PRIMARY CELL CULTURE OF AEDES ALBOPICTUS MIDGUT CELLS: A PROSPECTIVE MODEL FOR IN VITRO STUDY OF ARBOVIRUSES

  Objective: Midgut cells play a key role in the propagation of mosquito borne Arboviruses. The existing mosquito cell lines for studying viral pathogenesis are derived either from larvae or from eggs since there is no cell line available from the mosquito midgut. Therefore, to delineate the in situ viral interaction which naturally occurs within the mosquito midgut and represent cellular pathogenesis in human beings, the present work was aimed to develop a primary cell line from the midgut cells of Aedes albopictus.Methods: The midgut cells of A. albopictus were collected, cultured and incubated at 28°C to study the growth after every 24 hrs for 7 days.Result: The primary cell culture showed an increasing growth pattern of columnar cells up to 48 hrs followed by decrease in cell population afterward. However, the number of stem cells increased significantly throughout the study period, and their population outnumbered the columnar cells after 72 hrs. There was no significant change of goblet cells and regenerating cells which were scanty in number throughout the experiment.Conclusion: The present method will help to develop the individual cell lines from mosquito midgut and study the host pathogen interaction in arboviral diseases in future

Understanding the Effect of Chikungunya Virus Infection on Bone Homeostasis

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes Chikungunya fever (CHIKF), characterized by high fever, headache, rash, and joint pain. With global travel on the rise, infections caused by CHIKV have emerged in new regions, expanding their geographic range and infecting susceptible hosts. Currently, no approved prophylactic or therapeutic measures exist to prevent or treat these infections, with prevention strategies primarily focused on vector control. However, these measures have proven inadequate due to the emergence of mutations that enhance viral transmission or vector range. Therefore, urgent attention is needed to develop therapies to treat the infection. Although CHIKV has been reported to cause bone pathology, the underlying molecular mechanisms are not well understood. This study aims to investigate the effects of CHIKV infection on the function of bone marrow-derived mesenchymal stem cells (BMSCs), which are known to contribute to bone formation and regulate immune responses during infection and inflammation. In the following studies, we hypothesize that CHIKV alters the properties and function of BMSCs, leading to bone pathology and suppression of immune responses during infection. We investigated the effect of infection on matrix mineralization using staining and enzymatic assays during in vitro differentiation of BMSCs into osteogenic cells. In the presence of CHIKV infection, we observed reduced production of alkaline phosphatase enzyme and deposition of calcium crystals, essential for matrix mineralization during bone formation. Thus, osteogenic differentiation of BMSCs is impaired during CHIKV infection, as evidenced by the decrease in matrix mineralization (production of alkaline phosphate and calcium crystals). Using NanoString gene expression, we demonstrate that CHIKV infection alters the expression of several host factors associated with bone homeostasis, including major osteogenic transcription factors. Validation of gene expression using qRT-PCR analysis showed significant alteration in CTGF, ATF4, and c-FOS expression. Notably, the expression of c-FOS was upregulated 20-fold in infected cells compared to mock cells. c-FOS is a transcription factor that is found to be involved in a few viral pathogenesis. So, we treated cells with T-5224, a known inhibitor of c-FOS, and detected the effect of T-5224 on viral nsP1 gene expression during CHIKV infection. We observed a decreased viral nsP1 gene expression in T-5224 treated cells, indicating a role of c-FOS on nsP1 expression. In our second project, flow cytometry analysis revealed that infection induces the immunosuppressive effect of BMSCs on PBLs, leading to a decreased PBL population, and increased expression of markers associated with immunosuppressive function, including IDO1, iNOS, PDL-1, TGFβ1, PDL-1, and GAL9. The expression of immunosuppressive genes i.,e. IDO1, iNOS, and PGE2 were upregulated during CHIKV infection, with a ∼20-fold induction in the expression of IDO1. IDO1 is an enzyme that plays a critical role in the modulation of immune responses and the induction of immunosuppression. We also observed a significantly high expression of surface marker GAL9 (∼40 fold) during infection. GAL9 is involved in immune regulation and inflammation modulation, interacting with immune cells, and regulating cell activation, apoptosis, and cytokine production. Thus, we developed a novel in vitro cell model for studying CHIKV pathogenesis and demonstrated a possible mechanism of bone pathology during CHIKV infection. Furthermore, we identified host factors as potential targets for antiviral/anti-inflammatory treatment modality in CHIKV infection

