Endocrine-disrupting chemical concentrations in follicular fluid and follicular reproductive hormone levels.

PURPOSE: To determine correlations between chemicals in follicular fluid (FF) and follicular reproductive hormone levels. METHODS: The analysis was part of a larger cohort study to determine associations between exposure to EDCs and in vitro fertilization (IVF) outcomes. FF was aspirated from a single leading follicle per participant. Demographics and data on exposure to EDCs were self-reported by the participants using a questionnaire. The concentrations of estradiol (E2), progesterone (PG), anti-Mullerian hormone (AMH), and inhibin B, as well as that of 12 phthalate metabolites and 12 phenolic chemicals were measured in each FF sample. Multivariate linear regression model was used to identify the drivers of hormone levels based on participant's age, BMI, smoking status, and chemical exposure for the monitored chemicals detected in more than 50% of the samples. Benjamini-Hochberg false discovery rate (FDR) correction was applied on the resulting p values (q value). RESULTS: FF samples were obtained from 72 women (mean age 30.9 years). Most of the phthalates and phenolic substances monitored (21/24, 88%) were identified in FF. Ten compounds (7 phthalate metabolites, 3 phenols) were found in more than 50% of samples. In addition, there were positive associations between E2 levels and mono-n-butyl phthalate (MnBP) (beta = 0.01) and mono-isobutyl phthalate (MiBP) (beta = 0.03) levels (q value < 0.05). CONCLUSION: Higher concentrations of several phthalate metabolites, present among others in personal care products, were associated with increased E2 levels in FF. The results emphasize the need to further investigate the mechanisms of action of such EDCs on hormonal cyclicity and fertility in women.

Threshold protective levels of serum IgG to Shigella lipopolysaccharide: re-analysis of Shigella vaccine trials data.

OBJECTIVES: Establishing a correlate of protection is essential for the development and licensure of Shigella vaccines. We examined potential threshold levels of serum IgG to Shigella lipopolysaccharide (LPS) that could predict protection against shigellosis. METHODS: We performed new analyses of serologic and vaccine efficacy (VE) data from two randomized vaccine-controlled trials of the Shigella sonnei-Pseudomonas aeruginosa recombinant exoprotein A (rEPA) conjugate conducted in young adults and children aged 1-4 years in Israel. Adults received either S. sonnei-rEPA (n = 183) or control vaccines (n = 277). Children received the S. sonnei-rEPA conjugate (n = 1384) or S. flexneri 2a-rEPA conjugate (n = 1315). VE against culture-proven shigellosis was determined. Sera were tested for IgG anti-S. sonnei LPS antibodies. We assessed the association of various levels of IgG anti-S. sonnei LPS antibodies with S. sonnei shigellosis risk using logistic regression models and the reverse cumulative distribution of IgG levels. RESULTS: Among adults, four vaccinees and 23 controls developed S. sonnei shigellosis; the VE was 74% (95% CI, 28-100%). A threshold of ≥1:1600 IgG anti-S. sonnei LPS titre was associated with a reduced risk of S. sonnei shigellosis and a predicted VE of 73.6% (95% CI, 65-80%). The IgG anti-S. sonnei LPS correlated with serum bactericidal titres. In children, a population-based level of 4.5 ELISA Units (EU) corresponding to 1:1072 titre, predicted VE of 63%, versus 71% observed VE in children aged 3-4 years. The predicted VE in children aged 2-4 years was 49%, consistent with the 52% observed VE. CONCLUSION: Serum IgG anti-S. sonnei LPS threshold levels can predict the degree of VE and can be used for the evaluation of new vaccine candidates.

Vaccination with Shigella flexneri 2a conjugate induces type 2a and cross-reactive type 6 antibodies in humans but not in mice.

