Search

Regulation of the immune response to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection is a complex process, influenced by the interaction between genetic and environmental factors. Different inbred strains of mice exhibit distinct levels of resistance to S. Typhimurium infection, ranging from susceptible (e.g., C57BL/6J) to resistant (e.g., DBA/2J) strains. However, the underlying molecular mechanisms contributing to the host response remain elusive. In this study, we present a comprehensive proteomics profiling of spleen tissue from C57BL/6J and DBA/2J strains with different doses of S. Typhimurium infection by tandem mass tag labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (TMT-LC/LC-MS/MS). We identified and quantified 3,986 proteins, resulting in 475 differentially expressed proteins (DEPs) between C57BL/6J and DBA/2J strains. Functional enrichment analysis unveiled that the mechanisms of innate immune responses to S. Typhimurium infection could be associated with several signaling pathways, including the interferon (IFN) signaling pathway. We experimentally validated the roles of the IFN signaling pathway in the innate immune response to S. Typhimurium infection using an IFN-γ neutralization assay. We further illustrated the importance of macrophage and proinflammatory cytokines in the mechanisms underlying the resistance to S. Typhimurium using quantitative reverse transcription-PCR (qRT-PCR). Taken together, our results provided new insights into the genetic regulation of the immune response to S. Typhimurium infection in mice and might lead to the discovery of potential protein targets for controlling salmonellosis.

An exuberant host response contributes to influenza A virus (IAV) (or influenza)-mediated lung injury. However, despite significant information on the host response to IAV, the cellular framework and molecular interactions that dictate the development of acute injury in IAV-infected lungs remain incompletely understood. We performed an unbiased single-cell RNA sequencing (scRNAseq) analysis to examine the cellular heterogeneity and regulation of host responses in the IAV model of acute lung injury. At the cellular level, IAV infection promoted the overwhelming recruitment of monocytes that exhibited the cell differentiation trajectory to monocyte-derived macrophages. Together, monocytes and monocyte-derived myeloid cells constituted over 50% of the total immune cells in IAV-infected lungs. In contrast, IAV infection resulted in a significant loss of nonhematopoietic cells. Molecularly, our data show the multidimensional cell-cell communication dynamics of interferon and chemokine signaling between immune and nonimmune cells and the cell-specific molecular pathways regulating the host responses during IAV-induced lung injury. Our data provide a foundation for further exploring the mechanistic association of the IAV host response with acute lung injury. IMPORTANCE A dysregulated host response develops acute lung injury during IAV infection. However, the pathological immune mechanism(s) associated with acute lung injury during IAV infection is yet to be elucidated. In this study, we performed scRNAseq to examine the dynamics of host responses during the peak of IAV-mediated lung injury. At the cellular level, our data reveal significant myelopoiesis predominated by monocytes and macrophages and the simultaneous disruption of the nonhematopoietic cell framework, crucial for regulating inflammation and barrier integrity in IAV-infected lungs. Molecularly, we observed a complex cellular network involving cell-cell communications and a number of unique regulons dictating the outcome of interferon and chemokine responses during peak lung injury. Our data present a unique atlas of cellular changes and the regulation of global and cell-specific host responses during IAV infection. We expect that this information will open new avenues to identify targets for therapeutic intervention against IAV lung injury.

Influenza A Virus (IAV) infection triggers an exuberant host response that promotes acute lung injury. However, the host response factors that promote the development of a pathologic inflammatory response to IAV remain incompletely understood. In this study, we identify an interferon (IFN)-γ-regulated subset of monocytes, CCR2(+) monocytes, as a driver of lung damage during IAV infection. IFN-γ regulates the recruitment and inflammatory phenotype of CCR2(+) monocytes, and mice deficient in CCR2 (CCR2(−/−)) or IFN-γ (IFN-γ(−/−)) exhibit reduced lung inflammation, pathology, and disease severity. Adoptive transfer of WT (IFN-γR1(+/+)), but not IFN-γR1(−)/(−) CCR2(+) monocytes, restore the wild-type (WT)-like pathological phenotype of lung damage in IAV-infected CCR2(−/−) mice. CD8(+) T cells are the main source of IFN-γ in IAV-infected lungs. Collectively, our data highlights the requirement of IFN-γ signaling in the regulation of CCR2(+) monocyte-mediated lung pathology during IAV infection.