Role of MicroRNAs in Bone Pathology during Chikungunya Virus Infection

Chikungunya virus (CHIKV) is an alphavirus, transmitted by mosquitoes, which causes Chikungunya fever with symptoms of fever, rash, headache, and joint pain. In about 30%–40% of cases, the infection leads to polyarthritis and polyarthralgia. Presently, there are no treatment strategies or vaccine for Chikungunya fever. Moreover, the mechanism of CHIKV induced bone pathology is not fully understood. The modulation of host machinery is known to be essential in establishing viral pathogenesis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate major cellular functions by modulating gene expression. Fascinatingly, recent reports have indicated the role of miRNAs in regulating bone homeostasis and altered expression of miRNAs in bone-related pathological diseases. In this review, we summarize the altered expression of miRNAs during CHIKV pathogenesis and the possible role of miRNAs during bone homeostasis in the context of CHIKV infection. A holistic understanding of the different signaling pathways targeted by miRNAs during bone remodeling and during CHIKV-induced bone pathology may lead to identification of useful biomarkers or therapeutics

Serum testosterone levels in type 2 diabetes mellitus

Introduction: Diabetes mellitus is a multifactorial disease which is characterised by hyperglycaemia, dyslipidaemia, involves various organ systems, and results in various long-term complications. Several studies have suggested that men with low testosterone levels are at a greater risk of developing type 2 diabetes mellitus, and that low testosterone levels may even predict the onset of diabetes. Recent studies have shown that a low serum testosterone level is strongly associated with an increased likelihood of the metabolic syndrome. Aim: To compare the serum total testosterone levels in type 2 diabetes mellitus patients with that of non-diabetic healthy controls. Material and Methods: The study was conducted in OPD of Medical College, Kolkata. In the present study 50 men aged 35-55 years who were diagnosed as type 2 diabetes mellitus patients and confirmed by the estimation of fasting plasma glucose (≥126mg/dl), post prandial blood glucose (≥200mg/dl) and HbA1C (≥6.5%) were selected, 50 healthy age and BMI matched individuals, were selected as controls. Patients with a known history of hypogonadism, panhypopituitarism, hyperthyroidism, patients taking exogenous testosterone and glucocorticoids, patients suffering from chronic debilitating disease, such as renal failure, cardiac failure, liver cirrhosis, or HIV, were excluded from the study. The laboratory investigations included evaluation of serum testosterone levels, fasting and postprandial blood glucose, with the levels of HbA1c and Creatinine. Statistical analysis was performed using SPSS 20.0. Results are represented as mean±SD and number (%). Pearson’s correlation test was performed to measure the linear dependence of the study parameters. Results: Serum Total Testosterone level of diabetic group was 3.51±1.26ng/ml, which was found significantly lower than control group with serum total testosterone level 5.88±2.34ng/ml, (p-value < 0.0001). Conclusion: This study has shown that there is a significant reduction in serum total testosterone levels in type 2 diabetes mellitus patients

An observation on direct changes in Aedes albopictus midgut cells by Rhus tox 6C in relation to dengue virus infection

Background and Objectives: In mosquito vectors, dengue virus (DENV) invasion occurs through midgut cells, but available mosquito cell lines for in vitro study of DENV are prepared from eggs or larvae, which are not appropriate models, to study its infectivity. Hence, we developed a new primary cell culture, from Aedes albopictus mosquito midgut, and standardized it for in vitro study of DENV, with an aim to find out any possible role of homoeopathic medicines, in preventing or reducing DENV invasiveness in these midgut cells. This midgut primary cell culture demonstrated prominent cytopathic effects on infection with wild DENV isolated from dengue-infected patients in viremic phase. Materials and Methods: In this paper, we observed the direct effect of homoeopathic medicine Rhus toxicodendron 6C (Rhus tox 6C) (ultra dilution of 10−12 ) on this primary cell culture, to find out significant changes, to be used as baseline data in future experiments to observe possible role of Rhus tox 6C against DENV infection in these cells. Hence, these direct changes may be a prerequisite for the action of this medicine against DENV invasion; as this is one of common repertoire homoeopathic medicines used against dengue fever. Conclusion and Discussion: In our experiments, we found that Rhus tox 6C could increase cell size and help organization of cells on the solid surface as observed under scanning electron microscope although the total number of cells was decreased. Moreover, Rhus tox 6C treated cells were healthier as indicated by less number of deformed, clump, and diploform cells