Shigella flexneri (S. flexneri) 6 has emerged as an important cause of shigellosis. Our efficacy study of Shigella sonnei and S. flexneri 2a O-specific polysaccharide (O-SP) conjugates in 1-4year-olds had too few S. flexneri 2a cases for efficacy evaluation but surprisingly showed protection of 3-4year-olds, S. flexneri 2a-recipients, from S. flexneri 6 infection. To investigate this cross-protection antibodies to both Shigella types were investigated in all sera remaining from previous studies. Twenty to 30% of 3-44year-old humans injected with S. flexneri 2a conjugate responded with ≥4-fold increases of IgG anti type 6, p<0.00001. The specificity of these antibodies was shown by inhibition studies. S. flexneri 6 infection of 2 children induced besides S. flexneri 6, also S. flexneri 2a antibodies, at levels of S. flexneri 2a vaccinees. S. flexneri 2a antibodies induced by S. flexneri 6 conjugates could not be studied since no such conjugate was assessed in humans and mice responded almost exclusively to the O-SP of the injected conjugate, with no cross-reactive antibodies. Our results indicate induction of cross-reactive protective antibodies. The O-acetylated disaccharide shared by S. flexneri 6 and 2a O-SPs, is the likely basis for their cross-reactivity. S. flexneri 6 O-SP conjugates, alone and in combination with S. flexneri 2a, merit further investigation for broad S. flexneri protection.

Doxorubicin-induced vascular toxicity--targeting potential pathways may reduce procoagulant activity.

INTRODUCTION: Previous study in mice using real-time intravital imaging revealed an acute deleterious effect of doxorubicin (DXR) on the gonadal vasculature, as a prototype of an end-organ, manifested by a reduction in blood flow and disintegration of the vessel wall. We hypothesized that this pattern may represent the formation of microthrombi. We aimed to further characterize the effect of DXR on platelets' activity and interaction with endothelial cells (EC) and to examine potential protectants to reduce DXR acute effect on the blood flow. METHODS: The effect of DXR on platelet adhesion and aggregation were studied in vitro. For in vivo studies, mice were injected with either low molecular weight heparin (LMWH; Enoxaparin) or with eptifibatide (Integrilin(©)) prior to DXR treatment. Testicular arterial blood flow was examined in real-time by pulse wave Doppler ultrasound. RESULTS: Platelet treatment with DXR did not affect platelet adhesion to a thrombogenic surface but significantly decreased ADP-induced platelet aggregation by up to 40% (p<0.001). However, there was a significant increase in GPIIbIIIa-mediated platelet adhesion to DXR-exposed endothelial cells (EC; 5.7-fold; p<0.001) reflecting the toxic effect of DXR on EC. The testicular arterial blood flow was preserved in mice pre-treated with LMWH or eptifibatide prior to DXR (P<0.01). CONCLUSIONS: DXR-induced acute vascular toxicity may involve increased platelet-EC adhesion leading to EC-bound microthrombi formation resulting in compromised blood flow. Anti-platelet/anti-coagulant agents are effective in reducing the detrimental effect of DXR on the vasculature and thus may serve as potential protectants to lessen this critical toxicity.

Pentraxin 3 is a marker of early joint inflammation in patients with juvenile idiopathic arthritis.

Pentraxin 3 (PTX3) is an acute phase protein produced in different body tissues. The aims of this study were to characterize PTX3 secretion in synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients and to analyze the correlation of PTX3 levels in SF with clinical characteristics and the course of the disease. SF-PTX3 levels were measured in a cohort of 75 consecutive JIA patients followed in a single center. Patients' clinical characteristics, disease course, and therapies were analyzed for their correlation with SF-PTX3 levels. A synovial cell line was used to study the kinetics of PTX3 secretion by synoviocytes. SF-PTX3 levels varied over a wide range. Elevated SF-PTX3 levels were detected in patients who subsequently required treatment with disease-modifying antirheumatic drugs during the follow-up period. SF-PTX3 levels were found to be inversely correlated with the length of time from onset of joint swelling. No correlation was found between synovial and serum PTX3 or C-reactive protein (CRP). Following in vitro stimulation of synovial cell line with TNFa or IL1, the secretion of PTX3 increases transiently in the first 48-72 h. A similar increase was obtained in patients' synovial fluids but not with IL6. Higher SF-PTX3 levels were found when tested closer to arthritis exacerbation and 48-72 h after in vitro stimulation of cells from a synovial cell line, implying that PTX3 plays a role in early stages of inflammation. Higher SF-PTX3 levels were associated with several clinical features reflecting disease severity and prognostic data. Measuring SF-PTX3 levels may help in providing a more focused and patient-adjusted treatment.

S-allylmercapto-N-acetylcysteine up-regulates cellular glutathione and protects vascular endothelial cells from oxidative stress.

