The Molecular Detection of Bacterial Infections of Public Health Importance in Hard Tick (Ixodidae) Nymphs Collected from the Forest Fringes of Western Ghats in the Goa, Karnataka and Maharashtra States of India.

A survey was conducted to determine the human tick-borne bacterial infections in the nymphs which were collected from Western Ghats' fringe forest areas. Tick nymphs were collected using the flagging method from the villages where cases Kyasanur Forest Disease (KFD) were previously reported in the states of Goa, Karnataka and Maharashtra. A total of 200 tick pools consisting of 4587 nymphs were tested by PCR for the detection of bacteria of public health importance, such as Coxiella burnetii and Rickettsia spp. Of these, four pools (4.8%) in Karnataka and three pools (4.4%) in Maharashtra were positive for Coxiella burnetii, while none of the samples from Goa state were positive. Rickettsia spp. were positively obtained from Maharashtra (51.5%), Goa (35.42%) and Karnataka (26.19%). The sequence results of Rickettsia spp. showed similarity to the spotted fever group Candidatus Rickettsia shennongii, Rickettsia conorii subsp. heilongjiangensis and Rickettsia spp. strain koreansis. Individuals are entering into the forest areas for various reasons are more likely to infect with Coxiella burnetii. and Rickettsia spp.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease.

BACKGROUND: The dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. METHODS: Cell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. RESULTS: In the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. CONCLUSION: In conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

Eosinophils Restrict Diesel Exhaust Particles-induced Cell Proliferation of Lung Epithelial A549 Cells via Interleukin-13 Mediated Mechanisms: Implications for Tissue Remodeling and Fibrosis.

BACKGROUND: Diesel exhaust particles (DEPs) affect lung physiology and cause serious damage to the lungs. A number of studies demonstrated that eosinophils play a very important role in the development of tissue remodeling and fibrosis of the lungs. However, the exact mechanism of pathogenesis of tissue remodeling and fibrosis is not known. METHODS: Both in vitro and in vivo models were used in the study. HL-60 and A549 cells were also utilized in the study. 8 to 12 weeks old BALB/c mice were used for the in vivo study. Cell viability by MTT assay and RNA isolation by tri reagent was accomplished. mRNA expression of inflammatory genes was accomplished by real-time PCR or qPCR. Immunohistochemistry was done to assess the localization and expressions of proteins. One-way ANOVA followed by a post hoc test was done for the statistical analysis. Graph-Pad prism 5 software was used for statistical analysis. RESULTS: For the first time, we demonstrate that interleukin-13 plays a very important role in the development of tissue remodeling and fibrosis. We report that diesel exhaust particles significantly induce eosinophils cell proliferation and interleukin-13 release in in vitro culture conditions. Supernatant collected from DEP-induced eosinophils cells significantly restricts cell proliferation of epithelial cells in response to exposure to diesel exhaust particles. Furthermore, purified interleukin-13 decreases the proliferation of A549 cells, highlighting the involvement of IL- 13 in tissue remodeling. Notably, Etoricoxib (selective COX-2 inhibitor) did not inhibit the DEPtriggered release of interleukin-13, suggesting another cell signaling pathway. The in vivo exposure of DEP to the lungs of mice resulted in a high level of eosinophils degranulation as depicted by the EPX-1 immunostaining and altered level of mRNA expressions of inflammatory genes. We also found that a-SMA, fibroblast specific protein (FSP-1), has been changed in response to DEP in the mice lungs along with the mediators of inflammation. CONCLUSION: Altogether, we elucidated the mechanistic role of eosinophils and IL-13 in the DEP-triggered proliferation of lungs cells, thus providing an insight into the pathophysiology of tissue remodeling and fibrosis of lungs.

The Involvement of Neuroinflammation in Dengue Viral Disease: Importance of Innate and Adaptive Immunity.

