Mast cells modulate early responses to Mycobacterium bovis Bacillus Calmette-Guerin by phagocytosis and formation of extracellular traps.

The early interactions between the vaccine Mycobacterium bovis Bacillus Calmette Guerin (BCG) and host peripheral innate immune cells like Mast cells (MCs) may pave the way for generating appropriate protective innate and adaptive immune responses. Mice on administration of BCG by intratracheal instillation showed a massive increase in MC numbers in the infected lung. In vitro co-culture of BCG and rodent Rat Basophilic Leukaemia (RBL-2H3) MCs led to significant killing of BCG. RBL-2H3 MCs were able to phagocytose BCG, take up BCG-derived antigens by macropinocytosis, generate Reactive Oxygen Species (ROS) and degranulate. Further, a few MCs died and released MC extracellular traps (MCETs) having DNA, histones and tryptase to trap BCG. This study highlights the multi-pronged effector responses of MCs on encountering BCG. These responses or their evasion may lead to success or failure of BCG vaccine to provide long term immunity to infections.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites.

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.

Ras signaling activates glycosylphosphatidylinositol (GPI) anchor biosynthesis via the GPI-N-acetylglucosaminyltransferase (GPI-GnT) in Candida albicans.

The ability of Candida albicans to switch between yeast to hyphal form is a property that is primarily associated with the invasion and virulence of this human pathogenic fungus. Several glycosylphosphatidylinositol (GPI)-anchored proteins are expressed only during hyphal morphogenesis. One of the major pathways that controls hyphal morphogenesis is the Ras-signaling pathway. We examine the cross-talk between GPI anchor biosynthesis and Ras signaling in C. albicans. We show that the first step of GPI biosynthesis is activated by Ras in C. albicans This is diametrically opposite to what is reported in Saccharomyces cerevisiae Of the two C. albicans Ras proteins, CaRas1 alone activates GPI-GnT activity; activity is further stimulated by constitutively activated CaRas1. CaRas1 localized to the cytoplasm or endoplasmic reticulum (ER) is sufficient for GPI-GnT activation. Of the six subunits of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) that catalyze the first step of GPI biosynthesis, CaGpi2 is the key player involved in activating Ras signaling and hyphal morphogenesis. Activation of Ras signaling is independent of the catalytic competence of GPI-GnT. This too is unlike what is observed in S. cerevisiae where multiple subunits were identified as inhibiting Ras2. Fluorescence resonance energy transfer (FRET) studies indicate a specific physical interaction between CaRas1 and CaGpi2 in the ER, which would explain the ability of CaRas1 to activate GPI-GnT. CaGpi2, in turn, promotes activation of the Ras-signaling pathway and hyphal morphogenesis. The Cagpi2 mutant is also more susceptible to macrophage-mediated killing, and macrophage cells show better survival when co-cultured with Cagpi2.

The development of the Autism Stigma and Knowledge Questionnaire, Second edition (ASK-Q-2), through a cross-cultural psychometric investigation.

LAY ABSTRACT: The Autism Stigma and Knowledge Questionnaire (ASK-Q) was developed to assess autism knowledge across cultural contexts. The current study aimed to examine cultural equivalence of the measure using a large, international database. These analyses identified 18 items in need of examination for removal or revision. A team of autism experts recommended several additional changes to reduce stigma and increase cross-cultural sensitivity and accuracy of the items on the measure. These changes resulted in a briefer measure with maintained statistical support.

Assessing Autism Knowledge Across the Global Landscape Using the ASK-Q.

Substantial variability exists with regard to autism service provision around the world. Service disparities observed in many low- and middle-income countries may be driven, in part, by limited autism knowledge; however, measurement limitations have made it difficult to quantify autism knowledge across countries. The current study uses the autism stigma and knowledge questionnaire (ASK-Q) to quantify autism knowledge and stigma between different countries and demographics. The current study compiled data from 6830 participants collected using adapted versions of the ASK-Q administered in 13 different countries, representing four different continents. Structural equation modeling was used to examine how autism knowledge varied across country and individual factors. Results reveal cross country variability with a large, 17-point difference between the countries with the highest knowledge (Canada) and the lowest knowledge (Lebanon). As expected, countries with higher economies had higher levels of knowledge. We also documented differences based on country worldview, participant occupation, gender, age, and education level. These results help to identify specific regions and populations that might most need greater information about autism.

Host Mast Cells in Leishmaniasis: Friend or Foe?

