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Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2 fl/fl xCd4 cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The gut microbiome and intestinal immune system are engaged in a dynamic interplay that provides myriad benefits to host health. However, the microbiome can also elicit damaging inflammatory responses, and thus establishing harmonious immune-microbiome interactions is essential to maintain homeostasis. Gut microbes actively coordinate the induction of anti-inflammatory responses that establish these mutualistic interactions. Despite this, the microbial pathways that govern this dialogue remain poorly understood. We investigated the mechanisms through which the gut symbiont Bacteroides thetaiotaomicron exerts its immunomodulatory functions on murine- and human-derived cells. Our data reveal that B. thetaiotaomicron stimulates production of the cytokine IL-10 via secreted factors that are packaged into outer membrane vesicles, in a TLR2- and MyD88-dependent manner. Using a transposon mutagenesis-based screen, we identified a key role for the B. thetaiotaomicron-encoded NADH:ubiquinone oxidoreductase (NQR) complex, which regenerates NAD+ during respiration, in this process. Finally, we found that disruption of NQR reduces the capacity of B. thetaiotaomicron to induce IL-10 by impairing biogenesis of outer membrane vesicles. These data identify a microbial pathway with a previously unappreciated role in gut microbe-mediated immunomodulation that may be targeted to manipulate the capacity of the microbiome to shape host immunity., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis., (© 2022. The Author(s).)

Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and platelets. The latter is important since this protein may work as negative regulator of granule release. Thus, we sought to begin to understand the structure of this protein in platelets. Flow cytometric analysis of this protein using region-specific (N-terminus, central region and C-terminus) monoclonal antibodies was performed. Antibody to the central region gave the strongest shift among all three antibodies, with the C-terminus having intermediate shift and N-terminus minimal shift. Western blotting using the same antibodies showed similar binding of all antibodies to α-synuclein. These results suggest a similar arrangement of this protein in platelets as seen in neurons. Future studies ought to look at the role that each protein region plays in platelets., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A pandemic of historic impact, coronavirus disease 2019 (COVID-19) has potential consequences on the cardiovascular health of millions of people who survive infection worldwide. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, can infect the heart, vascular tissues, and circulating cells through ACE2 (angiotensin-converting enzyme 2), the host cell receptor for the viral spike protein. Acute cardiac injury is a common extrapulmonary manifestation of COVID-19 with potential chronic consequences. This update provides a review of the clinical manifestations of cardiovascular involvement, potential direct SARS-CoV-2 and indirect immune response mechanisms impacting the cardiovascular system, and implications for the management of patients after recovery from acute COVID-19 infection.

Cardiac sarcoidosis is a component of an often multiorgan granulomatous disease of still uncertain cause. It is being recognized with increasing frequency, mainly as the result of heightened awareness and new diagnostic tests, specifically cardiac magnetic resonance imaging and 18 F-fluorodeoxyglucose positron emission tomography scans. The purpose of this case-based review is to highlight the potentially life-saving importance of making the early diagnosis of cardiac sarcoidosis using these new tools and to provide a framework for the optimal care of patients with this disease. We will review disease mechanisms as currently understood, associated arrhythmias including conduction abnormalities, and atrial and ventricular tachyarrhythmias, guideline-directed diagnostic criteria, screening of patients with extracardiac sarcoidosis, and the use of pacemakers and defibrillators in this setting. Treatment options, including those related to heart failure, and those which may help clarify disease mechanisms are included.

