PARP1-cGAS-NF-κB pathway of proinflammatory macrophage activation by extracellular vesicles released during Trypanosoma cruzi infection and Chagas disease.

Trypanosoma cruzi (T. cruzi) is the etiological agent of Chagas cardiomyopathy. In the present study, we investigated the role of extracellular vesicles (Ev) in shaping the macrophage (Mφ) response in progressive Chagas disease (CD). We purified T. cruzi Ev (TcEv) from axenic parasite cultures, and T. cruzi-induced Ev (TEv) from the supernatants of infected cells and plasma of acutely and chronically infected wild-type and Parp1-/- mice. Cultured (Raw 264.7) and bone-marrow Mφ responded to TcEV and TEv with a profound increase in the expression and release of TNF-α, IL-6, and IL-1ß cytokines. TEv produced by both immune (Mφ) and non-immune (muscle) cells were proinflammatory. Chemical inhibition or genetic deletion of PARP1 (a DNA repair enzyme) significantly depressed the TEv-induced transcriptional and translational activation of proinflammatory Mφ response. Oxidized DNA encapsulated by TEv was necessary for PARP1-dependent proinflammatory Mφ response. Inhibition studies suggested that DNA-sensing innate immune receptors (cGAS>>TLR9) synergized with PARP1 in signaling the NFκB activation, and inhibition of PARP1 and cGAS resulted in >80% inhibition of TEv-induced NFκB activity. Histochemical studies showed intense inflammatory infiltrate associated with profound increase in CD11b+CD68+TNF-α+ Mφ in the myocardium of CD wild-type mice. In comparison, chronically infected Parp1-/- mice exhibited low-to-moderate tissue inflammation, >80% decline in myocardial infiltration of TNF-α+ Mφ, and no change in immunoregulatory IL-10+ Mφ. We conclude that oxidized DNA released with TEv signal the PARP1-cGAS-NF-κB pathway of proinflammatory Mφ activation and worsens the chronic inflammatory pathology in CD. Small molecule antagonists of PARP1-cGAS signaling pathway would potentially be useful in reprogramming the Mφ activation and controlling the chronic inflammation in CD.

Acute monocyte pro inflammatory response predicts higher positive to negative acute phase reactants ratio and severe hemostatic derangement in dengue fever.

PURPOSE: The present study was intended to investigate whether monocyte immune activation shapes plasma positive to negative acute phase reactants (APRs) ratio and predicts disease severity in dengue infection. METHODS: Serum level of ferritin, ceruloplasmin and transferrin was measured by means of electrochemiluminescence and immunoturbidimetry, respectively. Gene expression and plasma level for TNF-α, IL-6 and IL1-ß was measured by means of RT-qPCR and ELISA. RESULTS: A significant increased serum ferritin to transferrin [6.6 (3-11.7) vs 3.4 (1.9-6.1)] and ceruloplasmin to transferrin ratio [0.48 (0.21-0.87) vs 0.22 (0.13-0.43)] has been detected among the subjects with secondary dengue infection (SDENI) compared to primarily infected (PDENI) subjects (P < 0.001). Significant increased expression for CD14+ monocyte TNF-α, IL-6 and IL-1ß has been detected in SDENI patients (vs PDENI and control, P < 0.001). Plasma ferritin to transferrin ratio was found in a significant association with high level of plasma TNF-α [ρ = 0.6522, 95% CI (0.4714-0.7805)], IL-6 [ρ = 0.6181, 95% CI (0.4257-0.7571)] and IL- 1ß [ρ = 0.4119, 95% CI (0.1689-0.6077)] level among SDENI patients at 5th day time point after progression of the disease, with significantly low platelet [P < 0.001] and prolonging prothrombin time [P < 0.001] compared to control and PDENI subjects, respectively. CONCLUSION: Acute proinflammatory cytokine response is significantly associated with increased positive to negative APRs ratio in SDENI patients, which predicts intense immune activation, and renders SDENI patients extremely susceptible to hemostatic derangement.