Investigation of natural products has moved toward marine environments as they are a source of many biologically active agents. The natural antioxidant compounds isolated from seaweeds provide a valuable contribution to the innovation of new drugs for chronic diseases associated with oxidative stress. While the antioxidant activity and nutritional benefits of various seaweed products are well recognized, their proper utilization as antioxidants remains at initial stages that require further investigative studies. This review provides a detailed study of isolated antioxidant compounds from seaweeds and their major mechanism of action by focusing on the reports from 2015 to 2019. The report discusses various active antioxidant compounds, including phenolics (e.g., phlorotannins), polysaccharides, and pigments with proven benefits against oxidative stress-related diseases, especially carotenoids, from the aspect of benefits to human health.

Regulation of the immune response to Salmonella typhimurium (S. Typhimurium) infection is a complex process, influenced by genetic and environmental factors. Different inbred mouse strains show distinct levels of resistance to S. Typhimurium infection, ranging from susceptible (e.g., C57BL/6J) to resistant (e.g., DBA/2J) strains. However, the underlying molecular mechanisms contributing to the host response remain elusive. In this study, we present a comprehensive proteomics profiling of the spleen tissue from C57BL/6J and DBA/2J strains with different doses of S. Typhimurium infection by tandem tag mass coupled with two-dimensional liquid chromatography-tandem mass spectrometry. We identified and quantified 3,986 proteins, resulting in 475 differentially expressed proteins between C57BL/6J and DBA/2J strains. Functional enrichment analysis revealed that the mechanisms of innate immune responses to S. Typhimurium infection are associated with several signaling pathways, including the interferon signaling pathway. Our proteomic data also discovered a plausible gene in a genomic region that control different levels of resistance to S. Typhimurium infection. We further revealed the roles of macrophage cells and pro-inflammatory cytokines in the mechanisms under the natural resistance to S. Typhimurium. In summary, our results provide new insights into the genetic regulation of the immune response to S. Typhimurium infection in mice.Author SummarySalmonella infection (salmonellosis) is a common zoonotic disease that mainly propagates through contaminated food and drink. Various mouse strains display prominent disparities in responses to Salmonella invasion. Elucidating the heterogeneous immune reactions between different mouse strains can shed light on the fundamental molecular mechanisms of the innate immune system. Here, we employed a combination of proteomics and systems biology approaches to provide an unprecedented panorama of the inextricably interlaced immune signaling pathways in response to Salmonella infection in mice. Our results revealed the dynamics of cell signaling molecules elicited by inflammation and established new connections among them. We also identified a new candidate gene involved in the combat with pathogens. Our proteomic data and results contribute to understanding the intricate interactions of immune responses to Salmonella infection from molecular to systemic levels.

Acute lung injury (ALI) caused by influenza A virus (IAV or influenza) manifests from dysregulated cellular interactions between hematopoietic and non-hematopoietic cells that develop into a pathologic host response. However, the lung’s diverse cellular framework that dictates the pathologic host response and acute lung injury remains incompletely understood. We performed a single-cell RNA-seq (scRNA-seq) analysis of total lung cells in mice from severe influenza to examine the cellular heterogeneity and cell-specific regulation of host response. We observed that IAV infection resulted in significant myelopoiesis, predominantly monocyte, and macrophage subsets, which constituted over 50% of total immune cells. IAV infection resulted in the significant loss of endothelial and fibroblast cells, representing the most predominant non-hematopoietic cells and crucial to regulating inflammatory response and barrier integrity. We also show the cell-cell communication dynamics of interferon and chemokine signaling and global regulation of these responses in transition from homeostatic to IAV infection state. These data highlight a robust application of scRNA-seq technology in establishing the atlas of cellular heterogeneity from its homeostatic transition to infection state and the host response regulation in IAV-mediated lung pathology.