Background: Accumulation of iron is a consistent feature of Alzheimer's disease (AD) brains. The underlying cause, however, remains debatable., Objective: To explore whether local hepcidin synthesized by brain cells contributes to iron accumulation in AD brains., Methods: Brain tissue from the cingulate cortex of 33 cases of AD pre-assigned to Braak stage I-VI, 6 cases of non-dementia, and 15 cases of non-AD dementia were analyzed for transcriptional upregulation of hepcidin by RT-qPCR and RT-PCR. Change in the expression of ferritin, ferroportin (Fpn), microglial activation marker Iba1, IL-6, and TGFβ2 was determined by western blotting. Total tissue iron was determined by colorimetry., Results: Significant transcriptional upregulation of hepcidin was observed in Braak stage III-VI relative to Braak stage I and II, non-AD dementia, and non-dementia samples. Ferritin was increased in Braak stage V, and a significant increase in tissue iron was evident in Braak stage III-VI. The expression of Iba1 and IL-6 was also increased in Braak stage III-VI relative to Braak stage I and II and non-AD dementia samples. Amyloid-β plaques were absent in most Braak stage I and II samples, and present in Braak stage III-VI samples with few exceptions., Conclusion: These observations suggest that upregulation of brain hepcidin is mediated by IL-6, a known transcriptional activator of hepcidin. The consequent downregulation of Fpn on neuronal and other cells results in accumulation of iron in AD brains. The increase in hepcidin is disease-specific, and increases with disease progression, implicating AD-specific pathology in the accumulation of iron.

This study investigated responses to Toll-like receptor 2 (TLR2)-driven extracellular signal-related kinase (ERK) signaling in dendritic cells (DCs) versus macrophages. TLR2 signaling was induced with Pam 3 Cys-Ser-Lys 4 , and the role of ERK signaling was interrogated pharmacologically with MEK1/2 inhibitor U0126 or genetically with bone marrow-derived macrophages or DCs from Tpl2 -/- mice. We assessed cytokine production via enzyme-linked immunosorbent assay (ELISA) or V-Plex, and mRNA levels were assessed via reverse transcriptase quantitative PCR (qRT-PCR). In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited interleukin 10 (IL-10) expression and increased expression of the p40 subunit shared by IL-12 and IL-23 (IL-12/23p40). In DCs, blockade of ERK signaling similarly inhibited IL-10 expression but decreased IL-12/23p40 expression, which is opposite to the effect of ERK signaling blockade on IL-12/23p40 in macrophages. This difference in IL-12/23p40 regulation correlated with the differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12 family members, including IL-12 and IL-23. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and DCs, potentially regulating their distinctive functions in the immune system. ERK-mediated suppression of IL-12/23p40 in macrophages may prevent excessive inflammation and associated tissue damage following TLR2-stimulation, while ERK-mediated induction of IL-12/23p40 in DCs may promote priming of T helper 1 (Th1) responses. A greater understanding of the role that ERK signaling plays in different immune cell types may inform the development of host-directed therapy and optimal adjuvanticity for a number of infectious pathogens., (Copyright © 2020 American Society for Microbiology.)

Coronavirus disease 2019 (COVID-19) has presented substantial challenges to patient care and impacted health care delivery, including cardiac electrophysiology practice throughout the globe. Based upon the undetermined course and regional variability of the pandemic, there is uncertainty as to how and when to resume and deliver electrophysiology services for arrhythmia patients. This joint document from representatives of the Heart Rhythm Society, American Heart Association, and American College of Cardiology seeks to provide guidance for clinicians and institutions reestablishing safe electrophysiological care. To achieve this aim, we address regional and local COVID-19 disease status, the role of viral screening and serologic testing, return-to-work considerations for exposed or infected health care workers, risk stratification and management strategies based on COVID-19 disease burden, institutional preparedness for resumption of elective procedures, patient preparation and communication, prioritization of procedures, and development of outpatient and periprocedural care pathways., (Copyright © 2020 The Heart Rhythm Society, the American Heart Association, Inc., and