGF level > 200 pg/ml were used, an EP could be distinguished from a normal pregnancy with a sensitivity of 51.4%, a specificity of 90.9%, and a positive predictive value of 90%. Between EP and miscarriage, the sensitivity was 51.4%, specificity 42.8%, and a positive predictive value of 69.2%. CONCLUSIONS: Serum VEGF could not distinguish an EP from a miscarriage. However, serum VEGF concentrations could discriminate a normal intrauterine pregnancy (IUP) from an unviable pregnancy (EP or miscarriage).

PP007. Effects of STAT1 suppression on ERK1/2 in trophoblastic cells.

INTRODUCTION: Migration and trophoblast invasion are controlled functionally along with the active participation of cytokines and growth factors. Two important intracellular signaling pathways are the Janus kinase/signal transducer and activator of transcription (JAK-STAT) and extracellular regulated kinase1/2 (ERK1/2). These pathways have been associated with the regulation of gene expression, cellular proliferation, differentiation, angiogenesis, embryo development and invasion in tumor and trophoblast cells. OBJECTIVES: The aim of our study is to characterize and analyze the regulation and crosstalks of STAT1 and ERK1/2 in trophoblast cells and the identification of activating cytokines. METHODS: The trophoblast derived cell line HTR-8/svneo and a choriocarcinoma cell line (JEG-3) were stimulated with interleukin-6 (IL-6), IL-11, granulocyte-macrophage colony-stimulating factor (GMC-SF), leukemia inhibitory factor (LIF) or oncostatine M (OSM). The the expression and phosphorylation of STAT1(tyr705) and ERK1/2 were analyzed by gel electrophoresis and Western blotting. Expression of STAT1 was inhibited by administration of 50µM fludarabine (2-fluoro-ara-AMP) for 2, 4, 8, 24, 48 or 72h or by using small interfering RNA (siRNA). The full activation of STAT1 was assessed by using an STAT1 DNA-binding assay. Finally, proliferation and invasion assays were performed (Grant Deutscher Akademischer Austausch Dienst A/10172477). RESULTS: LIF and OSM induce STAT1 and ERK1/2 phosphorylation in HTR-8 and JEG-3 cells. Fludarabine inhibits the so induced phosphorylation of STAT1 when administered 48 or 72h before stimulation. Simultaneously, ERK phosphorylation increases. In contrast, silencing of STAT1 by application of specific siRNA induces reduction of ERK1/2 phosphorylation. Fludarabine reduces STAT1 DNA-binding capacity. LIF and OSM increase proliferation. Silencing of STAT1 slightly decreases invasiveness of analyzed cells. CONCLUSION: STAT1 in trophoblast cells can be activated by placental cytokines. Suppression of STAT1 by fludarabine or siRNA influences activity of ERK1/2 which indicates a crosstalk between both pathways. Current studies will clarify the reason for the different effects on ERK1/2 in trophoblastic cells.

PP115. Relationship between angiogenic factors and preeclampsia: Preliminary results.

INTRODUCTION: The pathogenesis of preeclampsia (PE) is complex and involves many mechanisms, including impaired placental angiogenesis. Endoglin (Eng) promotes angiogenesis, but in its soluble form (sEng) it is antiangiogenic and adiponectin has pro-angiogenic and anti-inflammatory effects on the endothelium. The combined analysis of these factors seems to better reflect maternal vascular damage. OBJECTIVES: We aimed to evaluate adiponectin and soluble endoglin levels, to analyze adiponectin (+45) gene polymorphism and its relation with adiponectin serum levels in patients with PE. METHODS: This case-control study included 24 PE patients and 20 healthy pregnant women (C: control). Adiponectin and sEng serum levels were determined by ELISA. Polymorphism genotyping was obtained by PCR-RFLP. Data were analyzed by Mann-Whitney, Chi-square or Fisher's exact tests and significance was set at p<0.05. RESULTS: There were no differences in adiponectin levels between the groups (C×PE: 6772.4ng/mL×7763.2ng/mL, p=0.99), but women with PE had significantly higher sEng levels (23.45 ng/mL×3.35ng/mL, p<0.0001). Moreover, the ratio adiponectin/sEng was significantly lower in PE than in C women (325.02×2119.4, p<0.0001). There was no association between PE and the analyzed polymorphism, neither between adiponectin genotype/phenotype. CONCLUSION: Our findings confirm an association between PE and altered sEng levels. In addition, these results suggest that angiogenic mediators when analyzed together, can better reflect their involvement in the pathophysiology of PE. Financial support: FAPESP (09/54729-6 and 10/08082-8).

