Using high-throughput sequencing for investigating intra-host hepatitis C evolution over long retrospective periods.

Collections of biological samples held by hospitals represent invaluable resources for conducting retrospective evolutionary studies of chronic infections. Using high-throughput sequencing, those collections permit analysis of within-host genetic diversity over long follow-up periods, and allow a better understanding of resistance to treatment regimes during disease evolution. Here, we studied the evolution of hepatitis C virus (HCV) populations in two patients with an absence of response to dual therapies. We implemented amplicon sequencing to survey genomic variation at the Core and NS5B regions of HCV over a period of 13 years from blood samples obtained at multiple time points. We observed mixed infection by multiple HCV genotypes in both patients. Genetic heterogeneity and sample composition analysis provided information about the changes in viral population over the course of clinical treatment, with NS5B experiencing an increase in diversity after treatment initiation. Secondary infections were estimated to predate treatment year, and our results pointed towards diversifying selection occurring post-treatment, acting on standing genomic variation and maintaining high genetic heterogeneity during infection. For these two patients infected with multiple HCV genotypes, the maintenance of viral diversity was explained with the hypothesis of soft selective sweep started at the same time as antiviral treatment was initiated.

Cellular and Molecular Mechanisms of Autoimmunity and Lupus Nephritis.

Autoimmunity involves immune responses directed against self, which are a result of defective self/foreign distinction of the immune system, leading to proliferation of self-reactive lymphocytes, and is characterized by systemic, as well as tissue-specific, inflammation. Numerous mechanisms operate to ensure the immune tolerance to self-antigens. However, monogenetic defects or genetic variants that weaken immune tolerance render susceptibility to the loss of immune tolerance, which is further triggered by environmental factors. In this review, we discuss the phenomenon of immune tolerance, genetic and environmental factors that influence the immune tolerance, factors that induce autoimmunity such as epigenetic and transcription factors, neutrophil extracellular trap formation, extracellular vesicles, ion channels, and lipid mediators, as well as costimulatory or coinhibitory molecules that contribute to an autoimmune response. Further, we discuss the cellular and molecular mechanisms of autoimmune tissue injury and inflammation during systemic lupus erythematosus and lupus nephritis.

Effects of commonly used antidiabetic drugs on antioxidant enzymes and liver function test markers in type 2 diabetes mellitus subjects - pilot study.

OBJECTIVE: The present study analyzed the effects of antidiabetic drugs on antioxidant enzymes and liver function test (LFT) markers and their association with homeostatic model assessment of insulin resistance (HOMA-IR) in type 2 diabetic subjects. METHODS: We assessed healthy and diabetic subjects (100 each). Diabetic subjects were divided based on treatment with only metformin, metformin in combination with other antidiabetic drugs and insulin in combination with other antidiabetic drugs. LFT markers, antioxidant status and HOMA-IR were assessed in the subjects. RESULTS: Superoxide dismutase activity was higher (p<0.01) while catalase activity was lower (p<0.01) in the diabetic subjects as compared to controls. Serum glutamate-pyruvate transaminase (SGPT) (p<0.01) and bilirubin (p<0.05) levels were higher in diabetic male subjects while urea (p<0.05) levels were lower and SGPT (p<0.01) levels were higher in diabetic female subjects. In male subjects consuming only metformin, a positive association between HOMA-IR and insulin (p<0.05) was seen. A positive association between HOMA-IR and glucose (p<0.01), insulin (p<0.01), SOD (p<0.01) and SGPT (p<0.05) was seen in males receiving metformin with other drugs. Interestingly, the female subjects on metformin displayed a positive association between HOMA-IR and insulin (p<0.05) only. A positive association of HOMA-IR with glucose (p<0.01) and insulin (p<0.05) was seen in females on metformin in combination with other anti-diabetic drugs. CONCLUSION: The alterations in the antioxidant enzyme activities and liver function tests are dependent upon the gender and glycemic status of subjects while the variations in correlations of HOMA-IR with antioxidant enzymes, liver function tests and inflammatory markers are dependent on type of treatments.

