Single and mixture effects of bisphenol A and benzophenone-3 on in vitro T helper cell differentiation.

Immune homeostasis is key to guarantee that the immune system can elicit effector functions against pathogens and at the same time raise tolerance towards other antigens. A disturbance of this delicate balance may underlie or at least trigger pathologies. Endocrine disrupting chemicals (EDCs) are increasingly recognized as risk factors for immune dysregulation. However, the immunotoxic potential of specific EDCs and their mixtures is still poorly understood. Thus, we aimed to investigate the effect of bisphenol A (BPA) and benzophenone-3 (BP-3), alone and in combination, on in vitro differentiation of T helper (TH)17 cells and regulatory T (Treg) cells. Naïve T cells were isolated from mouse lymphoid tissues and differentiated into the respective TH population in the presence of 0.001-10 µM BP-3 and/or 0.01-100 µM BPA. Cell viability, proliferation and the expression of TH lineage specific transcription factors and cytokines was measured by flow cytometry and CBA/ELISA. Moreover, the transcription of hormone receptors as direct targets of EDCs was quantified by RT-PCR. We found that the highest BPA concentration adversely affected TH cell viability and proliferation. Moreover, the general differentiation potential of both TH populations was not altered in the presence of both EDCs. However, EDC exposure modulated the emergence of TH17 and Treg cell intermediate states. While BPA and BP-3 promoted the development of TH1-like TH17 cells under TH17-differentiating conditions, TH2-like Treg cells occurred under Treg polarization. Interestingly, differential effects could be observed in mixtures of the two tested compounds compared with the individual compounds. Notably, estrogen receptor ß expression was decreased under TH17-differentiating conditions in the presence of BPA and BP-3 as mixture. In conclusion, our study provides solid evidence for both, the immune disruptive potential and the existence of cumulative effects of real nature EDC mixtures on T cell in vitro differentiation.

Single and combined exposures to bisphenol A and benzophenone-3 during early mouse pregnancy have differential effects on fetal and placental development.

Endocrine disrupting chemicals (EDCs) possess the capability to interfere with the endocrine system by binding to hormone receptors, for example on immune cells. Specific effects have already been described for individual substances, but the impact of exposure to chemical mixtures during pregnancy on maternal immune regulation, placentation and fetal development is not known. In this study, we aimed to investigate the combined effects of two widespread EDCs, bisphenol A (BPA) and benzophenone-3 (BP-3), at allowed concentrations on crucial pregnancy processes such as implantation, placentation, uterine immune cell populations and fetal growth. From gestation day (gd) 0 to gd10, female mice were exposed to 4 µg/kg/d BPA, 50 mg/kg/d BP-3 or a BPA/BP-3 mixture. High frequency ultrasound and Doppler measurements were used to determine intrauterine fetal development and hemodynamic parameters. Furthermore, uterine spiral artery remodeling and placental mRNA expression were studied via histology and CHIP-RT-PCR, respectively. Effects of EDC exposure on multiple uterine immune cell populations were investigated using flow cytometry. We found that exposure to BP-3 caused intrauterine growth restriction in offspring at gd14, while BPA and BPA/BP-3 mixture caused varying effects. Moreover, placental morphology at gd12 and placental efficiency at gd14 were altered upon BP-3 exposure. Placental gene transcription was altered particularly in female offspring after in utero exposure to BP-3. Flow cytometry analyses revealed an increase in uterine T cells and NK cells in BPA and BPA/BP-3-treated dams at gd14. Doppler measurements revealed no effect on uterine hemodynamic parameters and spiral artery remodeling was not affected following EDC exposure. Our results provide evidence that exposure to BPA and BP-3 during early gestation affects fetal development in a sex-dependent manner, placental function and immune cell frequencies at the feto-maternal interface. These results call for inclusion of studies addressing pregnancy in the risk assessment of environmental chemicals.

An old friend with a new face: YB-1 and its role in healthy pregnancy and pregnancy-associated complications.

By promoting tissue invasion, cell growth and angiogenesis, the Y-box binding protein (YB-1) became famous as multifunctional oncoprotein. However, this designation is telling only part of the story. There is one particular time in life when actual tumorigenic-like processes become undoubtedly welcome, namely pregnancy. It seems therefore reasonable that YB-1 plays also a crucial role in reproduction, and yet this biological aspect of the cold-shock protein has been overlooked for many years. To overcome this limitation, we would like to propose a new perspective on YB-1 and emphasize its pivotal functions in healthy pregnancy and pregnancy-related complications. Moreover, we will discuss findings obtained from cancer research in the light of reproductive events to elucidate the importance of YB-1 at the feto-maternal interface.