Oxidative stress and/or low cellular glutathione (GSH) levels are associated with the development and progression of numerous pathological conditions. Cells possess various antioxidant protection mechanisms, including GSH and phase II detoxifying enzymes. N-acetylcysteine (NAC) supplies cells with cysteine to increase GSH level but its efficacy is relatively low because of its limited tissue penetration. Allicin (diallyl thiosulfinate), a reactive sulfaorganic compound, increases cellular GSH and phase II detoxifying enzymes in vascular endothelial cells (EC). A novel compound was designed: S-allylmercapto-N-acetylcysteine (ASSNAC), a conjugate of S-allyl mercaptan (a component of allicin) and NAC. Both ASSNAC and NAC increased cellular GSH of ECs, reaching a maximum of up to four- and threefold increase after exposure for 24 or 6 h at a concentration of 0.2 or 1 mM, respectively. ASSNAC induced nuclear translocation of the activated transcription factor Nrf2 and expression of phase II detoxifying enzymes. EC exposure to tBuOOH resulted in 75% cytotoxicity, and pretreatment of cultures with 0.2 mM ASSNAC or 2mM NAC reduced cytotoxicity to 20 and 42%, respectively. In conclusion, ASSNAC is superior to NAC in protecting cells from oxidative stress because of its ability to up-regulate both GSH and the expression of phase II detoxifying enzymes.

Age-related efficacy of Shigella O-specific polysaccharide conjugates in 1-4-year-old Israeli children.

BACKGROUND: Despite its high worldwide morbidity and mortality, there is yet no licensed vaccine for shigellosis. We reported the safety and immunogenicity of Shigella O-specific polysaccharide-protein conjugates in adults and young children and efficacy of Shigella sonnei conjugate in young adults. METHODS: A double-blinded, randomized and vaccine-controlled Phase 3 evaluation of S. sonnei and Shigella flexneri 2a O-SP-rEPA conjugates, 25 microg, injected IM twice, 6 weeks apart, into healthy 1-4 years old, is reported. The children were followed for 2 years by telephone every other week and stool cultures were obtained for each episode of acute diarrhea (> or =3 loose stools/day or a bloody/mucous stool). Sera were taken randomly from 10% of the participants for IgG anti-LPS and anti-carrier levels. RESULTS: Of the 2799 enrollees, 1433 received S. sonnei and 1366 S. flexneri 2a conjugates; 2699 (96.4%) completed the 2-year follow-up. Local reactions occurred in approximately 5% and approximately 4% had temperatures > or =38.0 degrees C lasting 1-2 days. There were no serious adverse events attributable to the vaccines. Of the 3295 stool cultures obtained, 125 yielded S. sonnei and 21 S. flexneri 2a. Immunogenicity and efficacy were age-related. The overall efficacy of the S. sonnei conjugate was 27.5%; 71.1% (P=0.043) in the 3-4 years old. The numbers for S. flexneri 2a were too few for meaningful analysis. Cross-protection by S. flexneri 2a for non-vaccine S. flexneri types was found, but the numbers were too few for statistical significance. There was an age-related rise of vaccine-specific IgG anti-LPS in both groups, peaking at about 10 weeks and declining thereafter, but remaining > or =4-fold higher than in the controls 2 years after the second dose. CONCLUSIONS: Shigella conjugates are safe and immunogenic in 1-4 years old. The S. sonnei conjugate elicited 71.1% efficacy in the 3-4 years old and can be predicted to be efficacious in individuals older than 3 years of age. These results urge studies with our improved conjugates.

Phacoemulsification-induced injury in corneal endothelial cells mediated by apoptosis: in vitro model.

PURPOSE: To characterize the mechanism of injury caused by phacoemulsification and examine the protective effect of ascorbic acid in cultured bovine corneal endothelial cells (CECs). SETTING: Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. METHODS: To minimize the direct mechanical effects of phacoemulsification on CECs, cells cultured on gas-permeable flexible membranes were exposed to phacoemulsification. Apoptosis was assayed by immunohistochemistry using anticaspase-3 antibody. RESULTS: Phacoemulsification caused a significantly higher rate of apoptosis (mean apoptotic nuclei per square millimeter, 334+/-29 [SD] versus 45+/-12) 48 hours after exposure (P<.001). Results were similar in CECs exposed to hydrogen peroxide for 48 hours as a positive control (mean 345+/-6 and 376+/-1 at hydrogen peroxide concentrations of 50 microM and 100 microM, respectively). Addition of ascorbic acid (1 or 10 mM) significantly decreased the 48-hour apoptotic nuclei count induced by phacoemulsification (mean 219+/-15 and 130+/-29, respectively) (P<.001). Application of shear forces up to 2000 seconds(-1) for 60 minutes did not increase the rate of apoptotic nuclei. CONCLUSIONS: Flexible membranes covered with cultured CECs, used as a new model that mimics in vivo conditions, minimized the mechanical damage caused by ultrasonic vibrations and turbulent currents, which destroy cells grown on hard surfaces. Phacoemulsification damage was not mediated by mechanical or shear forces but resulted from free-radical formation that apparently triggered cellular cascades, leading to apoptosis. Cell death was significantly reduced by the addition of ascorbic acid, probably via a free radical-scavenging mechanism.