Integrated Functional Analysis of the Nuclear Proteome of Classically and Alternatively Activated Macrophages.

Macrophages (Mφ) play a central role in coordinating host response to pathogens, cellular injury, and environmental stimuli. Herein, we report multidimensional, nuclear proteomic analyses of protein expression and posttranslational modifications (PTMs) that control biological processes during Mφ activation. For this, Mφ were incubated with IFN-γ/LPS and IL-4, and their differentiation to proinflammatory (M1) and anti-inflammatory (M2a, referred as M2 for simplicity throughtout the manuscript) phenotypes was confirmed by detection of CD64 and CD206 surface markers and TNF-α, arginase I, and iNOS-dependent nitrite levels. We used a sequential method of organellar enrichment and labeling of nuclear fractions with BODIPY FL-maleimide fluorescence dye followed by two-dimensional electrophoresis (2DE) to capture quantitative changes in abundance and S-nitrosylated (SNO) proteome signatures. Exact same gels were then labeled with Pro-Q Diamond to detect protein phosphorylation. MALDI-TOF/TOF MS analysis of the protein spots with fold change of ≥|1.5| in any of the groups yielded 229 identifications. We found that 145, 78, and 173 protein spots in M1 Mφ and 105, 81, and 164 protein spots in M2 Mφ were changed in abundance, S-nitrosylation, and phosphorylation, respectively, with respect to M0 controls (fold change: ≥|1.5|, p ≤ 0.05). Targeted analysis by immunoprecipitation and Western blotting was performed to verify the differential abundance and phosphorylation levels of two of the proteins in M1 and M2 (vs. M0) Mφ. Ingenuity Pathway Analysis of the nuclear proteome datasets showed that the abundance and posttranslational (SNO and Phosphor) modifications of the proteins predicted to be involved in cytoskeletal organization/cell movement, phagocytosis/endocytosis, and cell proliferation/cell death were differentially regulated with proinflammatory and anti-inflammatory activation of Mφ.

DETERIORARON OF OVARIAN FUNCTION AFTER TOTAL ABDOMINAL HYSTERECTOMY WITH PRESERVARON OF OVARIES.

OBJECTIVE: To evaluate ovarian function after total abdominal hysterectomy in premenopausal women. METHODS: In the present cross-sectional study, we enrolled 52 healthy female subjects having normal menstrual cycle as controls and 37 female patients (age <45 years) who had undergone total abdominal hysterectomy (TAH) with preservation of at least one ovary for the evaluation of ovarian function. Serum antimüllerian hormone (AMH) and follicle-stimulating hormone (FSH) were measured by enzyme-linked immunosorbent assay in both groups. Transvaginal Doppler ultrasonography was done to measure ovarian stromal blood flow indices (resistive index [RI] and pulsatility index [PI]). The means obtained from different sample groups were compared using the nonparametric Mann-Whitney U test, and correlations between two variables were evaluated using the Spearman nonparametric correlation test. A value of P<.05 was considered statistically significant. RESULTS: Mean postoperative duration of patients who had undergone hysterectomy was 2.5 years. Mean serum AMH level was 7.68 ± 6.70 ng/mL in the cases, significantly lower than the level in controls (10.98 ± 7.83 ng/mL) (P = .016). Serum FSH level in controls was 12.01 ± 6.27 µIU/mL, which was significantly higher in the cases (20.27 ± 12.91 µIU/mL) (P = .001). An inverse correlation between serum AMH and FSH was observed (P = .0006; r = -0.4583). However, the ovary RI and PI values in both groups were similar. CONCLUSION: TAH affects ovarian function, despite normal ovarian blood supply. ABBREVIATIONS: AMH = antimüllerian hormone FSH = follicle-stimulating hormone RI = resistive index PI = pulsatility index TAH = total abdominal hysterectomy.

Role of NF-κB activation and VEGF gene polymorphisms in VEGF up regulation in non-proliferative and proliferative diabetic retinopathy.