Association of Fasting Plasma Glucose and Serum Lipids in Type 2 Diabetics.

Dyslipidemia is a significant morbidity associated with diabetes and cardiovascular disorders. The present study was undertaken to assess the lipid profile of type 2 diabetic and age-gender matched healthy subjects and its association, if any, with fasting plasma glucose. Clinically diagnosed diabetic subjects were recruited for the study. The fasting plasma glucose and lipid profiles were analyzed for 99 diabetic and 101 healthy volunteers. The blood samples were analyzed for fasting plasma glucose, total cholesterol, triglycerides, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol and very low density lipoprotein-cholesterol. Correlation analysis of lipid profile with fasting plasma glucose and calculation of risk ratio was done. The levels of high density lipoprotein-cholesterol and low density lipoprotein-cholesterol were found to be significantly low in diabetics and subjects with lower low density lipoprotein-cholesterol were on statins. Inspite of lower lipid values, the risk ratio for diabetics was significantly higher. The correlation analysis indicated significant difference in relationship between fasting plasma glucose, lipid parameters and risk ratios in the two groups. Diabetics with lower high density lipoprotein-cholesterol and higher total cholesterol present with a higher risk ratio pointing to need of non-statin high density lipoprotein-raising medications decreasing their predisposition to cardiovascular disorders. The study highlights the altered pattern of correlation of lipid profile with fasting plasma glucose in diabetics and their increased risk of cardiovascular disorders. The dyslipidemia in the form of triglyceridemia and significantly low high density lipoprotein-cholesterol in diabetics point towards the need of non-statin high density lipoprotein-raising medications.

Comparative immunomodulation potential of Tinospora cordifolia (Willd.) Miers ex Hook. F., Tinospora sinensis (Lour.) Merrill and Tinospora cordifolia growing on Azadirachta indica A. Juss.

Guduchi has been widely used in the traditional medicine as an immunomodulator. Description of guduchi in Ayurvedic literature resemble with T. sinensis rather than with commonly available T. cordifolia and hence this may be used as substitutes for T. sinensis. T. cordifolia growing on Azadirachta indica commonly called Neem-guduchi has more immunomodulatory potential. Thus, immunomodulatory activity of three Tinospora spp. was assessed by checking humoral and cell mediated immune responses to the antigenic challenges with sheep RBCs and by neutrophil adhesion tests on albino Wistar rats using Guduchi-Satwa, a well known dosage form. Results revealed that Neem-guduchi possesses higher immunomodulatory potential at the dose of 300 mg/kg, po and validated the traditional claim. Hence, Neem-Guduchi can be employed in immunomodulatory formulation prepared using guduchi.

The "floating door sign" in Parkinson's disease.

Individuals with Parkinson's disease (PD) display micrographia. We report a new method to elicit micrographic drawing in individuals with PD and compare this to drawing in individuals with essential tremor (ET). We asked 81 individuals with PD and 19 individuals with ET to draw a house and write a sentence. We examined house height and whether vertical lines of the door connected to the house floor. If both vertical door lines failed to reach the floor by more than 1mm we designated this a "floating door sign". House height of <5 cm and letter height of <5 mm were considered micrographic drawing and writing. 45 of 81 PD patients displayed a "floating door sign" compared with only 4 of 19 ET patients (p = 0.0103). 24 of 81 PD patients compared with 1 of 19 ET patients had micrographic writing (p = 0.0224). 60 of 81 PD patients compared with 9 of 19 ET patients had micrographic drawing (p = 0.00526). The "floating door sign" correlated with micrographic writing (p = 0.0275) but not micrographic drawing. The "floating door sign" had a positive predictive value for PD but not ET. We believe it correlates with hypometeric hand movements which cause inadequate stroke size, a phenomenon described in PD.

