-Tetrachlorodibenzo-p-dioxin-Mediated Impairment of B Cell Differentiation Involves Dysregulation of Paired Box 5 (Pax5) Isoform, Pax5a

ABSTRACT The persistent environmental contaminant and immunotoxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), markedly suppresses humoral immune responses. We recently reported impaired down-regulation of paired box 5 (Pax5), a repressor of B cell differentiation and concomitant suppression of the IgM response by TCDD in the murine CH12.LX B cell line. The objectives of the current study were to determine the impact of TCDD treatment on molecular outcomes characteristic of terminal B cell differentiation and to assess the role that Pax5 isoforms plays in the suppression of B cell differentiation by TCDD. In this study, we show that the highly abundant fulllength Pax5 isoform, Pax5a, and at least two additional modestly expressed Pax5 isoforms were expressed in CH12.LX and splenic B cells. In lipopolysaccharide (LPS)-activated B cells, all of the identified Pax5 isoforms were synchronously down-regulated, and in the presence of TCDD cotreatment they were abnormally and synchronously elevated, suggesting a common mechanism of regulation. Furthermore, B cell differentiation markers X-box protein-1 and major histocompatibility complex class II showed that the levels to which Pax5 was derepressed by TCDD were sufficient to impair B cell differentiation and immunoglobulin gene expression. Confirming the involvement of Pax5, ectopic expression of Pax5a in the LPS-activated CH12.LX cells closely mimicked the suppression of the IgM response by TCDD. In summary, our results demonstrate that Pax5a has a critical role in both the TCDD-mediated impairment of B cell differentiation and the suppression of the humoral immune response. Suppression of primary humoral immune responses is one of the most sensitive sequela associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous environmental contaminant. This suppression is characterized by a striking reduction in plasma cell formation and IgM secretion, and it is mediated through a direct effect by TCDD on B cells Activation by antigen or via polyclonal activators (e.g., 463 lipopolysaccharide; LPS) induces B cells to undergo cycles of intense proliferation (i.e., clonal expansion) followed by terminal differentiation into plasma cells. Terminally differentiated plasma cells specialize in secretion of antigen-specific Ig. A number of phenotypic changes distinguish terminally differentiated plasma cells from the resting B cells Materials and Methods Chemicals. TCDD, in 100% dimethyl sulfoxide (DMSO), was purchased from AccuStandard (New Heaven, CT). DMSO and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Mice. Virus-free, female B6C3F1mice (six weeks old) were purchased from Charles River (Portage, MI). On arrival, mice were randomly grouped with five per plastic cage on sawdust bedding. Mice had free access to food (Purina Certified Laboratory Chow) and water at all times. The mouse holding rooms were maintained at 21 to 24°C with 40 to 60% relative humidity on a 12-h light/dark cycle. All of the experimental procedures and conditions were performed according to the guidelines of the All University Committee on Animal Use and Care at Michigan State University (East Lansing, MI). Cell Line. The CH12.LX B cell line was derived from the murine B cell lymphoma, CH12, which arose in B10.H-2aH-4bP/Wts mice (B10.A ϫ B10.129) and has been characterized previously Flow Cytometric Analysis. Cells were harvested from culture at the indicated times by centrifugation at 300g for 10 min at 4°C, washed twice in ice-cold 1ϫ Hanks' balanced-salt solution, and stained using the BD Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. In brief, cells were incubated with 1 g/10 6 cells of purified rat anti-mouse CD16/CD32 monoclonal antibody (BD Pharmingen) for 15 min at 4°C to prevent nonspecific binding and then stained with surface marker detection antibody [anti-fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse I-A P or anti-allophycocyanin-conjugated anti-mouse CD19] or a respective isotype control (BD Pharmingen). To exclude nonviable cells, 2 l of 7-aminoactinomycin D (7-AAD) solution (Sigma-Aldrich) containing 1 mg of 7-AAD, 50 l methanol, and 950 l of Hanks' balanced-salt solution were added simultaneously with detection antibodies to the cells in 50 l of staining buffer. The cells were then fixed, washed, and maintained in staining buffer containing 10 mM actinomycin D (Sigma-Aldrich) to prevent 7-AAD leakage from fixed cells. For the detection of nuclear Pax5 protein, cells were fixed and permeabilized before staining with anti-Pax5 antibody or isotype control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Fluorescence detection was performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) on 10,000 viable cells per sample. Data analysis was performed using BD CellQuest