The present study was aimed to investigate the relation between nuclear factor kappa beta (NFκB) activation and downstream up-regulation of vascular endothelial growth factor (VEGF) in diabetic retinopathy (DR). Moreover the study was intended to evaluate the role of VEGF gene single nucleotide polymorphisms (SNPs) in DR occurrence and to investigate the functional relevance of VEGF gene SNPs in terms of VEGF expression in DR. Serum level of VEGF, VEGF R1 (receptor 1), VEGF R 2 (receptor 2) and NFκB (p50/65) activity was measured by enzyme linked immune sorbent assay. Genotyping and allelic composition of different SNPs i.e., rs2010963, rs3025039, rs1570360 and rs 2071559 were investigated by Taqman SNP genotyping assay. VEGF, NFκB p50/p65, and VEGF R1 & R2 gene expressions were quantified by real time quantitative polymerase chain reaction. Increased NFκB p50/p65 activity and expressions were observed in non proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) subjects compared to type 2 diabetes mellitus without retinopathy (DNR) group. Significantly elevated levels of serum VEGF and highest VEGF expression were found among PDR subjects compared to DNR or NPDR subjects. CC genotype and C allele of rs2010963 and TT genotype and T allele of rs3025039 were significantly over represented among PDR subjects compared to DNR group. Increased activation of NFκß in NPDR and PDR subjects might involve increased up regulation of VEGF. VEGF SNPs i.e., rs2010963 C allele and rs3025039 T allele might be associated with PDR occurrence and in turn regulates VEGF expression among PDR subjects.

Role of N-ε- carboxy methyl lysine, advanced glycation end products and reactive oxygen species for the development of nonproliferative and proliferative retinopathy in type 2 diabetes mellitus.

PURPOSE: The aim of the present study was to evaluate the collective role of N-epsilon-carboxy methyl lysine (N(ε)-CML), advanced glycation end-products (AGEs), and reactive oxygen species (ROS) for the development of retinopathy among type 2 diabetic subjects. METHODS: Seventy type 2 diabetic subjects with nonproliferative diabetic retinopathy (NPDR), 105 subjects with proliferative diabetic retinopathy (PDR), and 102 patients with diabetes but without retinopathy (DNR) were enrolled in this study. In addition, 95 normal individuals without diabetes were enrolled as healthy controls in this study. Serum and vitreous N(ε)-CML and AGEs were measured by enzyme-linked immunosorbent assay. The peripheral blood mononuclear cell (PBMC) ROS level was measured by flow cytometric analysis. Serum and PBMC total thiols were measured by spectrophotometry. RESULTS: Serum AGEs and N(ε)-CML levels were significantly elevated in subjects with PDR (p<0.0001) and NPDR (p=0.0297 and p<0.0001, respectively) compared to DNR subjects. Further vitreous AGEs and N(ε)-CML levels were found to be significantly high among PDR subjects compared to the control group (p<0.0001). PBMC ROS production was found to be strikingly high among NPDR (p<0.0001) and PDR (p<0.0001) subjects as compared to the DNR group. Serum and PBMC total thiol levels were remarkably decreased in NPDR (p<0.0001 and p=0.0043, respectively) and PDR (p=0.0108 and p=0.0332 respectively) subjects than those were considered as DNR. CONCLUSIONS: Our findings suggest that N(ε)-CML and ROS are the key modulators for the development of nonproliferative retinopathy among poorly controlled type 2 diabetic subjects. Furthermore, AGEs under persistent oxidative stress and the deprived antioxidant state might instigate the pathogenic process of retinopathy from the nonproliferative to the proliferative state.

Role of hyperglycemia-mediated erythrocyte redox state alteration in the development of diabetic retinopathy.