the American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: SARS-CoV-2 enters cells by binding of its spike protein to angiotensin-converting enzyme 2 (ACE2). Angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) have been reported to increase ACE2 expression in animal models, and worse outcomes are reported in patients with co-morbidities commonly treated with these agents, leading to controversy during the COVID-19 pandemic over whether these drugs might be helpful or harmful., Methods: Animal, in vitro and clinical data relevant to the biology of the renin-angiotensin system (RAS), its interaction with the kallikrein-kinin system (KKS) and SARS-CoV-2, and clinical studies were reviewed., Findings and Interpretation: SARS-CoV-2 hijacks ACE2to invade and damage cells, downregulating ACE2, reducing its protective effects and exacerbating injurious Ang II effects. However, retrospective observational studies do not show higher risk of infection with ACEI or ARB use. Nevertheless, study of the RAS and KKS in the setting of coronaviral infection may yield therapeutic targets., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Coronavirus disease 2019 (COVID-19) has presented substantial challenges to patient care and impacted health care delivery, including cardiac electrophysiology practice throughout the globe. Based upon the undetermined course and regional variability of the pandemic, there is uncertainty as to how and when to resume and deliver electrophysiology services for arrhythmia patients. This joint document from representatives of the Heart Rhythm Society, American Heart Association, and American College of Cardiology seeks to provide guidance for clinicians and institutions reestablishing safe electrophysiological care. To achieve this aim, we address regional and local COVID-19 disease status, the role of viral screening and serologic testing, return-to-work considerations for exposed or infected health care workers, risk stratification and management strategies based on COVID-19 disease burden, institutional preparedness for resumption of elective procedures, patient preparation and communication, prioritization of procedures, and development of outpatient and periprocedural care pathways., (Copyright © 2020 The Heart Rhythm Society, the American Heart Association, Inc., and the American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: To report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals., Methods: Postmortem examination was completed following Centers for Disease Control and Prevention public guidelines. Numerous formalin-fixed paraffin-embedded (FFPE) tissue types from each case were surveyed for SARS-CoV-2 RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). SARS-CoV-2 viral genome was sequenced by next-generation sequencing (NGS) from FFPE lung tissue blocks., Results: Postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. Viral RNA was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qRT-PCR method. RNA sequencing using NGS in case 1 revealed mutations most consistent with Western European Clade A2a with ORF1a L3606F mutation., Conclusions: SARS-CoV-2 testing and viral sequencing can be performed from FFPE tissue. Detection and sequencing of SARS-CoV-2 in combination with morphological findings from postmortem tissue examination can aid in gaining a better understanding of the virus's pathophysiologic effects on human health., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Coronavirus disease 2019 (COVID-19) has presented substantial challenges to patient care and impacted healthcare delivery, including cardiac electrophysiology practice throughout the globe. Based upon the undetermined course and regional variability of the pandemic, there is uncertainty as to how and when to resume and deliver electrophysiology services for patients with arrhythmia. This joint document from representatives of the Heart Rhythm Society, American Heart Association, and American College of Cardiology seeks to provide guidance for clinicians and institutions reestablishing safe electrophysiological care. To achieve this aim, we address regional and local COVID-19 disease status, the role of viral screening and serological testing, return-to-work considerations for exposed or infected health care workers, risk stratification and management strategies based on COVID-19 disease burden, institutional preparedness for resumption of elective procedures, patient preparation and communication, prioritization of procedures, and development of outpatient and periprocedural care pathways.