Intestinal fibrosis is an excessive proliferation of myofibroblasts and deposition of collagen, a condition frequently seen in Crohn's disease (CD). The mechanism underlying myofibroblast hyper-proliferation in CD needs to be better understood. In this report, we found that mTOR inhibitor rapamycin or mTOR deletion in CX3Cr1+ mononuclear phagocytes inhibits expression of interleukin (IL)-23, accompanied by reduced intestinal production of IL-22 and ameliorated fibrosis in the TNBS-induced fibrosis mouse model. This inhibition of IL-23 expression is associated with elevated autophagy activity. Ablating the autophagy gene Atg7 increases the expression of IL-23, leading to increased expression of IL-22 and increased fibrosis. Both induction of IL-22 and intestinal fibrosis occurred in RAG-/- mice and depletion of innate lymphoid cells (ILCs) attenuates the fibrotic reaction, suggesting that the pro-fibrotic process is independent of T and B cells. Moreover, IL-22 facilitates the transformation of fibroblasts into myofibroblasts. Finally, the fibrotic reaction was attenuated upon neutralization of either IL-23 or IL-22. Altogether, this study elucidated a signaling cascade underlying intestinal fibrosis in which altered mTOR/autophagy in CX3Cr1+ mononuclear phagocytes up-regulates the IL-23/IL-22 axis, leading to an excessive fibrotic response. Thus, our findings suggest that this cascade could be a therapeutic target for alleviation of CD fibrosis.

BackgroundWe sought to determine the role of host interleukin 17A (IL-17A) response against colonizing Streptococcus pneumoniae, and its transition to a pathogen during coinfection with an influenza virus, influenza A H1N1 A/Puerto Rico/8/1934 (PR8).MethodWild-type (WT) C57BL/6 mice were intranasally inoculated with S. pneumoniae serotype 6A to establish colonization and later infected with the influenza strain, PR8, resulting in invasive S. pneumoniae disease. The role of the IL-17A response in colonization and coinfection was investigated in WT, RoRγt−/− and RAG1−/− mice with antibody-mediated depletion of IL-17A (WT) and CD90 cells (RAG1−/−).ResultsRAG1−/− mice did not clear colonization and IL-17A neutralization impaired 6A clearance in WT mice. RoRγt−/− mice also had reduced clearance. S. pneumoniae–PR8 coinfection elicited a robust IL-17A response in the nasopharynx; IL-17A neutralization reduced S. pneumoniae invasive disease. RoRγt−/− mice also had reduced S. pneumoniae disease in a coinfection model. Depletion of CD90+ cells suppressed the IL-17A response and reduced S. pneumoniae invasion in RAG1−/− mice.ConclusionOur data show that although IL-17A reduces S. pneumoniae colonization, coinfection with influenza virus elicits a robust innate IL-17A response that promotes inflammation and S. pneumoniae disease in the nasopharynx.

A massive bird dropping (BD) deposition on the common rectangular flat plate (RFP) of photovoltaic (PV) module is a matter of great concern in Western Rajasthan (WR) that diminish the overall energy production capacity of the system remarkably. In this research article, a prototype novel flat plate (NFP) design of a front glass cover of PV module is proposed to prevent the impact of BD settlement by the restriction of bird's sitting/movement on the front glass cover. In this regard, the performance analysis of PV module with common RFP and newly designed NFP glass covers has been assessed at the different inclination β° (0-90). The BD accumulation onto the both glass covers was explored by the optical transmittance profiles at the different tilt angles, i.e., explained by bird movement on each flat glass surfaces. Consequently, a significant amount of output electric energy has been gained in NFP design rather than RFP corresponding to particular tilt regions TR I (0° ≤ β ≤ 25°), TR II (25° ≤ β ≤ 60°), and TR III (60° ≤ β ≤ 90°). According to the results achieved, an excellent level of improvement in average power loss, ~ 97.85%, corresponding to optimal TR (III) has been detected by employing NFP glass collector.