PURPOSE: To evaluate erythrocyte redox state and its surrogates in patients with different stages of diabetic retinopathy and their association with cellular metabolic derangement developed in retinal microvascular cells. METHODS: Sixty type 2 diabetic patients with nonproliferative diabetic retinopathy (NPDR), 85 patients with proliferative diabetic retinopathy (PDR), and 70 patients with diabetes but without retinopathy were considered as diabetic control (DC) for the study. In addition, 65 normal individuals without diabetes were enrolled as healthy control in this study. Erythrocyte oxidized nicotinamide adenine dinucleotide phosphate / reduced nicotinamide adenine dinucleotide phosphate (NADP / NADPH), oxidized nicotinamide adenine dinucleotide / reduced nicotinamide adenine dinucleotide (NAD / NADH) glutathione, plasma and vitreous lactate, and pyruvate levels were determined by enzymatic reaction-based spectrophotometric assay for the patients and individuals. RESULT: Erythrocyte NADP+ to NADPH ratio to NADPH ratio was found to be significantly higher among NPDR and PDR patients compared with DC subjects (P < 0.0001). Erythrocyte-reduced glutathione was significantly decreased in patients of NPDR (P = 0.0004) and patients of PDR (P = 0.0157) compared to DC. Erythrocyte NAD to NADH ratio was also significantly decreased in patients of NPDR (P < 0.0001) and PDR (P < 0.0001) compared to DC subjects. Lactate to pyruvate ratio of plasma was elevated significantly in patients with NPDR compared with DC (P < 0.0001) and those having PDR (P = 0.0046). In the vitreous fluid, the lactate to pyruvate ratios were found to be significantly lower in normal individuals without diabetes compared with patients having PDR (P < 0.0001). CONCLUSION: Hyperglycemia-mediated erythrocyte redox state alterations might be a potential risk factor for the development of NPDR in poorly controlled diabetic subjects.

Subunit nanovaccine elicited T cell functional activation controls Trypanosoma cruzi mediated maternal and placental tissue damage and improves pregnancy outcomes in mice.

This study investigated a candidate vaccine effect against maternal Trypanosoma cruzi (Tc) infection and improved pregnancy outcomes. For this, TcG2 and TcG4 were cloned in a nanoplasmid optimized for delivery, antigen expression, and regulatory compliance (nano2/4 vaccine). Female C57BL/6 mice were immunized with nano2/4, infected (Tc SylvioX10), and mated 7-days post-infection to enable fetal development during the maternal acute parasitemia phase. Females were euthanized at E12-E17 (gestation) days. Splenic and placental T-cell responses were monitored by flow cytometry. Maternal and placental/fetal tissues were examined for parasites by qPCR and inflammatory infiltrate by histology. Controls included age/immunization-matched non-pregnant females. Nano2/4 exhibited no toxicity and elicited protective IgG2a/IgG1 response in mice. Nano2/4 signaled a splenic expansion of functionally active CD4+ effector/effector memory (Tem) and central memory (Tcm) cells in pregnant mice. Upon challenge infection, nano2/4 increased the splenic CD4+ and CD8+T cells in all mice and increased the proliferation of CD4+Tem, CD4+Tcm, and CD8+Tcm subsets producing IFNγ and cytolytic molecules (PRF1, GZB) in pregnant mice. A balanced serum cytokines/chemokines response and placental immune characteristics indicated that pregnancy prevented the overwhelming damaging immune response in mice. Importantly, pregnancy itself resulted in a significant reduction of parasites in maternal and fetal tissues. Nano2/4 was effective in arresting the Tc-induced tissue inflammatory infiltrate, necrosis, and fibrosis in maternal and placental tissues and improving maternal fertility, placental efficiency, and fetal survival. In conclusion, we show that maternal nano2/4 vaccination is beneficial in controlling the adverse effects of Tc infection on maternal health, fetal survival, and pregnancy outcomes.

Acute muscle mass loss was alleviated with HMGB1 neutralizing antibody treatment in severe burned rats.

