TLR Signaling Paralyzes Monocyte Chemotaxis through Synergized Effects of p38 MAPK and Global Rap-1 Activation

Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ∼0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP3 (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase

Genetic diversity of Mycobacterium tuberculosis isolates from central India

Background & objectives: There is a paucity of data available on genetic biodiversity of Mycobacterium tuberculosis isolates from central India. The present study was carried out on isolates of M. tuberculosis cultured from diagnostic clinical samples of patients from Bhopal, central India, using spoligotyping as a method of molecular typing. Methods: DNA was extracted from 340 isolates of M. tuberculosis from culture, confirmed as M. tuberculosis by molecular and biochemical methods and subjected to spoligotyping. The results were compared with the international SITVIT2 database. Results: Sixty five different spoligo international type (SIT) patterns were observed. A total of 239 (70.3%) isolates could be clustered into 25 SITs. The Central Asian (CAS) and East African Indian (EAI) families were found to be the two major circulating families in this region. SIT26/CAS1_DEL was identified as the most predominant type, followed by SIT11/EAI3_IND and SIT288/CAS[2]. Forty (11.8%) unique (non-clustered) and 61 (17.9%) orphan isolates were identified in the study. There was no significant association of clustering with clinical and demographic characteristics of patients. Interpretation & conclusions: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis

Transcriptional changes in the rat brain induced by repetitive transcranial magnetic stimulation

IntroductionTranscranial Magnetic Stimulation (TMS) is a noninvasive technique that uses pulsed magnetic fields to affect the physiology of the brain and central nervous system. Repetitive TMS (rTMS) has been used to study and treat several neurological conditions, but its complex molecular basis is largely unexplored.MethodsUtilizing three experimental rat models (in vitro, ex vivo, and in vivo) and employing genome-wide microarray analysis, our study reveals the extensive impact of rTMS treatment on gene expression patterns.ResultsThese effects are observed across various stimulation protocols, in diverse tissues, and are influenced by time and age. Notably, rTMS-induced alterations in gene expression span a wide range of biological pathways, such as glutamatergic, GABAergic, and anti-inflammatory pathways, ion channels, myelination, mitochondrial energetics, multiple neuron-and synapse-specific genes.DiscussionThis comprehensive transcriptional analysis induced by rTMS stimulation serves as a foundational characterization for subsequent experimental investigations and the exploration of potential clinical applications

A participatory intervention to improve the mental health of widows of injecting drug users in north-east India as a strategy for HIV prevention

BACKGROUND: Manipur and Nagaland, in the north-east of India, are classified as high prevalence states for HIV, and intravenous drug use is an important route of transmission. Most injecting drug users (IDUs) are men, an estimated 40% are married, and death rates have been high in the last five years, consequently the number of widows of IDUs has increased. Many of these widows and their children are HIV-infected and experience poor health, discrimination, and impoverishment; all factors likely to be compromising their mental health. People with poor mental health are more likely to engage in HIV risk behaviours. Mental health can be promoted by public health actions with vulnerable population groups. METHODS: We designed an intervention study to assess the feasibility and impact of a participatory action process to promote the mental health and well-being of widows of IDUs in Manipur and Nagaland, as a strategy for reducing the risk of engagement in HIV risk behaviours. This paper describes the background and rationale for the study, the intervention, and the study methods in detail. RESULTS: Pending analysis. CONCLUSION: This intervention study will make a significant contribution to the emerging evidence that supports associations between mental health and HIV. The concept of promoting mental health among women who are vulnerable to HIV infection or already infected as a strategy for HIV prevention in a development setting is breaking new ground

Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis

Background: Impaired signaling in the IFN-g/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. Objective: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. Methods: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-g/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. Results: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-g–induced gene expression, but we found impaired responses to IFN-g restimulation. Conclusion: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-g–mediated inflammationFil: Sampaio, Elizabeth P.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unidos. Instituto Oswaldo Cruz. Laboratorio de Leprologia; BrasilFil: Hsu, Amy P.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Pechacek, Joseph. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Hannelore I.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unidos. Erasmus Medical Center. Department of Medical Microbiology and Infectious Disease; Países BajosFil: Dias, Dalton L.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Paulson, Michelle L.. Clinical Research Directorate/CMRP; Estados UnidosFil: Chandrasekaran, Prabha. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Rosen, Lindsey B.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Carvalho, Daniel S.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unidos. Instituto Oswaldo Cruz, Laboratorio de Leprologia; BrasilFil: Ding, Li. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Vinh, Donald C.. McGill University Health Centre. Division of Infectious Diseases; CanadáFil: Browne, Sarah K.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Datta, Shrimati. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Allergic Diseases. Allergic Inflammation Unit; Estados UnidosFil: Milner, Joshua D.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Allergic Diseases. Allergic Inflammation Unit; Estados UnidosFil: Kuhns, Douglas B.. Clinical Services Program; Estados UnidosFil: Long Priel, Debra A.. Clinical Services Program; Estados UnidosFil: Sadat, Mohammed A.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Host Defenses. Infectious Diseases Susceptibility Unit; Estados UnidosFil: Shiloh, Michael. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: De Marco, Brendan. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: Alvares, Michael. University of Texas. Southwestern Medical Center. Division of Allergy and Immunology; Estados UnidosFil: Gillman, Jason W.. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: Ramarathnam, Vivek. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: de la Morena, Maite. University of Texas. Southwestern Medical Center. Division of Allergy and Immunology; Estados UnidosFil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moreira, Ileana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; ArgentinaFil: Uzel, Gulbu. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Johnson, Daniel. University of Chicago. Comer Children; Estados UnidosFil: Spalding, Christine. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Zerbe, Christa S.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados UnidosFil: Wiley, Henry. National Eye Institute. Clinical Trials Branch; Estados UnidosFil: Greenberg, David E.. University of Texas. Southwestern Medical Center. Division of Infectious Diseases; Estados UnidosFil: Hoover, Susan E.. University of Arizona. College of Medicine. Valley Fever Center for Excellence; Estados UnidosFil: Rosenzweig, Sergio D.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Host Defenses Infectious Diseases Susceptibility Unit; Estados Unidos. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Primary Immunodeficiency Clinic; Estados UnidosFil: Galgiani, John N.. University of Arizona. College of Medicine. Valley Fever Center for Excellence; Estados UnidosFil: Holland, Steven M.. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Clinical Infectious Diseases. Immunopathogenesis Section; Estados Unido

Some peace of mind: assessing a pilot intervention to promote mental health among widows of injecting drug users in north-east India

<p>Abstract</p> <p>Background</p> <p>HIV prevalence in north-east India is high and injecting drug use (IDU) is common. Due to HIV-related deaths there are increasing numbers of IDU widows, many of whom are HIV infected, and experiencing poor health, social isolation, discrimination and poverty, all factors likely to be compromising their mental health. There is increasing recognition of the links between HIV and mental health.</p> <p>Methods</p> <p>The aim of this study was to pilot a peer-facilitated, participatory action group (PAG) process and assess the impact of the intervention on the mental health of participants. The intervention consisted of 10 PAG meetings involving 74 IDU widows. Changes in quality of life (WHOQOL-BREF), mental health (GHQ12) and somatic symptoms were assessed. The value of the intervention from the perspective of the participants was captured using a qualitative evaluation method (Most Significant Change).</p> <p>Results</p> <p>Participants' quality of life, mental health and experience of somatic symptoms improved significantly over the course of the intervention, and the women told stories reflecting a range of 'significant changes'.</p> <p>Conclusion</p> <p>This pilot intervention study demonstrated that a participatory approach to mental health promotion can have a positive impact on the lives of vulnerable women, and the potential to contribute to HIV prevention. Further investigation is warranted.</p

Diagnostic utility of interferon-γ–induced protein of 10 kDa (IP-10) in tuberculous pleurisy

Tuberculous pleuritis (TP) is characterized by predominant Th1 immune response. We observed significantly high levels of interferon γ (IFN-γ) and chemokines such as IP-10, monokine induced by IFN-γ (MIG), interleukin 8 (IL-8), monocyte chemotactic protein (MCP)-1, and macrophage inflammatory protein (MIP)-1α in tuberculous pleural effusions. In the current study, we evaluated the diagnostic utility of IFN-γ–dependent chemokine especially IP-10. The receiver operating characteristics (ROC) curve analyses based on cytometric bead array values depicted high sensitivity only for IP-10 (76.3%) followed by IFN-γ (73.7%). The ELISA test further confirmed the significantly high levels of IFN-γ and IP-10 in TP. The ROC curve analysis again demonstrated high area under the curve (AUC) for IP-10 (0.966) than the referred diagnostic marker IFN-γ (0.930). The better sensitivity (84.2% for IFN-γ and 89.2% for IP-10) and equal specificity (95.7%) of IP- 10 assay compared with IFN-γ suggest that IP-10 is a potential diagnostic marker for evaluating TP

Benchmarking library recognition in tweets

Ministry of Education - Singapor

LPS induced phosphorylation of MAPKs.