Pro software (BD Biosciences). In Vitro Polyclonal IgM Antibody-Forming Cell Response. Single-cell suspensions of splenocytes from naive mice were adjusted to 1 ϫ 10 6 cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 10% bovine calf serum (HyClone Laboratories), 100 units/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol. Spleen cells were transferred to 48-well culture plates in 500-l aliquots with four wells per treatment group. TCDD (3, 10, or 30 nM) and/or vehicle (0.01% DMSO) were added directly to each well in 5-l aliquots. The splenocytes were sensitized with 10 g/ml LPS and cultured for 3 days in a pressurized chamber at 5.0 psi containing 10% O 2 , 7% CO 2 , and 83% N 2 gas mixture with continuous rocking at 37°C. Enumeration of the IgM antibody-forming cell (AFC) response was performed using trinitrophenol-haptenated sheep red blood cells as described previously the cell mixture was poured onto a 100 ϫ 15-mm Petri dish and immediately covered with a 45 ϫ 50-mm glass coverslip. Upon solidification of the agar, the Petri dishes were placed in a humidified 37°C incubator overnight to allow for plaque formation. The number of splenocytes from each spleen was determined using a Coulter Counter (Beckman Coulter, Fullerton, CA). Results are expressed as AFC/10 6 viable splenocytes Ϯ S.E. Splenocyte viability was measured using pronase (EMD Biosciences, San Diego, CA) as described previously Purification of Splenic B Cells. Spleens were removed aseptically and made into a single-cell suspension. B cells were then isolated using the B Cell Isolation Kit (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's protocol. In brief, 40 l of MACS buffer (phosphate-buffered saline solution, pH 7.2, supplemented with 0.5% bovine serum albumin and 2 mM EDTA, 4°C) per 10 7 cells was used to suspend the splenocytes, and 10 l of Biotin-Antibody Cocktail (Miltenyi Biotec Inc.) per 10 7 cells was added to label non-B cells. After incubation at 4 to 8°C for 10 min, 30 l of buffer and 20 l of Anti-Biotin Microbeads (Miltenyi Biotec Inc.) per 10 7 cells were added. The cell suspension was incubated for another 15 min at 4 to 8°C, washed with 10 to 20 times the labeling volume, and then centrifuged at 300g for 5 min. The cell pellet was finally resuspended in 500 l of buffer per 1 ϫ 10 8 cells, and passed through the prerinsed LS column (Miltenyi Biotec Inc.), followed by four washes of the column with 3 ml of buffer. The entire effluent containing the purified B cell fraction was collected. The purity of the isolated B cells was evaluated using flow cytometry to enumerate the percentage of CD19 ϩ cells through directly labeling with FITC-conjugated anti-CD19 antibody (BD Biosciences). Real-Time Polymerase Chain Reaction. Total RNA was isolated from naive or LPS-activated cells using a SV Total RNA Isolation kit (Promega, Madison, WI). To synthesize cDNA, 1000 ng/ sample of total RNA was incubated with 600 ng of random primer (Invitrogen) in 10 l of endonuclease-free water at 70°C for 10 min, cooled on ice for 10 min, and reverse transcribed in 20 l of 1ϫ First-Strand Synthesis buffer (Invitrogen), containing 0.2 mM deoxynucleotide triphosphates, 10 mM dithiothreitol, and 200 units of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42°C for 60 min, and the reaction was stopped by incubation at 75°C for 15 min. Real-time polymerase chain reaction (PCR) detection was performed using TaqMan primers and probes Isolation of Pax5a cDNA. cDNA was obtained by PCR amplification of total RNA isolated from CH12.LX cells. Total RNA was extracted with the SV Total RNA Isolation kit (Promega), and firststrand cDNA was synthesized by reverse transcription (RT) using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). PCR was carried out as follows: one initial 2-min denaturing step at 94°C followed by denaturation for 30 s at 94°C, annealing for 30 s at 58°C, and extension for 90 s at 72°C during 30 cycles in the presence of Pfu polymerase (Stratagene, La Jolla, CA) under the conditions suggested by the supplier (at a final primer concentration of 0.5 M). Sequences of the primers designed for this PCR reaction were as follows: 5Ј-CTGTCCATTTCATCAAGTCCTGA-3Ј and 5Ј-ACC-GTCACTACCCTCAGAG-3Ј. The resulting PCR product of 1.2 kilobase was separated in 1% agarose gel electrophoresis in 1ϫ TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3) and stained with ethidium bromide at 1 g/ml. The PCR fragment was eluted from agarose using the QIAquick gel extraction kit (QIAGEN, Valencia, CA). Cloning and Ectopic Expression for Pax5a cDNA. Pax5a cDNA, generated as described above, was inserted into the pcDNA 3.1 V5-His-TOPO vector (Invitrogen) according to the manufacturer's protocol. The new Pax5a-V5-His tag vector sequence was confirmed by sequencing. Additional PCR reactions, in which Pax5a-V5-His vector was used as template, were performed to generate a new Pax5a expression