Objectives: Type II heparin-induced thrombocytopenia (HIT) is mediated by formation of antibodies to platelet factor 4 (PF4)-heparin complexes. We evaluated anti-PF4-heparin-negative samples for the presence of additional anti-platelet and anti-red blood cell (RBC) antibodies using whole-cell platelet/ RBC ELISAs we developed., Methods: Seventy-three samples tested for anti-PF4-heparin by ELISA were included: 62 tested negative, 9 tested positive, and 2 had equivocal results. Plasma specimens from healthy donors were used as controls., Results: 100% (9/9) anti-PF4-positive samples had anti-platelet antibodies detected by whole-cell platelet ELISA. 42.2% (27/64) anti-PF4-heparin-negative samples were negative for anti-platelet and anti-RBC antibodies. 32.8% (21/64) negative samples showed reactivity to both platelets and RBC; 12.5% (8/64) negative samples were each reactive with either platelet or RBC ELISA, respectively. Additionally, two samples that tested equivocal by anti-PF4-heparin ELISA had antibodies to both platelets and RBC by whole-cell ELISA., Conclusions: Our study suggests that patients with thrombocytopenia testing negative for anti-PF4-heparin may still harbor antibodies to platelets. However, additional research is needed to determine the significance of these antibodies. Nevertheless, these findings may encourage clinicians to further investigate patients with possible immune-mediated etiologies of thrombocytopenia and anemia., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

People living with HIV/AIDS on antiretroviral therapy have increased risk of non-AIDS-defining cancers (NADCs). However, the underlying mechanism for development and progression of certain NADCs remains obscure. Here we show that exosomes released from HIV-infected T cells and those purified from blood of HIV-positive patients stimulate proliferation, migration and invasion of oral/oropharyngeal and lung cancer cells. The HIV transactivation response (TAR) element RNA in HIV-infected T-cell exosomes is responsible for promoting cancer cell proliferation and inducing expression of proto-oncogenes and Toll-like receptor 3 (TLR3)-inducible genes. These effects depend on the loop/bulge region of the molecule. HIV-infected T-cell exosomes rapidly enter recipient cells through epidermal growth factor receptor (EGFR) and stimulate ERK1/2 phosphorylation via the EGFR/TLR3 axis. Thus, our findings indicate that TAR RNA-containing exosomes from HIV-infected T cells promote growth and progression of particular NADCs through activation of the ERK cascade in an EGFR/TLR3-dependent manner.

Effects of α-synuclein deficiency on cellular blood components have not been extensively investigated. This study evaluated ultrastructural changes of leukocytes in α-synuclein knockout (KO) mice using electron microscopy (EM). The following ultrastructural characteristics were quantified in leukocytes: mitochondria, primary granules, specific granules (SG), Golgi apparatus (GA), inclusions, rough-endoplasmic reticulum (RER), smooth-endoplasmic reticulum (SER), and cellular projections (CP). EM showed increased numbers or amounts of SG, inclusions, and SER in KO group (5.3 ± 4.5 in WT vs. 14.1 ± 10.3 in KO, p = 0.02; 0.4 ± 0.9 in WT vs. 3.2 ± 2.8 in KO, p = 0.007; and 7.7 ± 6.7 in WT vs. 17.7 ± 12.2 in KO, p = 0.03, respectively). Although CP number was not significantly different between the two groups (13.4 ± 5.3 in WT vs. 16.3 ± 7.5 in KO, p = 0.32), their size and shapes were altered in KO mice. Notably, findings occurred in the setting of significant lymphopenia. α-Synuclein deficiency leads to changes in size and shape of secretory particles and increases in SER, SG, and inclusions, indicating a potential role for α-synuclein in vesicular trafficking in leukocytes. Further studies are needed to elucidate functions mediated by α-synuclein., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis causes persistent infection due to its ability to evade host immune responses. M. tuberculosis induces Toll-like receptor 2 (TLR2) signaling, which influences immune responses to M. tuberculosis TLR2 agonists expressed by M. tuberculosis include lipoproteins (e.g., LprG), the glycolipid phosphatidylinositol mannoside 6 (PIM6), and the lipoglycan lipomannan (LM). Another M. tuberculosis lipoglycan, mannose-capped lipoarabinomannan (ManLAM), lacks TLR2 agonist activity. In contrast, PILAM, from Mycobacterum smegmatis , does have TLR2 agonist activity. Our understanding of how M. tuberculosis lipoproteins and lipoglycans interact with TLR2 is limited, and binding of these molecules to TLR2 has not been measured directly. Here, we directly measured M. tuberculosis lipoprotein and lipoglycan binding to TLR2 and its partner receptor, TLR1. LprG, LAM, and LM were all found to bind to TLR2 in the absence of TLR1, but not to TLR1 in the absence of TLR2. Trimolecular interactions were revealed by binding of TLR2-LprG or TLR2-PIM6 complexes to TLR1, whereas binding of TLR2 to TLR1 was not detected in the absence of the lipoprotein or glycolipid. ManLAM exhibited low affinity for TLR2 in comparison to PILAM, LM, and LprG, which correlated with reduced ability of ManLAM to induce TLR2-mediated extracellular-signal-regulated kinase (ERK) activation and tumor necrosis factor alpha (TNF-α) secretion in macrophages. We provide the first direct affinity measurement and kinetic analysis of M. tuberculosis lipoprotein and lipoglycan binding to TLR2. Our results demonstrate that binding affinity correlates with the functional ability of agonists to induce TLR2 signaling., (Copyright © 2018 American Society for Microbiology.)