Podocytes produce homeostatic chemokine stromal cell-derived factor-1/CXCL12, which contributes to glomerulosclerosis, podocyte loss and albuminuria in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Chemokine (C-X-C motif) ligand 12 (CXCL12) (also known as stromal cell-derived factor-1 [SDF-1]-alpha) is a homeostatic chemokine with multiple roles in cell homing, tumour metastasis, angiogenesis and tissue regeneration after acute injuries. However, its role in chronic diseases remains poorly defined, e.g. in chronic glomerular diseases like diabetic glomerulosclerosis. We hypothesised that CXCL12 may have a functional role during the evolution of diabetic glomerulosclerosis, either by assisting glomerular repair or by supporting the maladaptive tissue remodelling in response to hyperglycaemia and glomerular hyperfiltration. METHODS: To define the functional role of CXCL12 in the progression of glomerular disease, we used the CXCL12-specific inhibitor NOX-A12, an L: -enantiomeric RNA oligonucleotide (Spiegelmer). A mouse model of type 2 diabetes (db/db mice) was used. Male db/db mice, uni-nephrectomised at 6 weeks of age, received subcutaneous injections with a PEGylated form of NOX-A12, non-functional control Spiegelmer or vehicle on alternate days from 4 to 6 months of age. RESULTS: Immunostaining localised renal CXCL12 production to glomerular podocytes in db/db mice with early or advanced diabetic nephropathy. CXCL12 inhibition significantly reduced the degree of glomerulosclerosis, increased the number of podocytes, prevented the onset of albuminuria and maintained the peritubular vasculature without affecting blood glucose levels, body weight or glomerular macrophage infiltration. CONCLUSIONS/INTERPRETATION: We conclude that podocytes produce CXCL12, which contributes to proteinuria and glomerulosclerosis in our mouse model of type 2 diabetes. This novel pathomechanism provides the first evidence that CXCL12 could be a therapeutic target in (diabetic) glomerulosclerosis.

[CCL2/MCP1: a novel target in systemic lupus erythematosus and lupus nephritis]. / CCL2/MCP-1 als neues Target beim systemischen Lupus erythematodes und der Lupusnephritis.

Cytokine blockade, a valid therapeutic concept, is not established in lupus nephritis as yet. In lupus nephritis CCL2/MCP-1 and its chemokine receptor CCR2 are of interest because CCL2/CCR2 mediate the recruitment of macrophages and T cells in the nephritic kidney. Lupus nephritis is markedly attenuated in CCL2- or CCR2-deficient autoimmune mice. Epidemiological studies addressing mutations in the CCL2 gene support the hypothesis that CCL2 mediates renal inflammation. Meanwhile experimental studies have shown that several classes of CCL2 antagonists can control established lupus nephritis. Interestingly, therapeutic CCL2 blockade does not affect the autoimmune lymphoproliferative syndrome and the production of lupus autoantibodies. This article briefly summarizes the potential role of therapeutic CCL2 blockade in lupus nephritis.

Tubular atrophy, interstitial fibrosis, and inflammation in type 2 diabetic db/db mice. An accelerated model of advanced diabetic nephropathy.

OBJECTIVE: Advanced diabetic nephropathy (DN) is difficult to address experimentally in mice because available models of DN lack global glomerulosclerosis and major tubulointerstitial pathology. Accelerating the development of DN in mice would be desirable for feasible experimental validation of potential targets that mediate the progression to late stage DN. METHODS: 6 week old male db/db mice underwent uninephrectomy and the development of nephropathy was compared to wild-type mice and sham-operated db/db mice. RESULTS: Uninephrectomy at young age was associated with increased albuminuria and severe glomerulosclerosis in 37% of glomeruli at 24 weeks of age as compared to sham-operated db/db mice (8%). Uninephrectomy also increased the number of glomerular macrophages in db/db mice. The uninephrectomy-related acceleration of glomerular damage was associated with significant tubulointerstitial injury as indicated by an increase in indices of tubular cell damage, tubular dilatation, and expansion of interstitial volume. Uninephrectomy markedly increased the renal mRNA expression of Mcp-1/Ccl2, Tgf-beta, and collagen I. CONCLUSION: Early uninephrectomy can accelerate the development of advanced DN in db/db mice which may be instrumental in the design of interventional studies that intend to focus on the molecular pathology of the progression to late stage DN.