Neuroinflammation (inflammation in brain) has been known to play an important role in the development of dengue virus disease. Recently, studies from both clinical and experimental models suggest the involvement of neuroinflammation in dengue viral disease. Studies in clinical setup demonstrated that, microglial cells are actively involved in the patients having dengue virus infection, showing involvement of innate immune response in neuroinflammation. It was further proved that, clinical isolates of dengue-2 virus were able to initiate the pathologic response when injected in the mice brain. Natural killer cells were also found to play a crucial role to activate adaptive immune response. Notably, CXCL10/IFN-inducible protein 10 and CXCR3 are involved in dengue virus-mediated pathogenesis and play an important role in the development of dengue virus-mediated paralysis. In a latest report, it was seen that intracranial injection of dengue virus increases the CD8+ T-cell infiltration in brain, showing an important mechanism of neuroinflammation during the dengue virus infection. A similar study has described that, when DENV-3 is injected into the mice, it enhances the infiltration of CD8+ and CD4+ T cells as well as neutrophils. Cells immune-reactive against NS3 antigen were found throughout the brain. In conclusion, we focus on the various molecular mechanisms which contribute to the basic understanding about the role of neuroinflammation in dengue fever. These mechanisms will help in better understanding dengue pathophysiology and thus help in the development of possible therapeutics.

Nonstructural protein-1 (NS1) of dengue virus type-2 differentially stimulate expressions of matrix metalloproteinases in monocytes: protective effect of paracetamol.

BACKGROUND: Dengue fever is a re-emerging viral disease and affects millions of population worldwide. Monocytes are involved in dengue viral disease, however, their exact role is still not clear. In the present study, we investigated, the effect of NS1 antigen of dengue virus and paracetamol on THP-1 monocytes associated to expressions of matrix metalloproteinases (MMPs) and cytokine release. METHODS: Assessment of cell morphology by bright field microscopy, cell viability by MTT assay, protein estimation by Bradford reagent were done in cells exposed to NS1 antigen in the presence and absence of paracetamol. Cytokines estimations were done by ELISA. Expression profile of matrix metalloproteinase genes was done using real-time PCR and reverse-transcriptase PCR. RESULTS: NS1 exposure of THP-1 monocytes cells, changed their cell morphology and activated them for release of proteins in 24 h. Expressions of MMP-2, MMP-8, MMP-9 and MMP-14 genes were upregulated by NS1 exposure. Further, exposure of NS1 to THP-1 monocytes cells increased expression profile of MMP-10 and MMP-13 genes to a lesser extent. Treatment with paracetamol (1 mg/ml and 2 mg/ml), significantly down-regulated the expression profile of MMP-2, MMP-8, MMP-9 and 14 in dose dependent manner. NS1 exposure also increased the release of cytokines IL-4, IL-6, and IL-10 but decreased the release of TNF-α and IL-15. Interestingly, paracetamol reversed NS1 induced changes in the release of these cytokine in dose dependent manner. CONCLUSION: Monocytes mediated expression of MMPs participates in the development of dengue pathogenesis in the severe cases of disease and paracetamol may have a protective effect in dengue viral disease.

In Silico analysis of voltage-gated sodium channel in relation to DDT resistance in vector mosquitoes.

The voltage-gated sodium channel (VGSC) is the target site for insecticides such as DDT and synthetic pyrethroids. A single base (A-T) change in the knock-down resistance (kdr) allele leads to an amino acid substitution at position 267 that confers the target-mediated resistance to DDT and synthetic pyrethroids in Anopheles gambiae. A theoretical model of the VGSC domain II that contains the site of mutation was constructed using the K;+ channel protein of Aeropyrum pernix as a template. The validated model with 88.6% residues in the favored region was subjected to the CASTp program that predicted 30 pockets in the modeled domain II for ligand interaction. In the model, at position 267, leucine was manually replaced with phenylalanine. When this altered model was subjected to the CASTp program, the search results showed the same number of pockets. The docking results indicate that DDT interacts with the modeled VGSC domain II at position 275 in the presence of leucine or in the presence of phenylalanine (binding energy =-5.32 kcal/mol, -6.21 kcal/mol). It appears from the results that the mutation at position 267 has no direct influence on the interaction of DDT with the target protein. Therefore, to understand the interaction affinity of DDT with the target and influence of the mutation on the existence of active sites/pockets in relation to ligand binding, a whole VGSC model is necessary.