Excessive activation of mammalian target of rapamycin (mTOR) signaling is epileptogenic in genetic epilepsy. However, the exact role of microglial mTOR in acquired epilepsy remains to be clarified. In the present study, we found that mTOR is strongly activated in microglia following excitatory injury elicited by status epilepticus. To determine the role of microglial mTOR signaling in excitatory injury and epileptogenesis, we generated mice with restrictive deletion of mTOR in microglia. Both male and female mice were used in the present study. We found that mTOR-deficient microglia lost their typical proliferative and inflammatory responses to excitatory injury, whereas the proliferation of astrocytes was preserved. In addition, mTOR-deficient microglia did not effectively engulf injured/dying neurons. More importantly, microglial mTOR-deficient mice displayed increased neuronal loss and developed more severe spontaneous seizures. These findings suggest that microglial mTOR plays a protective role in mitigating neuronal loss and attenuating epileptogenesis in the excitatory injury model of epilepsy.SIGNIFICANCE STATEMENTThe mammalian target of rapamycin (mTOR) pathway is strongly implicated in epilepsy. However, the effect of mTOR inhibitors in preclinical models of acquired epilepsy is inconsistent. The broad presence of mTOR signaling in various brain cells could prevent mTOR inhibitors from achieving a net therapeutic effect. This conundrum has spurred further investigation of the cell type-specific effects of mTOR signaling in the CNS. We found that activation of microglial mTOR is antiepileptogenic. Thus, microglial mTOR activation represents a novel antiepileptogenic route that appears to parallel the proepileptogenic route of neuronal mTOR activation. This may explain why the net effect of mTOR inhibitors is paradoxical in the acquired models of epilepsy. Our findings could better guide the use of mTOR inhibitors in preventing acquired epilepsy.

Worldwide COVID-19 infection poses an enormous risk to public health and an alarming global socioeconomic burden. The impact of the COVID-19 pandemic on individuals with underlying health conditions as well as on the elderly population is extensive and effective strategies are needed to understand the mechanism behind it. Cellular senescence defines as an irreversible cell cycle arrest due to DNA damage leading to accumulation of senescent cells in the elderly population and may result in worsening of COVID-19 mediated increased mortality. However, whether this variation in senescence levels, in different aged populations, translation to COVID-19 infection is unknown. The spike protein of SARS-CoV-2 has been recently identified to be responsible for inducing pathogenic signals, although a clear understanding of how the host receptor interacts with SARS-CoV-2 protein and mediates the immune responses is not clear. In this review, we address the epidemiology of SARS-CoV-2 and the cellular senescence responding immune response to pathogenic SARS-CoV-2. We provide a prospective summary of what to expect and how to brace the possible immunological strategy to protect against COVID-19 infection. The review majorly explores an underline mechanism of how senescent cells trigger a hyperimmune inflammatory response and cause high mortality in aging people could serve as a potential aid to alleviate the treatment for elderly battling COVID-19 infection.

Summary Microglia are well known to play a critical role in maintaining brain homeostasis. However, their role in epileptogenesis has yet to be determined. Here, we demonstrate that elevated mTOR signaling in mouse microglia leads to phenotypic changes, including an amoeboid-like morphology, increased proliferation, and robust phagocytosis activity, but without a significant induction of pro-inflammatory cytokines. We further provide evidence that these noninflammatory changes in microglia disrupt homeostasis of the CNS, leading to reduced synapse density, marked microglial infiltration into hippo-campal pyramidal layers, moderate neuronal degeneration, and massive proliferation of astrocytes. Moreover, the mice thus affected develop severe early-onset spontaneous recurrent seizures (SRSs). Therefore, we have revealed an epileptogenic mechanism that is independent of the microglial inflammatory response. Our data suggest that microglia could be an opportune target for epilepsy prevention., Graphical abstract Zhao et al. reveal that elevated mTOR signaling in microglia propels the cells to acquire a noninflammatory reactive-like phenotype, which leads to disruption of CNS homeostasis. The authors also report that reactive-like microglia promote epileptogenesis independent of the inflammatory response.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn's-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn's-associated intestinal fibrosis.