Selected commensals educate the intestinal vascular and immune system for immunocompetence.

BACKGROUND: The intestinal microbiota fundamentally guides the development of a normal intestinal physiology, the education, and functioning of the mucosal immune system. The Citrobacter rodentium-carrier model in germ-free (GF) mice is suitable to study the influence of selected microbes on an otherwise blunted immune response in the absence of intestinal commensals. RESULTS: Here, we describe that colonization of adult carrier mice with 14 selected commensal microbes (OMM12 + MC2) was sufficient to reestablish the host immune response to enteric pathogens; this conversion was facilitated by maturation and activation of the intestinal blood vessel system and the step- and timewise stimulation of innate and adaptive immunity. While the immature colon of C. rodentium-infected GF mice did not allow sufficient extravasation of neutrophils into the gut lumen, colonization with OMM12 + MC2 commensals initiated the expansion and activation of the visceral vascular system enabling granulocyte transmigration into the gut lumen for effective pathogen elimination. CONCLUSIONS: Consortium modeling revealed that the addition of two facultative anaerobes to the OMM12 community was essential to further progress the intestinal development. Moreover, this study demonstrates the therapeutic value of a defined consortium to promote intestinal maturation and immunity even in adult organisms. Video Abstract.

Perinatal exposure to endocrine disrupting chemicals and neurodevelopment: How articles of daily use influence the development of our children.

Substances that interfere with the body's hormonal balance or their function are called endocrine disrupting chemicals (EDCs). Many EDCs are ubiquitous in the environment and are an unavoidable aspect of daily life, including during early embryogenesis. Developmental exposure to these chemicals is of critical relevance, as EDCs can permanently alter developmental programs, including those that pattern and wire the brain. Of emerging interest is how these chemicals may also affect the immune response, given the cross-talk between the endocrine and immune systems. As brain development is strongly dependent on hormones including thyroid, androgens, and estrogens, and can also be affected by immunomodulation, this complicated interplay may have long-lasting neurodevelopmental consequences. This review focuses on data available from human cohorts, in vivo models, and in vitro assays regarding the impact of EDCs after a gestational and/or lactational exposure, and how they may impact the immune system and/or neurodevelopment.

Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer.

Emerging data demonstrate that the activity of immune cells can be modulated by microbial molecules. Here, we show that the short-chain fatty acids (SCFAs) pentanoate and butyrate enhance the anti-tumor activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells through metabolic and epigenetic reprograming. We show that in vitro treatment of CTLs and CAR T cells with pentanoate and butyrate increases the function of mTOR as a central cellular metabolic sensor, and inhibits class I histone deacetylase activity. This reprogramming results in elevated production of effector molecules such as CD25, IFN-γ and TNF-α, and significantly enhances the anti-tumor activity of antigen-specific CTLs and ROR1-targeting CAR T cells in syngeneic murine melanoma and pancreatic cancer models. Our data shed light onto microbial molecules that may be used for enhancing cellular anti-tumor immunity. Collectively, we identify pentanoate and butyrate as two SCFAs with therapeutic utility in the context of cellular cancer immunotherapy.

YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits.

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

Dietary cellulose induces anti-inflammatory immunity and transcriptional programs via maturation of the intestinal microbiota.

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was Alistipes which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including A. finegoldii showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.

High Prevalence of Acquired Thrombophilia Without Prognosis Value in Patients With Coronavirus Disease 2019.