Mast cells (MCs) are skin-resident immune cells whose role in leishmaniasis has been recently explored. Researchers report varying inferences, that is, mast cells promote, eliminate, or have no role in leishmaniasis. This article discusses this heterogeneity in mast cell roles to facilitate potential therapeutic and vaccine interventions for these diseases.

Immunodominant Mycobacterium tuberculosis Protein Rv1507A Elicits Th1 Response and Modulates Host Macrophage Effector Functions.

Mycobacterium tuberculosis (M. tb) persists as latent infection in nearly a quarter of the global population and remains the leading cause of death among infectious diseases. While BCG is the only vaccine for TB, its inability to provide complete protection makes it imperative to engineer BCG such that it expresses immunodominant antigens that can enhance its protective potential. In-silico comparative genomic analysis of Mycobacterium species identified M. tb Rv1507A as a "signature protein" found exclusively in M. tb. In-vitro (cell lines) and in-vivo experiments carried out in mice, using purified recombinant Rv1507A revealed it to be a pro-inflammatory molecule, eliciting significantly high levels of IL-6, TNF-α, and IL-12. There was increased expression of activation markers CD69, CD80, CD86, antigen presentation molecules (MHC I/MHCII), and associated Th1 type of immune response. Rv1507A knocked-in M. smegmatis also induced significantly higher pro-inflammatory Th1 response and higher survivability under stress conditions, both in-vitro (macrophage RAW264.7 cells) and in-vivo (mice). Sera derived from human TB patients showed significantly enhanced B-cell response against M. tb Rv1507A. The ability of M. tb Rv1507A to induce immuno-modulatory effect, B cell response, and significant memory response, renders it a putative vaccine candidate that demands further exploration.

Mycobacterium smegmatis Bacteria Expressing Mycobacterium tuberculosis-Specific Rv1954A Induce Macrophage Activation and Modulate the Immune Response.

Mycobacterium tuberculosis (M. tb), the intracellular pathogen causing tuberculosis, has developed mechanisms that endow infectivity and allow it to modulate host immune response for its survival. Genomic and proteomic analyses of non-pathogenic and pathogenic mycobacteria showed presence of genes and proteins that are specific to M. tb. In silico studies predicted that M.tb Rv1954A is a hypothetical secretory protein that exhibits intrinsically disordered regions and possess B cell/T cell epitopes. Treatment of macrophages with Rv1954A led to TLR4-mediated activation with concomitant increase in secretion of pro-inflammatory cytokines, IL-12 and TNF-α. In vitro studies showed that rRv1954A protein or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) activates macrophages by enhancing the expression of CD80 and CD86. An upregulation in the expression of CD40 and MHC I/II was noted in the presence of Rv1954A, pointing to its role in enhancing the association of APCs with T cells and in the modulation of antigen presentation, respectively. Ms_Rv1954A showed increased infectivity, induction of ROS and RNS, and apoptosis in RAW264.7 macrophage cells. Rv1954A imparted protection against oxidative and nitrosative stress, thereby enhancing the survival of Ms_Rv1954A inside macrophages. Mice immunized with Ms_Rv1954A showed that splenomegaly and primed splenocytes restimulated with Rv1954A elicited a Th1 response. Infection of Ms_Rv1954A in mice through intratracheal instillation leads to enhanced infiltration of lymphocytes in the lungs without formation of granuloma. While Rv1954A is immunogenic, it did not cause adverse pathology. Purified Rv1954A or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) elicited a nearly two-fold higher titer of IgG response in mice, and PTB patients possess a higher IgG titer against Rv1954A, also pointing to its utility as a diagnostic marker for TB. The observed modulation of innate and adaptive immunity renders Rv1954A a vital protein in the pathophysiology of this pathogen.

Modulation of azole sensitivity and filamentation by GPI15, encoding a subunit of the first GPI biosynthetic enzyme, in Candida albicans.