<p>p44/42 ERK (<b>A1–A3</b>) and p38 MAPK (<b>B1–B3</b>) were phosphorylated after CXCL12 (20 nM) stimulation or LPS (2 ng/ml) treatment. Human monocytes (2×10⁶ cells for each time point) treated with or without CXCL12 were collected at 1.5, 2, 3 and 5 min. Monocytes (5×10⁶ for each time point) treated with or without LPS were collected 0, 15, 30, 60 and 100 min. Half of the cells treated with or without LPS and all the cells treated with or without CXCL12 were extracted with RIPA buffer and proteins were resolved by 4–20% gradient SDS-PAGE. Phospho-ERK and total ERKs were detected by immunoblotting with phospho-ERK specific mAb, E10 and rabbit antibody against total ERK (<b>A1</b> and <b>A2</b>). Reacting with anti phospho-p38 MAPK (T180/Y182) mAb and rabbit antibody against total p38 detected phospho-p38 and total p38 respectively (<b>B1</b> and <b>B2</b>). Immunoblots are representative of results with monocytes from 3 donors. The remaining half of the cells treated with or without were stained with a mixture of Alexa-647 E10 mAb against phospho ERK (T202/Y204) and Alexa-488 28B10 mAb against phospho-p38 MAPK (T180/Y182) and analyzed by flow cytometry. Ratios of MFVs for the respective phosho-MAPKs in LPS treated vs. untreated cells are plotted as histograms (n = 5, * p<0.03, ** p<0.01). The remaining cells were extracted with RIPA buffer and proteins resolved by 4–20% gradient SDS-PAGE. Phospho-ERK and total ERKs were detected by immunoblotting with phospho-ERK specific mAb, E10 and rabbit antibody against total ERK. Reacting with anti phospho-p38 MAPK (T180/Y182) mAb and rabbit antibody against total p38 detected phospho-p38 and total p38 respectively (A3 and B3) Immunoblots are representative of results with monocytes from 5 donors.</p

Simultaneous inhibition of Rap1 and p38 MAPK or PI3K inhibition reversed LPS induced block of F-actin polarization and chemotaxis in moncoytes.

<p><b>A</b>) Monocytes in RPMI with 5% FBS were treated with 20 µM Rap1 inhibitor GGTI-298 (Calbiochem, EMD Biosci Corp), 10 µM 42/44ERK MAPK inhibitor PD98059 (Cell signaling), 10 µM p38 MAPK inhibitor SB203580 (Tocris Corp), 20 µM GGTI-298+10 µM PD98059 and 20 µM GGTI-298+10 µM SB203580, or DMSO control for 30 min at 37°C. Cells were plated on cover slips and treated with or without LPS (2 ng/ml) at 37°C for 15 min, followed by stimulation with 20 nM CCL2 for 2 min. Cells were fixed and stained with phalloidin-488, and images were collected with Leica TCS SP5. <b>B</b>) 10⁷ monocytes were treated with inhibitors as described above for 30 min followed by a 15 min incubation with or without (control) LPS (2 ng/ml). ∼0.5×10⁶ LPS treated or untreated cells in 100 µl of RPMI with 1% FBS were loaded in the upper Transwell chambers (Nunc 5.0 µm) challenged with or without 20 nM CCL2 in the bottom chambers, and incubated at 37°C CO₂ incubator for 2 h. Chemotaxis was measured as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030404#s4" target="_blank">Methods</a>. Migrated cells for control and LPS treatments after respective inhibitors are plotted pair wise in the histograms (with error bars). n = 3, ^***p<0.01,^**p<0.04, ^*p<0.05. <b>C</b>) LPS induced Rap1 activation was inhibited in monocytes pretreated with GGTI-298 alone or with GGTI-298 and p38 MAPK inhibitor, SB203580, but not with SB203580 alone. Activated (GTP+) Rap1 was extracted from cytoplasmic extracts using a commercial pull-down kit followed by SDS/PAGE and imunoblotting with anti-Rap1 mAb. Numbers represent relative fraction (%) of Rapi that was activated (n = 3). <b>D</b>) PI3K inhibitors reversed LPS induced block of F-actin polarization in monocytes stimulated with CCL2. Monocytes in 5%FBS/RPMI were treated with 50 µM LY2940002, 1 µM wortmannin, or DMSO control for 30 min 37°C. Cells were plated on cover slips and treated with or without LPS (2 ng/ml) at 37°C for 20 min, followed by 20 nM CCL2 for 2 min. Cells were fixed and stained with phalloidin-488, and images were collected with Leica TCS SP5.</p