Excessive activation of mTOR in microglia impairs CNS homeostasis and causes severe epilepsy. Autophagy constitutes an important part of mTOR signaling. The contribution of microglial autophagy to CNS homeostasis and epilepsy remains to be determined. Here we report that ATG7KO mice deficient for autophagy in microglia display a marked increase of myelination markers, a higher density of mature oligodendrocytes, and altered lengths of the nodes of Ranvier. Moreover, we found that deficiency of microglial autophagy (ATG7KO) leads to increased seizure susceptibility in three seizure models (pilocarpine, kainic acid, and amygdala kindling). We demonstrated that ATG7KO mice develop severe generalized seizures and display nearly 100% mortality to convulsions induced by pilocarpine and kainic acid. In the amygdala kindling model, we observed significant facilitation of contralateral propagation of seizures, a process underlying the development of generalized seizures. Taken together, our results reveal impaired microglial autophagy as a novel mechanism underlying altered homeostasis of oligodendrocytes and increased susceptibility to severe and fatal generalized seizures.Significance statement Microglia play a critical role in CNS homeostasis, predominantly by impinging on neurons and astrocytes. A role for microglia in the development of oligodendrocytes has begun to be recognized, but the mechanism remains largely unknown. The present study uncovered a novel role of microglial autophagy in oligodendrocyte homeostasis. Importantly, we found that deficiency of microglial autophagy causes severe forms of generalized seizures and high mortality, pointing to a strong association of altered oligodendrocyte homeostasis and epilepsy. Our findings have implications in understanding severe forms of epilepsy.

Allergic asthma consists of diverse immune phenotypes exhibiting differential lung pathology, remodeling of the respiratory tract and mucociliary bronchial clearance. These diverse inflammatory immune mechanisms may promote or resist microbial infections in allergic human and mouse models. IL-6 governs optimal T-cell and inflammatory response leading to the resolution of bacterial and viral infections in respiratory airways. Additionally, IL-6 plays an important role in maintaining the integrity of airway barrier response during microbial infections. We hypothesized that lung IL-6 responses alleviate Streptococcus pneumoniae (Spn) pathogenesis during fungal allergic asthma caused by Aspergillus fumigatus. Using a murine Aspergillus fumigatus asthma model, we show that IL-6 deficiency exacerbates lung inflammation and promotes Spn disease pathogenesis in allergic host. IL-6 deficiency was associated with increased lung damage, which correlated with higher levels of lung eosinophilia, inflammatory cytokines and chemokines. Additionally, IL-6 deficiency correlated with increased TGF-β1 cytokine levels, increased epithelial-mesenchymal transition and dysregulated epithelial barrier proteins in asthmatic mice. We conclude that IL-6 offers a significant protective role in the control of Spn pathogenesis during allergic asthma by regulation of immune cell recruitment and maintenance of lung epithelial barrier integrity.

CD40 plays dual immunoregulatory roles in Leishmania major infection and tumor regression. The functional duality emerges from CD40-induced reciprocal p38MAPK and ERK-1/2 phosphorylations. Because phosphotyrosine-based signaling in hematopoietic cells is regulated by the phosphotyrosine phosphatase SHP-1, which is not implied in CD40 signaling, we examined whether SHP-1 played any roles in CD40-induced reciprocal signaling and anti-leishmanial function. We observed that a weaker CD40 stimulation increased SHP-1 activation. ERK-1/2 inhibition or p38MAPK overexpression inhibited CD40-induced SHP-1 activation. An ultra-low-dose, CD40-induced p38MAPK phosphorylation was enhanced by SHP-1 inhibition but reduced by SHP-1 overexpression. A reverse profile was observed with ERK-1/2 phosphorylation. SHP-1 inhibition reduced syk phosphorylation but increased lyn phosphorylation; syk inhibition reduced but lyn inhibition enhanced CD40-induced SHP-1 phosphorylation. Corroborating these findings, in L. major–infected macrophages, CD40-induced SHP-1 phosphorylation increased and SHP-1 inhibition enhanced CD40-induced p38MAPK activation and inducible NO synthase expression. IL-10 enhanced SHP-1 phosphorylation and CD40-induced ERK-1/2 phosphorylation but reduced the CD40-induced p38MAPK phosphorylation, whereas anti–IL-10 Ab exhibited reverse effects on these CD40-induced functions, identifying IL-10 as a crucial element in the SHP-1-MAPK feedback system. Lentivirally overexpressed SHP-1 rendered resistant C57BL/6 mice susceptible to the infection. Lentivirally expressed SHP-1 short hairpin RNA enhanced the CD40-induced L. major parasite killing in susceptible BALB/c mice. Thus, we establish an SHP-1–centered feedback system wherein SHP-1 modulates CD40-induced p38MAPK activation threshold and reciprocal ERK-1/2 activation, establishing itself as a critical regulator of CD40 signaling reciprocity and mechanistically re-emphasizing its role as a potential target against the diseases where CD40 is involved.