Platelets enhance CD4+ lymphocyte adhesion to extracellular matrix under flow conditions: role of platelet aggregation, integrins, and non-integrin receptors.

The purpose of this study was to examine the role of platelets in CD4+ T lymphocyte adhesion to subendothelial extracellular matrix (ECM). Herpesvirus saimiri (HVS)-infected CD4+ T cells were incubated on ECM. An image analysis was used to evaluate T cell adhesion. Under static condition, T cell activation with 4-alpha-Phorbol 12-myristate 13-acetate (PMA) resulted in a 2.6-fold increase in cell adhesion. However, adhesion was not affected by platelets. In contrast, under flow (200s(-1)), platelets markedly enhanced both resting and PMA-activated T cell adhesion (33- and 48-fold), forming lymphocyte-platelet co-aggregates that contain approximately 90% of the adherent T cells. Abrogation of platelet aggregation with tirofiban inhibited formation of platelet-T cell co-aggregates under flow and reduced T cell adhesion by 74%. Separate and combined blockade of CD40L and P-selectin glycoprotein-1 (PSGL-1) on PMA-activated lymphocytes reduced adhesion under flow in the presence of platelets by 28%, 33%, and 55%, respectively. Blockade of beta1-integrins decreased adhesion under both static and flow conditions (by 35% and 44%, respectively), while blockade of beta2-integrin reduced adhesion only under static condition (by 23%). A similar adhesion pattern was observed using CD4+ T cells isolated from normal donor peripheral blood. In conclusion, platelets support CD4+ lymphocyte adhesion to ECM under flow by formation of heterotypic platelet-lymphocyte coaggregates involving alphaIIbbeta3 integrin and beta1-related integrins, as well as CD40L and PSGL-1.

Safety and immunogenicity of Shigella sonnei-CRM9 and Shigella flexneri type 2a-rEPAsucc conjugate vaccines in one- to four-year-old children.

BACKGROUND AND OBJECTIVE: Shigella conjugate vaccines have been shown to be safe, immunogenic and efficacious in adult volunteers. We have now investigated the safety and immunogenicity of investigational Shigella sonnei and Shigella flexneri 2a conjugate vaccines in 1- to 4-year-old children, the age group at greatest risk for shigellosis. METHODS: The O-specific polysaccharides of S. sonnei and S. flexneri 2a, the two most common shigellae from patients in Israel, were bound to medically useful carrier proteins to form conjugates. Eighty healthy 1- to 4-year-olds were randomized to receive two 0.5-ml im injections 6 weeks apart of either S. sonnei-CRM(9) or S. flexneri 2a-rEPA(succ). Blood was taken before, 6 weeks after the first injection, 4 weeks after the second injections and 2 years after immunization for assay of IgG anti-lipopolysaccharide, diphtheria toxin and Pseudomonas aeruginosa exotoxin A antibodies by enzyme-linked immunosorbent assay. RESULTS: During an 8-day surveillance period after each immunization, low fever (37.8-39.0 degrees C) lasting only 24 to 48 h occurred in 2 of 40 recipients after the first injection and 4 of 40 recipients after the second injection of S. flexneri 2a-rEPA(succ) and in 2 of 38 of S. sonnei-CRM(9) after the second injection; no fever was detected after the first injection. Liver function tests were normal in all vaccinees. S. sonnei-CRM(9) elicited a >4-fold rise in IgG anti-LPS in 92.1% and S. flexneri 2a-rEPA(succ) in 85% (P < 0.0001) after the second injection; both conjugates elicited type-specific booster responses. At 2 years the geometric mean concentrations of both IgG anti-lipopolysaccharides were significantly higher than preimmunization levels. A >4-fold rise of IgG anti-diphtheria (65.8%) and IgG anti-ETA (77.5%) was observed. CONCLUSION: These experimental Shigella conjugate vaccines were safe and immunogenic in 1- to 4-year-old children.