Increased Vitamin B12 levels are associated with mortality in critically ill medical patients.

BACKGROUND & AIMS: We describe an observational study in critically ill medical patients showing the association between serum Vitamin B12 levels measured on or near admission and the outcome in these patients. METHODS: We used the database of patients admitted to the Medical Intensive Care Unit (MICU) at the Hadassah-Hebrew University Medical Center in Jerusalem, Israel, to analyze associations between patient demographics, background, diagnoses and serum Vitamin B12 levels with hospital and 90 day outcomes. RESULTS: Higher mean Vitamin B12 levels were found in patients who did not survive their hospital stay (1719 pg/ml vs 1003 pg/ml, p < 0.01). Those who had died by 90 days after admission to the MICU also had higher Vitamin B12 levels than survivors (1593 pg/ml vs 990 pg/ml). Regression analysis showed that elevated Vitamin B12 levels were associated with increased 90 day mortality, even after controlling for other variables. Survival analysis also showed an increased mortality rate in patients with Vitamin B12 levels over 900 pg/ml (p < 0.0002). CONCLUSIONS: Our data show that high serum Vitamin B12 levels are associated with increased mortality in critically ill medical patients. We suggest that Vitamin B12 levels should be included in the work-up of all medical intensive care patients, particularly those with a chronic health history and increased severity of illness.

Moxonidine improves cardiac structure and performance in SHR through inhibition of cytokines, p38 MAPK and Akt.

BACKGROUND AND PURPOSE: Regression of left ventricular hypertrophy by moxonidine, a centrally acting sympatholytic imidazoline compound, results from a sustained reduction of DNA synthesis and transient stimulation of DNA fragmentation. Because apoptosis of cardiomyocytes may lead to contractile dysfunction, we investigated in spontaneously hypertensive rats (SHR), time- and dose-dependent effects of in vivo moxonidine treatment on cardiac structure and function as well as on the inflammatory process and signalling proteins involved in cardiac cell survival/death. EXPERIMENTAL APPROACH: 12 week old SHR received moxonidine at 0, 100 and 400 µg·kg(-1)·h(-1) , s.c., for 1 and 4 weeks. Cardiac function was evaluated by echocardiography; plasma cytokines were measured by elisa and hearts were collected for histological assessment of fibrosis and measurement of cardiac proteins by Western blotting. Direct effects of moxonidine on cardiac cell death and underlying mechanisms were investigated in vitro by flow cytometry and Western blotting. KEY RESULTS: After 4 weeks, the sub-hypotensive dose of moxonidine (100 µg) reduced heart rate and improved global cardiac performance, reduced collagen deposition, regressed left ventricular hypertrophy, inhibited Akt and p38 MAPK phosphorylation, and attenuated circulating and cardiac cytokines. The 400 µg dose resulted in similar effects but of a greater magnitude, associated with blood pressure reduction. In vitro, moxonidine inhibited norepinephrine-induced neonatal cardiomyocyte mortality but increased fibroblast mortality, through I(1)-receptor activation and differential effects on downstream Akt and p38 MAPK. CONCLUSIONS AND IMPLICATIONS: While the antihypertensive action of centrally acting imidazoline compounds is appreciated, new cardiac-selective I(1)-receptor agonists may confer additional benefit.

IFPA Meeting 2010 Workshop Report I: Immunology; ion transport; epigenetics; vascular reactivity; epitheliochorial placentation; proteomics.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.

Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women.