plasmid, Pax5a-V5-His-GFP. The primer used for 5Ј-end cloning was as follows: 5Ј-CTC ACT ATA GGG AGA TCT AAG CTG GCT AGT-3Ј (BglII restriction site is underlined); and the primer used for the 3Јend cloning was as follows: 5Ј-TGA TCA GCG GGT TTA AAA GCT TTG GGA TGG TG-3Ј (HindIII restriction site is underlined). PCR products of 1.38 kilobase were obtained and isolated as described above. The Pax-5a-V5-His PCR fragment was then cloned into the vector phCMV-C-GFP (Genlantis, San Diego, CA) by a standard ligation reaction using T4-DNA ligase (New England Biolabs, Ipswich, MA). The phCMV vector from Genlantis contains an optimized cytomegalovirus promoter and intron sequences and a kanamycin/neomycin resistance gene for generation of stable cell lines. Transfection of Pax5a-V5-His-GFP. CH12.LX cells (2.5 ϫ 10 6 ) were transfected with 5 g of each plasmid using Amaxa Nucleofector (Amaxa Inc., Gaithersburg, MD). Cells, DNA, and 100 l of 90:20 (solution V supplement) were mixed and electroporated using program A-20 following the manufacturer's recommendations. A total of TCDD Impairment of B Cell Differentiation by Altered Pax5 465 2.5 ϫ 10 7 cells was used in each experiment. The backbone plasmid, phCMV-C-GFP, was used as a control in all of the transfection experiments. After electroporation, cells were incubated at 37°C in an atmosphere of 5% CO 2 for 8 h. Eight hours after transfection, cells were collected and sorted by flow cytometry for green fluorescent protein (GFP)-expressing cells. Cells that showed fluorescence above the level of fluorescence of naive CH12.LX cells were isolated using a BD FACSCalibur. The isolated cells were counted and LPS-activated for assessments of IgM secretion as determined by enzymelinked immunosorbent assay (ELISA). An aliquot of the sorted cells was also used to prepare protein extracts to determine endogenous/ ectopic Pax5a levels by Western blotting and by flow cytometry. IgM ELISA. IgM ELISA was performed as described previously Western Blotting. Proteins were extracted from transfected cells before and after sorting by flow cytometry. Protein extracts were resolved on 4 to 12% Nu-Page gradient gel (Invitrogen) and then transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Immunoblotting was performed using anti-␤-actin and anti-Pax5 antibodies (Santa Cruz Biotechnology, Inc.). SuperSignal West Pico reagent (Pierce, Rockford, IL) was used for protein detection. Statistical Analysis of Data. Mean Ϯ S.E. was determined for each treatment group of a given PCR or ELISA experiment. Statistical differences between groups in each experiment were determined by a two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for PCR experiments and by a one-way ANOVA followed by Bonferroni's post hoc test for ELISA experiments. Results TCDD Decreases the Levels of XBP-1 in LPS-Activated B Cells. Previous studies in LPS-activated CH12.LX cells demonstrated robust suppression of the humoral IgM response by TCDD, which correlated with a marked reduction in XBP-1 protein levels Additional studies were performed to determine whether LPS and/or TCDD treatment affects the post-transcriptional modification of XBP-1. Transcriptional activity of XBP-1 is known to be enhanced by a splicing event, which removes a 26-nucleotide-long fragment from XBP-1 mRNA, resulting in an open reading frame shift and yielding a larger XBP-1 466 Schneider et al. protein that is more potent as a transcription factor TCDD Attenuates Cell Surface MHC Class II DownRegulation in LPS-Activated CH12.LX Cells. An additional event closely associated with terminal differentiation of B cells is the down-regulation of MHC class II on the cell membrane. Levels of surface MHC class II in LPS-activated CH12.LX cells and in splenic B cells (i.e., CD19 ϩ ), in the presence and absence of TCDD, were monitored by flow cytometry. LPS activation induced a down-regulation of MHC class II on the CH12.LX cell surface between 24 and 72 h of culture compared with the time-matched naive control TCDD Attenuates the LPS-Induced Down-Regulation of Pax5 Protein in LPS-Activated CH12.LX Cells. We also examined the impact of TCDD on Pax5, a repressor of B cell differentiation. Expression of Pax5 protein was characterized in splenic B cells and CH12.LX cells by flow cytometry facilitating the evaluation of individual cells in contrast to prior Western blot studies that we performed exclusively in CH12.LX cells Characterization of Pax5 Isoforms in CH12.LX Cells. In light of the altered Pax5 expression observed by flow cytometry and by real-time PCR, studies were undertaken to further characterize the effect of TCDD on Pax5 regulation. In particular, we examined the role of alternative splicing in the deregulation of Pax5 by TCDD Additional studies performed in CH12.LX cells showed that the levels of amplicons I, II, and III were down-regulated by LPS between 24 and 72 h, as determined by real-time quantitative PC