Microglia play a pivotal role in maintaining homeostasis of the CNS. There is growing interest in understanding how microglia influence normal brain function and disease progression. Several microglia-specific Cx3cr1-Cre lines have been developed and have become indispensable tools in many investigations of microglial function. However, some recent studies have reported that these lines may have significant leakage into neurons. Other studies have reported that Cx3cr1 is expressed in non-microglial cells, including neurons and astrocytes, in vitro or in vivo either during brain development or upon neurological insult. All these reports raise serious concerns about the trustworthiness of these Cre-lines and whether the conclusions drawn from previous studies are valid. Here, we found that a floxed fluorescent reporter mouse line which has been frequently used to verify Cre lines displayed spontaneous expression of the GFP reporter, independent of Cre recombinase, thus revealing a potential caveat in assessing cre lines. We further confirmed that two Cx3cr1-Cre mouse lines can drive fluorescent reporter expression largely restrictively in microglia. Finally, we clarified that these two mouse lines maintain microglia-specific expression even following excitatory injury. Together, our findings confirm that two previously created Cx3cr1-Cre lines remain as invaluable tools for studying microglia. Moreover, to ensure the quality of data generated and the soundness of conclusions drawn from such data, it should be compulsory to thoroughly examine reporter lines for spontaneous leakiness when labeling cells to study CNS function and diseases.

Summary Toll-like receptor 11 (TLR11) recognizes T. gondii profilin (TgPRF) and is required for interleukin-12 production and induction of immune responses that limit cyst burden in Toxoplasma gondii -infected mice. However, TLR11 only modestly affects survival of T. gondii -challenged mice. We report that TLR12, a previously uncharacterized TLR, also recognized TgPRF. TLR12 was sufficient for recognition of TgPRF by plasmacytoid dendritic cells (pDCs), whereas TLR11 and TLR12 were both required in macrophages and conventional DCs. In contrast to TLR11, TLR12-deficient mice succumb rapidly to T. gondii infection. TLR12-dependent induction of IL-12 and IFN-α in pDCs led to production of IFN-γ by NK cells. Consistent with this observation, the partial resistance of Tlr11 −/− mice is lost upon pDC or NK cell depletion. Thus, TLR12 is critical for the innate immune response to T. gondii , and this TLR may promote host resistance by triggering pDC and NK cell function.

We have reported that TLR11 contributes to the murine response to flagellin and resistance to infection with Salmonella spp., including those responsible for typhoid fever in humans (Mathur et al., 2012). Here, we respond to the Correspondence in this issue of Cell by Song et al. (2016), which claims that mice lacking TLR11 are not more susceptible to infection with Salmonella species and that TLR11 does not recognize Salmonella flagellin.

Summary Salmonella spp. are gram-negative flagellated bacteria that can cause food- and waterborne gastroenteritis and typhoid fever in humans. We now report that flagellin from Salmonella spp. is recognized in mouse intestine by Toll-like receptor 11 (TLR11). Absence of TLR11 renders mice more susceptible to infection by S. Typhimurium, with increased dissemination of the bacteria and enhanced lethality. Unlike S. Typhimurium, S. Typhi, a human obligatory pathogen that causes typhoid fever, is normally unable to infect mice. TLR11 is expressed in mice, but not in humans, and remarkably, we find that tlr11 −/− mice are efficiently infected with orally administered S. Typhi. We also find that tlr11 −/− mice can be immunized against S. Typhi. Therefore, tlr11 −/− mice represent a small-animal model for the study of the immune response to S. Typhi and for the development of vaccines against this important human pathogen.