Hyperglycemia occurs in a variety of conditions such as overt diabetes, gestational diabetes and mild hyperglycemia, all of which are generally defined based on the oral glucose tolerance test and glucose profiles. Whereas diabetes has received considerable attention in recent decades, few studies have examined the mechanisms of mild hyperglycemia and its associated disturbances. Mild gestational hyperglycemia is associated with macrosomia and a high risk of perinatal mortality. Morphologically, the placenta of these women is characterized by an increase in the number of terminal villi and capillaries, presumably as part of a compensatory mechanism to maintain homeostasis at the maternal-fetal interface. In this study, we analised the expression of VEGF and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in placentas from mildly hyperglycemic women. This expression was compared with that of normoglycemic women and women with gestational and overt diabetes. Immunohistochemistry revealed strong staining for VEGF and VEGFR-2 in vascular and trophoblastic cells of mildly hyperglycemic women, whereas the staining for VEGFR-1 was discrete and limited to the trophoblast. The pattern of VEGF and VEGF-receptor reactivity in placentas from women with overt diabetes was similar to that of normoglycemic women. In women with gestational diabetes, strong staining for VEGFR-1 was observed in vascular and trophoblastic cells whereas VEGF and VEGFR-2 were detected only in the trophoblast. The expression of these proteins was confirmed by western blotting, which revealed the presence of an additional band of 75 kDa. In the decidual compartment, only extravillous trophoblast reacted with all antibodies. Morphological analysis revealed collagen deposition around large arteries in all groups with altered glycemia. These findings indicate a placental response to altered glycemia that could have important consequences for the fetus. The change in the placental VEGF/VEGFR expression ratio in mild hyperglycemia may favor angiogenesis in placental tissue and could explain the hypercapillarization of villi seen in this gestational disturbance.

Could exercise be a new strategy to revert some patients with atrial fibrillation?

BACKGROUND: This study is the result of the anecdotal observation that a number of patients with atrial fibrillation (AF) had noted reversion to sinus rhythm (SR) with exercise.We aimed to evaluate the potential role of exercise stress test (EST) for the reversion of AF. METHODS: Patients with AF who were scheduled to undergo electrical cardioversion (DCR) underwent EST using a modified Bruce protocol. RESULTS: Eighteen patients (16 male); aged 36-74 years (mean 58 years) were studied. Five patients (27.7%) had successful reversion with exercise (group 1). Thirteen patients remained in AF (group 2). No patient who failed to revert with exercise did so spontaneously before DCR 3 h to 7 months later (median 20 days). Comparison between group 1 and group 2 did not reveal any significant difference CONCLUSION: This small preliminary study suggests that in some patients it may be possible to revert AF to SR with exercise and avoid DCR and concomitant general anaesthesia. The authors suggest that a larger multicentre randomized trial is warranted to confirm or refute these initial results and if correct identify those who might benefit.

Control of left ventricular mass by moxonidine involves reduced DNA synthesis and enhanced DNA fragmentation.

BACKGROUND AND PURPOSE: Left ventricular hypertrophy (LVH) is a maladaptive process associated with increased cardiovascular risk. Regression of LVH is associated with reduced complications of hypertension. Moxonidine is an antihypertensive imidazoline compound that reduces blood pressure primarily by central inhibition of sympathetic outflow and by direct actions on the heart to release atrial natriuretic peptide, a vasodilator and an antihypertrophic cardiac hormone. This study investigated the effect of moxonidine on LVH and the mechanisms involved in this effect. EXPERIMENTAL APPROACH: Spontaneously hypertensive rats were treated with several doses of moxonidine (s.c.) over 4 weeks. Blood pressure and heart rate were continuously monitored by telemetry. Body weight and water and food intake were measured weekly. Measurements also included left ventricular mass, DNA content, synthesis, fragmentation, and apoptotic/anti-apoptotic pathway proteins. KEY RESULTS: The decrease in mean arterial pressure stabilized at approximately -10 mm Hg after 1 week of treatment and thereafter. Compared to vehicle-treated rats (100%), left ventricular mass was dose- and time-dependently reduced by treatment. This reduction remained significantly lower after normalizing to body weight. Moxonidine reduced left ventricular DNA content and inhibited DNA synthesis. DNA fragmentation transiently, but significantly increased at 1 week of moxonidine treatment and was paralleled by elevated active caspase-3 protein. The highest dose significantly decreased the apoptotic protein Bax and all doses stimulated anti-apoptotic Bcl-2 after 4 weeks of treatment. CONCLUSIONS AND IMPLICATIONS: These studies implicate the modulation of cardiac DNA dynamics in the control of left ventricular mass by moxonidine in a rat model of hypertension.