Burn injury is associated with muscle wasting, though the involved signaling mechanisms are not well understood. In this study, we aimed to examine the role of high mobility group box 1 (HMGB1) in signaling hyper-inflammation and consequent skeletal muscle impairment after burn. Sprague Dawley rats were randomly assigned into three groups: (1) sham burn, (2) burn, (3) burn/treatment. Animals in group 2 and group 3 received scald burn on 30% of total body surface area (TBSA) and immediately treated with chicken IgY and anti-HMGB1 antibody, respectively. Muscle tissues and other samples were collected at 3-days after burn. Body mass and wet/dry weights of the hind limb muscles (total and individually) were substantially decreased in burn rats. Acute burn provoked the mitochondrial stress and cell death and enhanced the protein ubiquitination and LC3A/B levels that are involved in protein degradation in muscle tissues. Further, an increase in muscle inflammatory infiltrate associated with increased differentiation, maturation and proinflammatory activation of bone marrow myeloid cells and αß CD4+ T and γδ T lymphocytes was noted in in circulation and spleen of burn rats. Treatment with one dose of HMGB1 neutralizing antibody reduced the burn wound size and preserved the wet/dry weights of the hind limb muscles associated with a control in the markers of cell death and autophagy pathways in burn rats. Further, anti-HMGB1 antibody inhibited the myeloid and T cells inflammatory activation and subsequent dysregulated inflammatory infiltrate in the muscle tissues of burn rats. We conclude that neutralization of HMGB1-dependent proteolytic and inflammatory responses has potential beneficial effects in preventing the muscle loss after severe burn injury.

A molecular approach to identification and profiling of first-line-drug-resistant mycobacteria from sputum of pulmonary tuberculosis patients.

Conventional and molecular techniques were applied to detect and characterize drug resistance of mycobacteria in the sputum samples of clinically confirmed tuberculosis. The sensitivities of mycobacterium detection by ZN staining, culture, multiplex PCR, and restriction fragment length polymorphism (RFLP) were 27.7%, 19.9%, 92.9%, and 95.7%, respectively, but all were 100% specific. The conventional and multiple-allele-specific PCR (MAS-PCR) methods enabled establishment of the drug resistance in 19.3% and 86.9% cases, respectively. We demonstrated that molecular techniques have potential in the accurate diagnosis of tuberculosis.

Association of vascular endothelial growth factor, transforming growth factor beta, and interferon gamma gene polymorphisms with proliferative diabetic retinopathy in patients with type 2 diabetes.

PURPOSE: Chronic hyperglycemia and hypoxemia are believed to be causal factors in the development of proliferative diabetic retinopathy (PDR) among individuals with type 2 diabetes. It is hypothesized that formation of new blood vessels in the retina due to prolonged hypoxia is associated with increased expression of several growth factors and angiogenic cytokines. In the present study, we investigated the association of genetic polymorphisms in vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-ß), and interferon γ (IFN-γ) genes, which may be responsible for the hypoxia-induced VEGF-mediated neovascularization pathway for the pathogenesis of PDR. METHODS: Our case-control association study composed of 493 ethnically matched volunteers (253 with PDR [cases] and 240 diabetic controls [DC]). Gene polymorphisms were determined with Taqman-based real-time PCR and amplification refractory mutation analysis system PCR. RESULTS: The VEGF-460C (rs833061C; p=0.0043) and IFN-γ +874T (rs2430561T; p=0.0011) alleles were significantly associated with PDR. CONCLUSIONS: Genetic variations at VEGF-460C and IFN-γ +874T might accelerate the pathogenesis of retinal neovascularization in PDR.

Association of tumor necrosis factor α, interleukin 6, and interleukin 10 promoter polymorphism with proliferative diabetic retinopathy in type 2 diabetic subjects.

PURPOSE: New blood vessel formation in the retina because of prolonged hypoxia is believed to be directly associated with increased expression of several growth factors and angiogenic cytokines. In the present study, we made an attempt to investigate the possible association of the promoter polymorphisms of interleukin 6, tumor necrosis factor α, and interleukin 10 for the pathogenesis of proliferative diabetic retinopathy (PDR). METHODS: This case-control study comprised 493 volunteers (253 PDR cases and 240 diabetic controls). Cases and controls were ascertained such that age, sex, nutrition, and glycemic status were matched. Genotypes were determined by polymerase chain reaction-based methods. RESULTS: Interleukin 10-1082GG (P = 0.0037; odds ratio [OR] = 2.232), tumor necrosis factor α-238AA (P = 0.0001; OR = 5.791), and GA (P = 0.0015; OR = 1.909) genotypes were significantly associated with PDR occurrence. The interleukin 10-1082G allele (P = 0.0048, OR = 1.4442) and the tumor necrosis factor α-238A allele (P = 0.0001; OR = 2.2897) were significantly increased among PDR cases. CONCLUSION: From our study, it may be concluded that the genetic variation, that is, tumor necrosis factor α-238A and interleukin 10-1082G alleles are the potent risk factors for the pathogenesis of PDR.