Objectives The relationship between quinolone resistance acquisition and invasion impairment has been studied in some Salmonella enterica serovars. However, little information has been reported regarding the invasive human-restricted pathogen Salmonella Typhi. The aim of this study was to investigate the molecular mechanisms of quinolone resistance acquisition and its impact on virulence in this serovar. Methods Two antibiotic-resistant mutants (Ty_c1 and Ty_c2) were generated from a Salmonella Typhi clinical isolate (Ty_wt). The three strains were compared in terms of antimicrobial susceptibility, molecular mechanisms of resistance, gene expression of virulence-related factors, ability to invade eukaryotic cells (human epithelial cells and macrophages) and cytokine production. Results Multidrug resistance in Ty_c2 was attributed to AcrAB/TolC overproduction, decreased OmpF (both mediated by the mar regulon) and decreased OmpC. The two mutants showed a gradually reduced expression of virulence-related genes (invA, hilA, hilD, fliC and fimA), correlating with decreased motility, reduced infection of HeLa cells and impaired uptake by and intracellular survival in human macrophages. Moreover, Ty_c2 also showed reduced tviA expression. Additionally, we revealed a significant reduction in TNF-α and IL-1β production and decreased NF-κB activation. Conclusions In this study, we provide an in-depth characterization of the molecular mechanisms of antibiotic resistance in the Salmonella Typhi serovar and evidence that acquisition of antimicrobial resistance is concomitantly detected with a loss of virulence (epithelial cell invasion, macrophage phagocytosis and cytokine production). We suggest that the low prevalence of clinical isolates of Salmonella Typhi highly resistant to ciprofloxacin is due to poor immunogenicity and impaired dissemination ability of these isolates.

Various species of Mycobacteria produce a major cell wall-associated lipoglycan, called Lipoarabinomannan (LAM), which is involved in the virulence of Mycobacterial species. In this study, we tried to establish the role of the increased IL-10 secretion under Arabinosylated-LAM (Ara-LAM) treatment, the LAM that induces apoptosis in host macrophages or PBMC. We have studied the survival and apoptotic factors by western blotting, and estimated nitrite generation by Griess reaction, quantified iNOS mRNA by semi-quantitative RT-PCR, and ultimately the fate of the cells were studied by Flow Cytometric Analysis of AnnexinV-FITC binding. As per our observations, neutralization of released IL-10 in C57BL/6 peritoneal macrophages prior to Ara-LAM treatment, as well as macrophages from IL-10 knockout (KO) mice treated with Ara-LAM, showed significant down regulation of pro-apoptotic factors and up regulation of survival factors. These effects were strikingly similar to those when peritoneal macrophages were subjected to TNF-alpha and IL-12 neutralization followed by Ara-LAM-treatment. However, under similar conditions virulent Mannosylated-LAM (from Mycobacterium tuberculosis) treatment of macrophages clearly depicts the importance of IL-10 in the maintenance of pathogenesis, proving its usual immunosuppressive role. Thus, from our detailed investigations we point out an unusual pro-inflammatory action of IL-10 in Ara-LAM treated macrophages, where it behaves in a similar manner as the known Th1 cytokines TNF- alpha and IL-12.

T cells regulate the immune responses to pathogens and autoantigens. The immune responses are tolerizing or anti-inflammatory against autoantigens but are inflammatory against pathogens and allografts. Such contradictory immune responses have been attributed to two counteracting effector cell types or to two counterregulatory sets of molecules: cell-surface expressed or secreted. By contrast, recent reports suggest that CD40, a co-stimulatory molecule on antigen-presenting cells, is a crucial controller of these counteractive immune responses, and emphasize reciprocal inhibition as an essential feature of biological responses. The molecular mechanism of such reciprocity in CD40 functions is the basis of immunotherapy in many diseases.

Macrophages play host to Leishmania major, a parasite that causes leishmaniasis in 500,000 people annually. Macrophage-expressed CD40, a costimulatory molecule1, induces interleukin-12 (IL-12)-dependent and interferon-γ (IFN-γ)-dependent host-protective immune responses to Leishmania and other intracellular pathogens2,3,4,5,6. Paradoxically, IL-10, another CD40-induced cytokine in macrophages7, promotes Leishmania infection8. How CD40 signaling regulates the secretion of these two counteractive cytokines remains unknown. Here we show that weak CD40 signals induce extracellular stress–related kinase-1/2 (ERK-1/2)-dependent IL-10 expression, whereas stronger signals induce p38 mitogen-activated protein kinase (p38MAPK)-dependent IL-12 production. p38MAPK and ERK-1/2 therefore have counter-regulatory actions. Leishmania skews CD40 signaling toward ERK-1/2, inducing IL-10, which inhibits activation of CD40-induced p38MAPK and expression of inducible nitric oxide synthase-2 (iNOS-2) and IL-12. ERK-1/2 inhibition or IL-10 neutralization restores CD40-induced p38MAPK activation and parasite killing in macrophages and the BALB/c mouse, a susceptible host. These data uncover a new immune evasion strategy, whereby Leishmania differentially modulates CD40-engaged, reciprocally functioning signaling modules, and provide a new conceptual framework for immune homeostasis.