Objectives: In platelets, α-synuclein is important in calcium-dependent granule release. Notably, cells release α-synuclein in setting of cell damage or death. Therefore, we investigated α-synuclein levels in plasma of single donor platelet (SDP) units during storage., Methods: Aliquots were obtained from same SDP units for 7 days from day of donation. Additionally, randomly sampled SDP units at same storage time points were also assayed by enzyme-linked immunosorbent assay., Results: α-Synuclein in SDP plasma increased continuously over time at each assayed time point. Significant increases were measured on day 3 (11.7 ± 9.6 ng/mL, P = 0.025), day 5 (15.3 ± 5.9 ng/mL, P = 0.002), and highest on day 7 (23.7 ± 5.6 ng/mL, P < 0.0001) compared to day 0 (1.1 ± 0.8 ng/mL). Similar significant results were obtained in randomly sampled SDP units at same corresponding time points. Flow cytometry showed that platelets had strong expression of α-synuclein and lacked expression of other synucleins., Conclusions: Increases of α-synuclein during SDP storage is a steady and continuous process that increases with time. Our findings indicate that α-synuclein may represent a biomarker of platelet biological state during storage. Further research will be needed to determine how α-synuclein increases correlate with platelets' function., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose-capped lipoarabinomannan (ManLAM) inhibits CD4 + T-cell activation by inhibiting proximal T-cell receptor (TCR) signaling when activated by anti-CD3. To understand the impact of ManLAM on CD4 + T-cell function when both the TCR-CD3 complex and major costimulator CD28 are engaged, we performed label-free quantitative MS and network analysis. Mixed-effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti-CD3- and anti-CD28-activated CD4 + T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF-κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x-Akt-mTOR pathway. Thus, ManLAM induced global changes in the CD4 + T-cell proteome by affecting Akt-mTOR signaling, resulting in broad functional impairment of CD4 + T-cell activation beyond inhibition of proximal TCR-CD3 signaling., (© 2017 The Authors, Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Physician-scientists are needed to continue the great pace of recent biomedical research and translate scientific findings to clinical applications. MD-PhD programs represent one approach to train physician-scientists. MD-PhD training started in the 1950s and expanded greatly with the Medical Scientist Training Program (MSTP), launched in 1964 by the National Institute of General Medical Sciences (NIGMS) at the National Institutes of Health. MD-PhD training has been influenced by substantial changes in medical education, science, and clinical fields since its inception. In 2014, NIGMS held a 50th Anniversary MSTP Symposium highlighting the program and assessing its outcomes. In 2016, there were over 90 active MD-PhD programs in the United States, of which 45 were MSTP supported, with a total of 988 trainee slots. Over 10,000 students have received MSTP support since 1964. The authors present data for the demographic characteristics and outcomes for 9,683 MSTP trainees from 1975-2014. The integration of MD and PhD training has allowed trainees to develop a rigorous foundation in research in concert with clinical training. MSTP graduates have had relative success in obtaining research grants and have become prominent leaders in many biomedical research fields. Many challenges remain, however, including the need to maintain rigorous scientific components in evolving medical curricula, to enhance research-oriented residency and fellowship opportunities in a widening scope of fields targeted by MSTP graduates, to achieve greater racial diversity and gender balance in the physician-scientist workforce, and to sustain subsequent research activities of physician-scientists.