Platelets, Macrophages, and Thromboinflammation in Chagas Disease.

Chagas disease (CD) is a major health problem in the Americas and an emerging health problem in Europe and other nonendemic countries. Several studies have documented persistence of the protozoan parasite Trypanosoma cruzi, and oxidative and inflammatory stress are major pathogenic factor. Mural and cardiac thrombi, cardiac arrhythmias, and cardiomyopathy are major clinical features of CD. During T. cruzi infection, parasite-released factors induce endothelial dysfunction along with platelet (PLT) and immune-cell activation. PLTs have a fundamental role in maintaining hemostasis and preventing bleeding after vascular injury. Excessive activation of PLTs and coagulation cascade can result in thrombosis and thromboembolic events, which are recognized to occur in seropositive individuals in early stages of CD when clinically symptomatic heart disease is not apparent. Several host and parasite factors have been identified to signal hypercoagulability and increase the risk of ischemic stroke in early phases of CD. Further, PLT interaction with immune cells and their role in host defense against pathogens and inflammatory processes have only recently been recognized and evolving. In the context of parasitic diseases, PLTs function in directly responding to T. cruzi infection, and PLT interactions with immune cells in shaping the proinflammatory or immunoregulatory function of monocytes, macrophages, and neutrophils remains elusive. How T. cruzi infection alters systemic microenvironment conditions to influence PLT and immune-cell interactions is not understood. In this review, we discuss the current literature, and extrapolate the mechanistic situations to explain how PLT and innate immune cell (especially monocytes and macrophages) interactions might be sustaining hypercoagulability and thromboinflammation in chronic CD.

Impact of interleukin-6 promoter polymorphism and serum interleukin-6 level on the acute inflammation and neovascularization stages of patients with Eales' disease.

PURPOSE: To evaluate the role of interleukin-6 (IL-6) in the inflammatory and proliferative stages of Eales' disease (ED) and to determine the influence of IL-6-174G/C polymorphism in the IL-6 and IL-6-regulated protein expression, as well as the development of ED. METHODS: One hundred and twenty-one patients diagnosed with ED, 223 matched healthy controls, and 16 control patients with macular holes were recruited from the eastern Indian population. Serum and vitreous levels of IL-6 and vascular endothelial growth factors (VEGF) were measured by enzyme-linked immunosorbent assay. Serum levels of high-sensitivity C-reactive protein (hsCRP) were measured by enzyme immunoassay. Subjects were genotyped for the IL-6-174G/C polymorphism (rs1800795) by a custom TaqMan single-nucleotide polymorphism (SNP) Genotyping Assays system. RESULTS: Serum IL-6 (p<0.0001), hsCRP (p<0.0001), and VEGF (p=0.0031) levels were significantly higher in the inflammatory stage of ED than in healthy controls. Serum IL-6 also significantly correlated with hsCRP (Spearman's correlation coefficient; r=0.4992, p=0.0009), but not with VEGF in this stage in ED patients. At the proliferative stage of ED, significantly higher levels of vitreous IL-6 (p=<0.0001) and VEGF (p=<0.0001) were found compared with the vitreous of patients with macular holes. A significant correlation was observed between vitreous IL-6 and VEGF in ED patients (Spearman's correlation coefficient; r=0.5834, p=0.0087). A statistically significant association was found between the -174GG genotype (p=0.006) and occurrence of ED. Mean serum and vitreous concentrations of IL-6 were also higher in the subjects with the GG genotype than in those with the GC or CC genotype in this population. CONCLUSIONS: IL-6 expression, regulated by the allelic distribution of -174 loci and the enhanced level of IL-6, modulates CRP and VEGF concentration depending respectively on the acute inflammatory stimulation at the initial stage and angiogenic stimulation at the advanced stage of ED.