2 nd Brazilian Consensus on Chagas Disease, 2015

Abstract Chagas disease is a neglected chronic condition with a high burden of morbidity and mortality. It has considerable psychological, social, and economic impacts. The disease represents a significant public health issue in Brazil, with different regional patterns. This document presents the evidence that resulted in the Brazilian Consensus on Chagas Disease. The objective was to review and standardize strategies for diagnosis, treatment, prevention, and control of Chagas disease in the country, based on the available scientific evidence. The consensus is based on the articulation and strategic contribution of renowned Brazilian experts with knowledge and experience on various aspects of the disease. It is the result of a close collaboration between the Brazilian Society of Tropical Medicine and the Ministry of Health. It is hoped that this document will strengthen the development of integrated actions against Chagas disease in the country, focusing on epidemiology, management, comprehensive care (including families and communities), communication, information, education, and research

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development’s (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security

Involvement of Blimp-1 and AP-1 Dysregulation in the 2,3,7,8-Tetrachlorodibenzo-p-dioxin–mediated Suppression of the IgM Response by B Cells

B cell differentiation and humoral immune responses are markedly suppressed by the persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The suppression of humoral immune responses by TCDD occurs by direct actions on the B cell and involves activation of the aryl hydrocarbon receptor. Transcriptional regulation of paired box gene 5 (Pax5), an important regulator of B cell differentiation, is altered by TCDD in concordance with the suppression of B cell differentiation and humoral immunoglobulin M response. We hypothesized that TCDD treatment leads to dysregulation of Pax5 transcription by interfering with the basic B cell differentiation mechanisms and aimed to determine the effects of TCDD on upstream regulators of Pax5. A critical regulator of B cell differentiation, B lymphocyte–induced maturation protein-1 (Blimp-1) acts as a transcriptional repressor of Pax5. In lipopolysaccharide (LPS)-activated murine B cell lymphoma, CH12.LX, Blimp-1 messenger RNA, and DNA-binding activity within the Pax5 promoter were suppressed by TCDD. Furthermore, LPS activation of CH12.LX cells upregulated DNA-binding activity of activator protein 1 (AP-1) at three responsive element–like motifs within the Blimp-1 promoter. TCDD treatment of LPS-activated CH12.LX cells suppressed AP-1 binding to these motifs between 24 and 72 h, in concordance with the suppression of Blimp-1 by TCDD. A more comprehensive analysis at 72 h demonstrated that the suppression of AP-1 binding within the Blimp-1 promoter by TCDD was concentration dependent. In summary, our findings link the TCDD-mediated suppression of Blimp-1 through AP-1 to the dysregulation of Pax5, which ultimately leads to the suppression of B cell differentiation and humoral immune responses

Incidence of congenital toxoplasmosis among infants born to HIV-coinfected mothers: case series and literature review

Introduction:There is a paucity of data on the occurrence of congenital toxoplasmosis in children born to mothers dually infected with HIV and Toxoplasma gondii.Objective:To evaluate aspects of the mother–infant pairs associated with vertical transmission of toxoplasmosis in women co-infected with HIV in a referral center for perinatally acquired infections in Belo Horizonte, Brazil.Methods:Descriptive study of HIV vertically exposed children, with congenital toxoplasmosis, followed at a referral center (cohort/Belo Horizonte). Prenatal and post-natal variables for the mother–infant pairs were evaluated. A literature review with no filtering for time and language was performed to identify reports of congenital toxoplasmosis in HIV vertically exposed children.Results:Among 2007 HIV vertically exposed children evaluated in the period from 1998 to 2011, 10 cases of congenital toxoplasmosis were identified (incidence: 0.5%, 95% confidence interval: 0.24–0.91). In searching the literature 22 additional cases in 17 reports were found. Combining the findings of our cohort with other reported cases, 50% (16/32) of congenital toxoplasmosis in HIV vertically exposed children were from Brazil. The cases of congenital toxoplasmosis in HIV vertically exposed children identified in Brazil occurred mainly in the post-Highly Active Antiretroviral Therapy era (p = 0.002) and presented a lower death rate (p = 0.003) than those from other countries. In the cohort/Belo Horizonte, HIV infection was identified mainly during gestation; T. gondii vertical transmission was observed in pregnant women with CD4+>500 cells/mm3 and latent toxoplasmosis. High rates of ocular lesions (87.5%) and central nervous system involvement (70%) were detected.Conclusions:The risk of vertical transmission of T. gondii in HIV-infected women is low and has been usually associated with maternal immunosuppression and elevated viral load. However, our findings of congenital toxoplasmosis in children born to HIV-infected mothers with latent toxoplasmosis and not immunosuppressed emphasize the need for careful follow-up in these cases