Mycobacterium tuberculosis utilizes multiple mechanisms to evade host immune responses, and inhibition of effector CD4 + T cell responses by M. tuberculosis may contribute to immune evasion. TCR signaling is inhibited by M. tuberculosis cell envelope lipoglycans, such as lipoarabinomannan and lipomannan, but a mechanism for lipoglycans to traffic from M. tuberculosis within infected macrophages to reach T cells is unknown. In these studies, we found that membrane vesicles produced by M. tuberculosis and released from infected macrophages inhibited the activation of CD4 + T cells, as indicated by reduced production of IL-2 and reduced T cell proliferation. Flow cytometry and Western blot demonstrated that lipoglycans from M. tuberculosis -derived bacterial vesicles (BVs) are transferred to T cells, where they inhibit T cell responses. Stimulation of CD4 + T cells in the presence of BVs induced expression of GRAIL, a marker of T cell anergy; upon restimulation, these T cells showed reduced ability to proliferate, confirming a state of T cell anergy. Furthermore, lipoarabinomannan was associated with T cells after their incubation with infected macrophages in vitro and when T cells were isolated from lungs of M. tuberculosis -infected mice, confirming the occurrence of lipoarabinomannan trafficking to T cells in vivo. These studies demonstrate a novel mechanism for the direct regulation of CD4 + T cells by M. tuberculosis lipoglycans conveyed by BVs that are produced by M. tuberculosis and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids.

Objective: To assess CD4 T-cell responsiveness to IL-7 and IFN-α in HIV-infected patients who experience poor recovery of CD4 T-cell counts during therapy (immune failure patients)., Design: Responses to IL-7 and IFN-α were compared between HIV-infected immune failure (CD4 cell counts <379 cells/μl) patients and immune success (CD4 cell counts >500 cells/μl) as well as healthy control patients., Methods: Flow cytometry was used to assess peripheral blood mononuclear cells for IL-7-induced proliferation, CD25 expression, and signaling (signal transducer and activator of transcription 5 phosphorylation and Akt phosphorylation) in CD4 T cells. Freshly isolated cells were characterized by expression of IL-7Rα (CD127) among CD4 T-cell maturation subsets by flow cytometry and sorted CD3 T cells were assessed for expression of IFN-α and interferon stimulated genes (2'-5'-oligoadenylate synthetase-1 and myxovirus resistance A protein) by quantitative real-time PCR. Responses to IFN-α were assessed by induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of IL-7-induced CD4 T-cell proliferation., Results: IL-7-induced proliferation and CD25 expression were decreased in CD4 T cells from immune failure patients. CD127 expressing CD4 T cells were decreased, whereas expression of 2'-5'-oligoadenylate synthetase-1, myxovirus resistance A protein, and IFN-α mRNA were increased in total CD3 T cells from immune failure patients. CD127 expression correlated with CD25 induction but not proliferation, whereas T-cell IFN-α mRNA was associated with reduced proliferation in CD4 T cells from immune failure patients. IFN-α-mediated induction of signal transducer and activator of transcription 1 phosphorylation and inhibition of proliferation were not diminished in CD4 T cells from immune failure patients., Conclusion: IL-7 responsiveness is impaired in immune failure patients and may be related to expression of CD127 and IFN-α., Competing Interests: There are no conflicts of interest.