Background Recent literature reports a strong thrombotic tendency in patients hospitalized for a coronavirus disease 2019 (COVID-19) infection. This characteristic is unusual and seems specific to COVID-19 infections, especially in their severe form. Viral infections can trigger acquired thrombophilia, which can then lead to thrombotic complications. We investigate for the presence of acquired thrombophilia, which could participate in this phenomenon, and report its prevalence. We also wonder if these thrombophilias participate in the bad prognosis of severe COVID-19 infections. Methods and Results In 89 consecutive patients hospitalized for COVID-19 infection, we found a 20% prevalence of PS (protein S) deficiency and a high (ie, 72%) prevalence of antiphospholipid antibodies: mainly lupus anticoagulant. The presence of PS deficiency or antiphospholipid antibodies was not linked with a prolonged activated partial thromboplastin time nor with D-dimer, fibrinogen, or CRP (C-reactive protein) concentrations. These coagulation abnormalities are also not linked with thrombotic clinical events occurring during hospitalization nor with mortality. Conclusions We assess a high prevalence of positive tests detecting thrombophilia in COVID-19 infections. However, in our series, these acquired thrombophilias are not correlated with the severity of the disease nor with the occurrence of thrombotic events. Albeit the strong thrombotic tendency in COVID-19 infections, the presence of frequent acquired thrombophilia may be part of the inflammation storm of COVID-19 and should not systematically modify our strategy on prophylactic anticoagulant treatment, which is already revised upwards in this pathological condition. Registration URL: https://www.clini​caltr​ials.gov; Unique identifier: NCT04335162.

Monitoring unfractionated heparin therapy. 4 hour-stability of anti-Xa activity in unspun citrated tubes.

Current guidelines recommend performing laboratory tests aimed at monitoring unfractionated heparin (UFH) treatments within a delay not exceeding 1 to 2 h(s) after sampling when blood is collected into citrated tubes. As such a short delay could be an issue, we evaluated the potential impact of longer delays. For that purpose, two citrated tubes were obtained from patients on UFH: one was centrifuged and tested for anti-Xa activity and aPTT within 1 h after collection (T1 h) and one was stored for 4 h at room temperature (T4 h) before being processed. A total of 123 paired tubes were investigated. Anti-Xa activity was significantly lower at T4 h than at T1 h, with a mean bias, calculated according to Bland-Altman, of 0.05 IU/mL. Considering 0.30 to 0.70 IU/mL as the therapeutic range, there were 12 cases of discrepant test results (9.8%). Most of them being around the lower limit of the therapeutic range had no impact on patients' management. APTT was significantly shortened (p < 0.0001) at T4 h vs. T1 h, with a mean bias of -7.9 s. Considering anti-Xa correlated aPTT therapeutic range, 29 cases of discrepant test results (23.6%) were found, 10% would have induce dosage changes. The concordance between anti-Xa activities measured at T4 h and T1 h was excellent (kappa = 0.813) and good for aPTT (kappa = 0.661). In conclusion, extending the delay between blood collection and measurement of tests prescribed for monitoring UFH therapy up to 4 h was found to lead to a systematic reduction in both anti-Xa activity and aPTT in unspun citrated tubes. As changes at T4 h were limited and had few clinically relevance than the ones observed with aPTT testing, a 4 h-delay was found to be acceptable for anti-Xa activity. The maximum delay for aPTT should remain around 1-2 h as changes were more relevant.

The NF-κB transcription factor c-Rel controls host defense against Citrobacter rodentium.

Mice lacking CD4+ T cells or B cells are highly susceptible to Citrobacter rodentium infection. In this study, we show that the activity of the transcription factor c-Rel in lymphocytes is crucial for clearance of C. rodentium. Mice deficient for c-Rel fail to generate protective antibodies and to eradicate the pathogen.

Intestinal development and homeostasis require activation and apoptosis of diet-reactive T cells.

The impact of food antigens on intestinal homeostasis and immune function is poorly understood. Here, we explored the impact of dietary antigens on the phenotype and fate of intestinal T cells. Physiological uptake of dietary proteins generated a highly activated CD44+Helios+CD4+ T cell population predominantly in Peyer patches. These cells are distinct from regulatory T cells and develop independently of the microbiota. Alimentation with a protein-free, elemental diet led to an atrophic small intestine with low numbers of activated T cells, including Tfh cells and decreased amounts of intestinal IgA and IL-10. Food-activated CD44+Helios+CD4+ T cells in the Peyer patches are controlled by the immune checkpoint molecule PD-1. Blocking the PD-1 pathway rescued these T cells from apoptosis and triggered proinflammatory cytokine production, which in IL-10-deficient mice was associated with intestinal inflammation. In support of these findings, our study of patients with Crohn's disease revealed significantly reduced frequencies of apoptotic CD4+ T cells in Peyer patches as compared with healthy controls. These results suggest that apoptosis of diet-activated T cells is a hallmark of the healthy intestine.