Oxidative stress implications for therapeutic vaccine development against Chagas disease.

INTRODUCTION: Pathogenesis of Chagas disease (CD) caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) involves chronic oxidative and inflammatory stress. In this review, we discuss the research efforts in therapeutic vaccine development to date and the potential challenges imposed by oxidative stress in achieving an efficient therapeutic vaccine against CD. AREAS COVERED: This review covers the immune and nonimmune mechanisms of reactive oxygen species production and immune response patterns during T. cruzi infection in CD. A discussion on immunotherapy development efforts, the efficacy of antigen-based immune therapies against T. cruzi, and the role of antioxidants as adjuvants is discussed to provide promising insights to developing future treatment strategies against CD. EXPERT OPINION: Administration of therapeutic vaccines can be a good option to confront persistent parasitemia in CD by achieving a rapid, short-lived stimulation of type 1 cell-mediated immunity. At the same time, adjunct therapies could play a critical role in the preservation of mitochondrial metabolism and cardiac muscle contractility in CD. We propose combined therapy with antigen-based vaccine and small molecules to control the pathological oxidative insult would be effective in the conservation of cardiac structure and function in CD.

Associations of insulin-induced lipodystrophy in children, adolescents, and young adults with type 1 diabetes mellitus using recombinant human insulin: a cross-sectional study.

OBJECTIVE: Insulin-induced lipodystrophy is of two types, lipohypertrophy and lipoatrophy. Lipodystrophy often leads to worsening of glycemic control in type 1 diabetes mellitus. Our objective was to identify the clinical, immunological, and other factor(s) associated with the development of lipodystrophy. METHODS: In this observational cross-sectional hospital-based study, 95 children, adolescents, and young adults with type 1 diabetes mellitus were observed for the development of lipodystrophy. Injection technique, insulin dose, and glycemic parameters were noted. Serum TNF-α, IL-1ß, and anti-insulin antibody levels were measured. Histopathological examination of the lipodystrophic area was done in a small number of people. RESULTS: Among the participants, 45.2% of participants had lipohypertrophy and 4.2% had lipoatrophy exclusively; 3.1% of participants had coexisting lipohypertrophy and lipoatrophy. Improper injection site rotation technique was more common in participants with lipohypertrophy in comparison to those without lipodystrophy. The age of onset of diabetes, duration of insulin use, and the number of times of needle reuse were not significantly different between the lipohypertrophy and nonlipodystrophy groups. Serum TNF-α, IL-1ß, and anti-insulin antibody levels; HbA1c; rate of hypoglycemia; and body weight-adjusted dose requirement were higher among the participants with lipohypertrophy. On histopathology, scant, or no inflammatory infiltrate was found in lipoatrophic and lipohypertrophic areas, respectively. CONCLUSION: Improper insulin injection technique and higher levels of proinflammatory cytokines and anti-insulin antibody are associated with lipodystrophy in type 1 diabetes mellitus. HbA1c and rate of hypoglycemia are higher in people with lipodystrophy.

Prognostic Performance of Peripheral Blood Biomarkers in Identifying Seropositive Individuals at Risk of Developing Clinically Symptomatic Chagas Cardiomyopathy.