Exosomes and other extracellular microvesicles (ExMVs) have important functions in intercellular communication and regulation. During the course of infection, these vesicles can convey pathogen molecules that serve as antigens or agonists of innate immune receptors to induce host defense and immunity, or that serve as regulators of host defense and mediators of immune evasion. These molecules may include proteins, nucleic acids, lipids, and carbohydrates. Pathogen molecules may be disseminated by incorporation into vesicles that are created and shed by host cells, or they may be incorporated into vesicles shed from microbial cells. Involvement of ExMVs in the induction of immunity and host defense is widespread among many pathogens, whereas their involvement in immune evasion mechanisms is prominent among pathogens that establish chronic infection and is found in some that cause acute infection. Because of their immunogenicity and enrichment of pathogen molecules, exosomes may also have potential in vaccine preparations and as diagnostic markers. Additionally, the ability of exosomes to deliver molecules to recipient cells raises the possibility of their use for drug/therapy delivery. Thus, ExMVs play a major role in the pathogenesis of infection and provide exciting potential for the development of novel diagnostic and therapeutic approaches.

Alpha-synuclein is highly expressed in the central nervous system and plays an important role in pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia. Previous studies have demonstrated the expression of α-synuclein in hematopoietic elements and peripheral blood mononuclear cells, although its roles in hematopoiesis and adaptive immunity are not studied. Using an α-synuclein knock out (KO) mouse model, we have recently shown that α-synuclein deficiency is associated with a mild defect in late stages of hematopoiesis. More importantly, we demonstrated a marked defect in B lymphocyte development and IgG, but not IgM production in these mice. Here we show a marked defect in development of T lymphocytes in α-synuclein KO mice demonstrated by a significant increase in the number of CD4 and CD8 double negative thymocytes and significant decreases in the number of CD4 single positive and CD8 single positive T cells. This resulted in markedly reduced peripheral T lymphocytes. Interestingly, splenic CD4(+) and CD8(+) T cells that developed in α-synuclein KO mice had a hyperactivated state with higher expression of early activation markers and increased IL-2 production. Moreover, splenic CD4(+) T cells from α-synuclein KO mice produced lower levels of IL-4 upon antigenic stimulation suggesting a defective Th2 differentiation. Our data demonstrate an important role for α-synuclein in development of T lymphocytes and regulation of their phenotype and function., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Mycobacterium tuberculosis cell wall glycolipid, lipoarabinomannan, can inhibit CD4(+) T cell activation by downregulating the phosphorylation of key proximal TCR signaling molecules: Lck, CD3ζ, ZAP70, and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an Ag encounter, yet remain viable and hyporesponsive. We tested whether mannose-capped lipoarabinomannan (LAM)-induced inhibition of CD4(+) T cell activation resulted in CD4(+) T cell anergy. The presence of LAM during primary stimulation of P25 TCR-transgenic murine CD4(+) T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25 TCR-transgenic CD4(+) T cells primed in the presence of LAM also exhibited decreased response upon restimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hyporesponsiveness to restimulation was not due to apoptosis, generation of Foxp3-positive regulatory T cells, or inhibitory cytokines. Acquisition of the anergic phenotype correlated with upregulation of gene related to anergy in lymphocytes (GRAIL) protein in CD4(+) T cells. Inhibition of human CD4(+) T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA-mediated knockdown of GRAIL before LAM treatment abrogated LAM-induced hyporesponsiveness. In addition, exogenous IL-2 reversed defective proliferation by downregulating GRAIL expression. These results demonstrate that LAM upregulates GRAIL to induce anergy in Ag-reactive CD4(+) T cells. Induction of CD4(+) T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2(flox/flox) Lyz2(Cre/Cre) mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice was enriched for CD11b+ myeloid cells, CD11b(hi) Gr-1(hi) neutrophils, Lin- c-Kit+ Sca-1+ hematopoietic stem cells, and Lin- c-Kit+ CD34+ CD16/32+ granulocyte-macrophage progenitors. Culture of bone marrow Lin- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Mycobacterium tuberculosis