Draft Genome Sequence of Lactobacillus delbrueckii Strain #22 Isolated from a Patient with Short Bowel Syndrome and Previous d-Lactic Acidosis and Encephalopathy.

d-Lactic acidosis with associated encephalopathy caused by overgrowth of intestinal lactic acid bacteria is a rarely diagnosed neurological complication of patients with short bowel syndrome. Here, we report the draft genome sequence of Lactobacillus delbrueckii strain #22 isolated from a patient with short bowel syndrome and previous d-lactic acidosis/encephalopathy.

Is anti-factor Xa chromogenic assay for Rivaroxaban appropriate in clinical practice? Advantages and comparative drawbacks.

INTRODUCTION: No routine monitoring is required with factor Xa inhibitor rivaroxaban. Yet, its titration must be adapted, and its misuse may lead to increased risk of bleeding; therefore, therapeutic rivaroxaban monitoring might help in specific situations. MATERIAL AND METHODS: As asked by clinicians of our medical center, we measured rivaroxaban plasma concentrations in real conditions of use and checked their corresponding prescriptions. Measurement of 112 samples from 94 consecutive patients was performed with a Biophen LRT® anti-Xa chromogenic assay and compared blindly to the HPLC-MSMS "gold standard" method. Rivaroxaban was effectively given to 80 out of 94 patients but a mere 57% through an adequate prescription (within the scope of indications/titration). All chromogenic measurements were over the pre-specified 30ng/ml LOQ, whereas only 98 /114 samples had quantifiable rivaroxaban with HPLC-MSMS (LOQ 1ng/ml). Correlation between the two methods and linear regression were highly significant (p<0.0001). However, chromogenic values (mean 141.6ng/ml[96.6]) overestimated HPLC-MSMS values (119.7ng/ml[79.5]) by 22ng/ml according to Bland-Altman analysis (p<0.001). After re-assessing the chromogenic LOQ at 52ng/ml, 83 quantifiable samples had a mean concentration of 176.9ng/ml as compared to 158.5ng/ml with HPLC-MSMS, with no false positive anymore. CONCLUSIONS: In our medical center, rivaroxaban concentrations could be assessed by a rapid chromogenic method. Its pre-specified LOQ proved too high after being checked "on site" against HPLC-MSMS. Prescriptions for rivaroxaban were not optimal. An overestimated LOQ may impair observance monitoring or predispose patients to either risky thrombolysis or otherwise adjournable surgery in clinical practice.

Evaluation and performance characteristics of the Q Hemostasis Analyzer, an automated coagulation analyzer.

The Q Hemostasis Analyzer (Grifols, Barcelona, Spain) is a fully-automated random-access multiparameter analyzer, designed to perform coagulation, chromogenic and immunologic assays. It is equipped with a cap-piercing system. The instrument was evaluated in a hemostasis laboratory of a University Hospital with respect to its technical features in the determination of coagulation i.e. prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time, fibrinogen and single coagulation factors V (FV) and VIII (FVIII), chromogenic [antithrombin (AT) and protein C activity] and immunologic assays [von Willebrand factor antigen (vWF:Ag) concentration], using reagents from the analyzer manufacturer. Total precision (evaluated as the coefficient of variation) was below 6% for most parameters both in normal and in pathological ranges, except for FV, FVIII, AT and vWF:Ag both in the normal and pathological samples. No carryover was detected in alternating aPTT measurement in a pool of normal plasma samples and in the same pool spiked with unfractionated heparin (>1.5 IU/mL). The effective throughput was 154 PT, 66 PT/aPTT, 42 PT/aPTT/fibrinogen, and 38 PT/aPTT/AT per hour, leading to 154 to 114 tests performed per hour, depending of the tested panel. Test results obtained on the Q Hemostasis Analyzer were well correlated with those obtained on the ACL TOP analyzer (Instrumentation Laboratory), with r between 0.862 and 0.989. In conclusion, routine coagulation testing can be performed on the Q Hemostasis Analyzer with satisfactory precision and the same apply to more specialized and specific tests.

Functional recovery in rats with chronic spinal cord injuries after exposure to an enriched environment.