Leishmania, a protozoan parasite, lives and multiplies as amastigote within macrophages. It is proposed that the macrophage expressed CD40 interacts with CD40 ligand on T cells to induce IFN-γ, a Th1-type cytokine that restricts the amastigote growth. Here, we demonstrate that CD40 cross-linking early after infection resulted in inducible nitric oxide synthetase type-2 (iNOS2) induction and iNOS2-dependent amastigote elimination. Although CD40 expression remained unaltered on L. major–infected macrophages, delay in the treatment of macrophages or of mice with anti-CD40 antibody resulted in significant reduction in iNOS2 expression and leishmanicidal function suggesting impaired CD40 signaling in Leishmania infection. The inhibition of CD40-induced iNOS2 expression by SB203580, a p38-mitogen activated protein kinase (p38MAPK)-specific inhibitor, and the reversal of the inhibition by anisomycin, a p38MAPK activator, suggested a crucial role of p38MAPK in CD40 signaling. Indeed, the CD40-induced p38MAPK phosphorylation, iNOS2 expression and anti-leishmanial function were impaired in Leishmania-infected macrophages but were restored by anisomycin. Anisomycin's effects were reversed by SB203580 emphasizing the role of p38MAPK in CD40-induced iNOS2-dependent leishmanicidal function. Anisomycin administration in L. major–infected BALB/c mice resulted in significant reduction in the parasite load and established a host-protective Th1-type memory response. Also implicated in these findings is a scientific rationale to define novel anti-parasite drug targets and to bypass the problem of drug resistance.

To a certain extent recording of speech samples and analysis of exemplars depends on the quality and contents of the material prepared for experiments. Therefore, preparing speech materials seems an important part of the research methodology. For this particular case study, various texts have been prepared in Standard Hindi using devnagri script, including the words or phrases commonly found in telephonic interception of criminal acts. Later, these prepared texts were transliterated into each dialect chosen for the study considering the accent as well as the uses.

In Chap. 1, we discussed the basics of voice identification process, which is very useful to forensic expert, in order to correctly identify the speaker. In this study, we are trying to establish the identity of a person on the basis of his/her dialectal accent. Therefore, it is recommended to have a basic knowledge of the native language and its dialects.

Speech of person is characterized using some phonetic features like stylistic impression, phonation, nasality, dynamic of loudness, and flow of speech. Based on these features, the linguistic characteristics of a person in specimen are compared with that of the person in questioned. A conventional method, combined aural and spectrographic method, is used for analysis of speech exemplars. Although analysis is performed equally on all the speech exemplars of all chosen regional dialects, due to limited space, Khariboli and Bhojpuri dialects are selected to discuss the results in this chapter. Bhojpuri dialect is selected randomly, whereas Khariboli is considered as a base language in this study. A prosodic analysis is also performed in order to study the intonation tonal pattern of the dialects. This chapter will go through the details of each method used and the results obtained through it.

Summary Immune tolerance against enteric commensal bacteria is important for preventing intestinal inflammation. Deletion of phosphoinositide-dependent protein kinase 1 ( Pdk1 ) in T cells via Cd4-Cre induced chronic inflammation of the intestine despite the importance of PDK1 in T cell activation. Analysis of colonic intraepithelial lymphocytes of PDK1-deficient mice revealed markedly increased CD8α + T cell receptor (TCR)γδ + T cells, including an interleukin-17 (IL-17)-expressing population. TCRγδ + T cells were responsible for the inflammatory colitis as shown by the fact that deletion of Tcrd abolished spontaneous colitis in the PDK1-deficient mice. This dysregulation of intestinal TCRγδ + T cells was attributable to a reduction in the number and functional capacity of PDK1-deficient T regulatory (Treg) cells. Adoptive transfer of wild-type Treg cells abrogated the spontaneous activation and proliferation of intestinal TCRγδ + T cells observed in PDK1-deficient mice and prevented the development of colitis. Therefore, suppression of intestinal TCRγδ + T cells by Treg cells maintains enteric immune tolerance.