survives within macrophages and employs immune evasion mechanisms to persist in the host. Protective T helper type 1 (Th1) responses are induced, and the immune response in most individuals is sufficient to restrict M. tuberculosis to latent infection, but most infections are not completely resolved. As T cells and macrophages respond, a balance is established between protective Th1-associated and other proinflammatory cytokines, such as interleukin-12 (IL-12), interferon gamma (IFN-γ), and tumor necrosis factor alpha, and anti-inflammatory cytokines, such as IL-10. The mechanisms by which M. tuberculosis modulates host responses to promote its survival remain unclear. In these studies, we demonstrate that M. tuberculosis induction of IL-10, suppression of IL-12, and inhibition of class II major histocompatibility complex (MHC-II) molecules in infected macrophages are all driven by Toll-like receptor 2 (TLR2)-dependent activation of the extracellular signal-regulated kinases (ERK). Elimination of ERK signaling downstream of TLR2 by pharmacologic inhibition with U0126 or genetic deletion of Tpl2 blocks IL-10 secretion and enhances IL-12 p70 secretion. We demonstrate that M. tuberculosis regulation of these pathways in macrophages affects T cell responses to infected macrophages. Thus, genetic blockade of the ERK pathway in Tpl2(-/-) macrophages enhances Th1 polarization and IFN-γ production by antigen-specific CD4(+) T cells responding to M. tuberculosis infection. These data indicate that M. tuberculosis and its potent TLR2 ligands activate ERK signaling in macrophages to promote anti-inflammatory macrophage responses and blunt Th1 responses against the pathogen., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Persistent type I IFN production occurs during chronic viral infections, such as HIV disease. As type I IFNs have antiproliferative activity, it is possible that chronic exposure to these cytokines could adversely affect T cell homeostasis. We investigated the capacity of IFN-α to impair T cell proliferation induced by the homeostatic cytokine, IL-7, or another common γ-chain cytokine, IL-2, in cells from healthy human donors. We found that IL-7- or IL-2-induced proliferation of CD4(+) T cells was partially inhibited in the presence of IFN-α. The CD4(+) T cells that were exposed to IFN-α also displayed attenuated induction of IL-2 and CD40L following TCR stimulation. Analyses of signaling pathways indicated that IL-7 and IL-2 induced a delayed and sustained P-Akt signal that lasted for several days and was partially inhibited by IFN-α. In contrast, IL-7-induced P-STAT5 was not affected by IFN-α. Furthermore, IFN-α had no detectable effect on P-Akt that was induced by the chemokine SDF-1. Both inhibitors of P-Akt and P-STAT5 blocked IL-7-induced T cell proliferation, confirming that both signaling pathways are important for IL-7-induced T cell proliferation. These results demonstrate that IFN-α can selectively inhibit cytokine-induced P-Akt as a potential mechanism to disrupt homeostasis of T lymphocytes., (© Society for Leukocyte Biology.)

The dwindling repertoire of antibiotics to treat methicillin-resistant Staphylococcus aureus (MRSA) calls for novel treatment options. Quorum-quenching agents offer an alternative or an adjuvant to antibiotic therapy. Three biaryl hydroxyketone compounds discovered previously (F1, F12, and F19; G. Yu, D. Kuo, M. Shoham, and R. Viswanathan, ACS Comb Sci 16:85-91, 2014) were tested for efficacy in MRSA-infected animal models. Topical therapy of compounds F1 and F12 in a MRSA murine wound infection model promotes wound healing compared to the untreated control. Compounds F1, F12, and F19 afford significant survival benefits in a MRSA insect larva model. Combination therapy of these quorum-quenching agents with cephalothin or nafcillin, antibiotics to which MRSA is resistant in monotherapy, revealed additional survival benefits. The quorum-quenching agents sensitize MRSA to the antibiotic by a synergistic mode of action that also is observed in vitro. An adjuvant of 1 μg/ml F1, F12, or F19 reduces the MIC of nafcillin and cephalothin about 50-fold to values comparable to those for vancomycin, the antibiotic often prescribed for MRSA infections. These findings suggest that it is possible to resurrect obsolete antibiotic therapies in combination with these novel quorum-quenching agents., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein(-/-) (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220(+)IgM(+) B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×10(5) vs. KO: 27±5×10(5)). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response., (Copyright © 2014 Elsevier GmbH. All rights reserved.)