BACKGROUND/OBJECTIVE: The objective of this study was to determine the effect of environmental enrichment on the sensorimotor function of rats with chronic spinal cord injuries. DESIGN: Adult Sprague-Dawley rats received a contusive injury of moderate severity at vertebral level T8 using a weight-drop device. Three months after injury, 1 randomized group (n = 16) of rats was placed in an enriched environment, whereas the control group (n = 16) remained housed in standard laboratory cages (2/cage). METHODS: Animals were placed in an enriched environment for 4 weeks beginning at 3 months after injury. The enriched environment consisted of a large cage (5-6 rats/cage) with access to items such as tubes, ramps, and running wheel, with items changed daily. MAIN OUTCOME MEASURES: Functional evaluation consisted of the open field Basso, Beattie and Bresnahan (BBB) locomotor test and the tests that form the combined behavioral score (CBS). The CBS includes motor score, toe spread, placing, withdrawal, righting, inclined plane, hot plate, and swim tests. Behavioral testing was repeated 7 times before and after the period of intervention. RESULTS: The group placed in the enriched environment scored significantly better on the BBB (ANOVA repeated-measures, P < 0.01) test and CBS (ANOVA repeated-measures, P < 0.01). CONCLUSIONS: Environmental enrichment results in significant functional improvement in animals with spinal cord injury even with a substantial delay in initiating treatment after injury. The features of an enriched environment that may be responsible for the improvement include social interactions, exercise, and novel items in an interesting environment. These findings suggest a continued plasticity of the chronically injured rat spinal cord and a possible therapeutic intervention for people with spinal cord injury.

Evaluation and performance characteristics of the automated coagulation analyzer ACL TOP.

INTRODUCTION: The ACL TOP is a fully-automated random-access multiparameter coagulation analyzer equipped with a photo-optical clot-detection unit. It is designed to perform coagulation, chromogenic and immunologic assays with continuous loading capabilities for samples, reagents and disposables. MATERIALS AND METHODS: The instrument was evaluated in a coagulation laboratory of a university hospital with respect to its technical features in the determination of routine coagulation (prothrombin time, activated partial thromboplastin time, fibrinogen and single coagulation factor levels), chromogenic (anti-activated factor X, antithrombin and protein C activities) and immunologic assays (free protein S and von Willebrand factor antigen concentrations). RESULTS: Using fresh and lyophilized plasma samples, the intra-assay and inter-assay coefficients of variation were below 5% for most of the parameters both in the normal and in the pathological ranges. For clotting assays performed at 671 nm, no significant interference could be demonstrated with hemolytic, icteric and lipemic samples as demonstrated by results similar to those obtained using a mechanical clot-detection-based analyzer (STAR). No sample carryover was detected in measuring alternatively heparinized (1.0 IU/mL unfractionated heparin) and normal plasma samples. The results of the different coagulation, chromogenic and immunologic assays obtained on the ACL TOP were well correlated with those obtained on the STAR analyzer with the correlation coefficient (r) in the range from 0.876 to 0.990. CONCLUSIONS: Our results demonstrated that using the ACL TOP analyzer, routine hemostasis testing can be performed with satisfactory precision and the same applied to more specialized and specific tests such as single factor activity or antigen concentration.

Evaluation of the automated coagulation analyzer Sysmex CA-7000.

The Sysmex CA-7000 is a fully automated multiparameter hemostasis analyzer equipped with a photo-optical clot detection unit and a cap-piercing system. It is designed to perform coagulation tests as well as chromogenic and immunologic assays. It was evaluated in a coagulation laboratory of a university hospital with respect to its technical characteristics in the determination of routine coagulation (prothrombin time, activated partial thromboplastin time, fibrinogen and single coagulation factors), chromogenic (antithrombin, and anti-FXa activity) and immunologic assays (von Willebrand factor). The intra-assay and inter-assay coefficients of variation (CV) were below 5% for most parameters both in the normal and in the pathological range (exceptions: intra-assay CV=5.2% for the fibrinogen and 5.1% for antithrombin in the low range of concentrations; and inter-assay CV=5.7% and 7.2% for clotting factors V and VII levels in the normal ranges, and in the range from 6.1% to 7.8% for anti-FXa activity). No significant interference could be demonstrated with hemolytic and icteric samples as demonstrated by results similar to those obtained using a mechanical clot detection-based analyzer (STAR). No carryover was detected in alternating measurements of heparinized (1.0 IU/mL unfractionated heparin) and normal plasma samples. The results of the different coagulation, chromogenic and immunologic assays obtained with the CA-7000 analyzer were well correlated with those obtained on the STAR analyzer (r in the range from 0.885 to 0.990). Our results demonstrated that using the CA-7000 analyzer, routine coagulation testing can be performed with satisfactory precision and the same applied to more specialized and specific tests such as single factor activity or antigen concentration.