Environmental risk factors for canine toxoplasmosis in a deprived district of Botucatu, SP, Brazil

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii that can infect a large variety of animals, including humans. The present study aimed to evaluate the frequency of anti-T.gondii antibodies in dogs from a peripheral district of Botucatu and to establish the association among some epidemiological variables in order to evaluate risk factors for toxoplasmosis infection. Serum samples from dogs were screened using an indirect fluorescent antibody (IFA) test. Anti-T.gondii antibody prevalence was 56 percent. The highest titer was 1024 (1.79 percent) and the most frequent titers were 16 (57.14 percent) and 64 (33.93 percent). The chi-square (X²) test revealed significant association among variables such as dog access to street, ingestion of raw meat and presence of synantropic animals in the domestic environment. These results demonstrate that toxoplasmosis is present in dogs from Jardim Santa Elisa district.

Regulation of gene expression in mouse trophoblast cells by interferon-gamma.

We have previously shown that interferon-gamma (IFN-gamma) activates phagocytosis and induces nitric oxide production in cultured mouse trophoblast cells. In the present study we examined the effect of this cytokine on ectoplacental cone and gene expression in trophoblast cells. Ectoplacental cones were obtained during the postimplantation period on gestational day 7.5 from CD-1 mice and exposed to 100U/mL IFN-gamma. Ectoplacental cone morphology, cell proliferation and death were also determined upon IFN-gamma treatment. Complementary DNA macroarray and semiquantitative RT-PCR were used to analyze gene expression. IFN-gamma treatment did not alter ectoplacental cone morphology, trophoblast cell proliferation or death. However, using gene array technology, we observed that IFN-gamma affected the developing trophoblast, altering the level of mRNA expression, which resulted in upregulation of 35 genes and downregulation of seven others. The upregulation of transcription factors and immune response-associated genes suggests that IFN-gamma is involved in processes beyond immunological homeostasis and plays an important role in placental development and function.

Pregnancy alters nitric oxide synthase and natriuretic peptide systems in the rat left ventricle.

Cyclic guanosine monophosphate (cGMP), which is implicated in cardiac cell growth and function, is synthesized by cytoplasmic soluble guanylyl cyclase (GC) stimulated via nitric oxide (NO) and by particulate membrane-bound GC activated via natriuretic peptides. We investigated possible cGMP elevation in the left ventricle (LV) of rats developing physiologic LV hypertrophy during gestation. Furthermore, expression of estrogen receptors (ER) and oxytocin receptors (OTR) was evaluated because their activation stimulates NO and atrial natriuretic peptide (ANP) release from the heart. Compared with nonpregnant controls, Sprague-Dawley rats on day 7 of gestation had similar heart weights, but, on days 14 and 21, ventricular mass increased by 12% and 28% respectively (P< 0.05). LV cGMP concentration was elevated at day 14 of gestation (3.25 +/- 0.12 vs 4.65 +/- 0.17 pmol/g wet weight, P< 0.01) but decreased at day 21 (2.45 +/- 0.09 pmol/g, P< 0.05) to increase again on postpartum day 1 (6.01 +/- 0.15 pmol/g) and day 4 (9.21 +/- 1.79 pmol/g). Changes in endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), OTR and ERalpha, but not ERbeta, proteins paralleled the pregnancy-related cGMP changes in the LV. In contrast, ANP mRNA of the LV remained at control level throughout gestation but increased postpartum, whereas brain natriuretic peptide (BNP) expression declined at term and increased postpartum. The particulate GC natriuretic peptide receptors (GC-A and GC-B) transcripts were already lower at day 14 of gestation. Natriuretic peptide clearance receptor (NPR-C) transcript was not altered on days 7 and 14, but increased at term. We conclude that cGMP concentration in the rat LV is influenced by both NOS and natriuretic peptide systems and may be involved in the changes of LV contractility and hypertrophy that occur during rat gestation.