Glycosylphosphatidylinositol (GPI)-anchored proteins are important for virulence of many pathogenic organisms including the human fungal pathogen, Candida albicans. GPI biosynthesis is initiated by a multi-subunit enzyme, GPI-N-acetylglucosaminyltransferase (GPI-GnT). We showed previously that two GPI-GnT subunits, encoded by CaGPI2 and CaGPI19, are mutually repressive. CaGPI19 also co-regulates CaERG11, the target of azoles while CaGPI2 controls Ras signaling and hyphal morphogenesis. Here, we investigated the role of a third subunit. We show that CaGpi15 is functionally homologous to Saccharomyces cerevisiae Gpi15. CaGPI15 is a master activator of CaGPI2 and CaGPI19. Hence, CaGPI15 mutants are azole-sensitive and hypofilamentous. Altering CaGPI19 or CaGPI2 expression in CaGPI15 mutant can elicit alterations in azole sensitivity via CaERG11 expression or hyphal morphogenesis, respectively. Thus, CaGPI2 and CaGPI19 function downstream of CaGPI15. One mode of regulation is via H3 acetylation of the respective GPI-GnT gene promoters by Rtt109. Azole sensitivity of GPI-GnT mutants is also due to decreased H3 acetylation at the CaERG11 promoter by Rtt109. Using double heterozygous mutants, we also show that CaGPI2 and CaGPI19 can independently activate CaGPI15. CaGPI15 mutant is more susceptible to killing by macrophages and epithelial cells and has reduced ability to damage either of these cell lines relative to the wild type strain, suggesting that it is attenuated in virulence.

Blocking dephosphorylation at Serine 120 residue in t-SNARE SNAP-23 leads to massive inhibition in exocytosis from mast cells.

Mast cells (MCs) respond to allergen challenge by release of pre-stored inflammatory mediators from their secretory granules, on cross-linking of Fc(epsilon) receptor I (Fc(epsilon)RI) receptors. The target-SNARE (t-SNARE) SNAP-23 has been shown to play an important role in MC exocytosis and undergoes transient phosphorylation at Serine 95 (S95) and Serine 120 (S120), concomitant with mediator release. During current study we explored the importance of transient nature of phosphorylation at S120 in MC exocytosis. A phosphomimetic SNAP-23-S120D mutant of rodent SNAP-23 was cloned into EGFP vector and its effect on the exocytosis and the mechanisms involved was studied in RBL-2H3 MC line. Secretion reporter assay with SNAP-23-S120D transfected MCs revealed a very significant inhibition of exocytosis, and reduced ruffling in response to Fc(epsilon)RI cross-linking. Further, the effect of this mutation on localization of SNAP-23 in MCs was studied. Immunofluorescence microscopy studies and membrane-cytosol fractionation of green fluorescent protein-tagged SNAP- 23-S120D (GFP-SNAP-23-S120D) transfected MCs showed that a large proportion of GFP-SNAP-23-S120D was residing in cytosol unlike wild-type SNAP-23, in resting and activated MCs and even the membrane associated portion was on internal lysosomal membranes than plasma membrane. These studies imply that dephosphorylation of S120 is important for SNAP-23 membrane association dynamics and subsequently MC degranulation.

Role of Mast Cells in clearance of Leishmania through extracellular trap formation.

Mast Cells (MCs) are one of the first immune cells encountered by invading pathogens. Their presence in large numbers in the superficial dermis, where Leishmania is encountered, suggests that they may play a critical role in immune responses to Leishmania. In this study the interactions of Leishmania donovani, the causative agent of visceral Leishmaniasis, and Leishmania tropica, the causative agent of cutaneous Leishmaniasis with MCs were studied. Co-culture of Leishmania with Peritoneal Mast Cells (PMCs) from BALB/c mice and Rat Basophilic Leukaemia (RBL-2H3) MCs led to significant killing of L. tropica and to a lesser extent of L. donovani. Also, while there was significant uptake of L. tropica by MCs, L. donovani was not phagocytosed. There was significant generation of Reactive Oxygen Species (ROS) by MCs on co-culture with these species of Leishmania which may contribute to their clearance. Interactions of MCs with Leishmania led to generation of MC extracellular traps comprising of DNA, histones and tryptase probably to ensnare these pathogens. These results clearly establish that MCs may contribute to host defences to Leishmania in a differential manner, by actively taking up these pathogens, and also by mounting effector responses for their clearance by extracellular means.

Development and Psychometric Evaluation of the Autism Stigma and Knowledge Questionnaire (ASK-Q).

ASD knowledge deficits contribute to disparities in the timing and quality of ASD services. To address the limitations with existing measures of ASD knowledge, we developed and examined the Autism Stigma and Knowledge Questionnaire (ASK-Q), which comprehensively assesses multiple subdomains of ASD knowledge while maintaining strong psychometric support and cross-cultural utility. ASK-Q items derived from the published research are organized into four subscales: (i) diagnosis, (ii) etiology, (iii) treatment, and (iv) stigma. ASK-Q items were selected based on ratings of face, construct, and cross-cultural validity by a group of 16 international researchers. Using Diagnostic Classification Modeling we confirmed the proposed factor structure and evaluated the statistical validity of each item among a lay sample of 617 participants.