Biomarkers for prognosis-based detection of Trypanosoma cruzi-infected patients presenting no clinical symptoms to cardiac Chagas disease (CD) are not available. In this study, we examined the performance of seven biomarkers in prognosis and risk of symptomatic CD development. T. cruzi-infected patients clinically asymptomatic (C/A; n = 30) or clinically symptomatic (C/S; n = 30) for cardiac disease and humans who were noninfected and healthy (N/H; n = 24) were enrolled (1 - ß = 80%, α = 0.05). Serum, plasma, and peripheral blood mononuclear cells (PBMCs) were analyzed for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), vimentin, poly(ADP-ribose) polymerase (PARP1), 8-hydroxy-2-deoxyguanosine (8-OHdG), copeptin, endostatin, and myostatin biomarkers by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Secreted hnRNPA1, vimentin, PARP1, 8-OHdG, copeptin, and endostatin were increased by 1.4- to 7.0-fold in CD subjects versus N/H subjects (P < 0.001) and showed excellent predictive value in identifying the occurrence of infection (area under the receiver operating characteristic [ROC] curve [AUC], 0.935 to 0.999). Of these, vimentin, 8-OHdG, and copeptin exhibited the best performance in prognosis of C/S (versus C/A) CD, determined by binary logistic regression analysis with the Cox and Snell test (R2C&S = 0.492 to 0.688). A decline in myostatin and increase in hnRNPA1 also exhibited good predictive value in identifying C/S and C/A CD status, respectively. Furthermore, circulatory 8-OHdG (Wald χ2 = 15.065), vimentin (Wald χ2 = 14.587), and endostatin (Wald χ2 = 17.902) levels exhibited a strong association with changes in left ventricular ejection fraction and diastolic diameter (P = 0.001) and predicted the risk of cardiomyopathy development in CD patients. We have identified four biomarkers (vimentin, 8-OHdG, copeptin, and endostatin) that offer excellent value in prognosis and risk of symptomatic CD development. Decline in these four biomarkers and increase in hnRNPA1 would be useful in monitoring the efficacy of therapies and vaccines in halting CD. IMPORTANCE There is a lack of validated biomarkers for diagnosis of T. cruzi-infected individuals at risk of developing heart disease. Of the seven potential biomarkers that were screened, vimentin, 8-OHdG, copeptin, and endostatin exhibited excellent performance in distinguishing the clinical severity of Chagas disease. A decline in these four biomarkers can also be used for monitoring the therapeutic responses of infected patients to established or newly developed drugs and vaccines and precisely inform the patients about their progress. These biomarkers can easily be screened using the readily available plasma/serum samples in the clinical setting by an ELISA that is inexpensive, fast, and requires low-tech resources at the facility, equipment, and personnel levels.

Chronic inflammation in polycystic ovary syndrome: A case-control study using multiple markers.

BACKGROUND: Polycystic ovary syndrome (PCOS) is associated with insulin resistance and elevated risk of cardiovascular disease and diabetes. Chronic inflammation has been observed in PCOS in several studies but there is also opposing evidence and a dearth of research in Indians. OBJECTIVE: To estimate chronic inflammation in PCOS and find its relationship with appropriate anthropometric and biochemical parameters. MATERIALS AND METHODS: Chronic inflammation was assessed in 30 women with PCOS (Group A) and 30 healthy controls (Group B) with highly sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6), tumour necrosis factor alpha (TNFα), and platelet microparticles (PMP). In group A, the relationship of chronic inflammation with insulin resistance, waist hip ratio (WHR) serum testosterone, and serum glutamate pyruvate transaminase (SGPT) were examined. RESULTS: In group A, the hsCRP, TNFα, and PMP were significantly elevated compared to group B. However, IL-6 level was similar between the groups. In group A, PMP showed a significant positive correlation with waist-hip ratio and serum testosterone. IL-6 showed a significant positive correlation with insulin sensitivity and significant negative correlation with insulin resistance and serum glutamate pyruvate transaminase. CONCLUSION: PCOS is associated with chronic inflammation and PMP correlates positively with central adiposity and biochemical hyperandrogenism in women with PCOS.

Trypanosoma cruzi Induces the PARP1/AP-1 Pathway for Upregulation of Metalloproteinases and Transforming Growth Factor ß in Macrophages: Role in Cardiac Fibroblast Differentiation and Fibrosis in Chagas Disease.

Chagas disease (CD), caused by Trypanosoma cruzi, is a degenerative heart condition. In the present study, we investigated the role of poly [ADP-ribose] polymerase 1/activator protein 1 (PARP1/AP-1) in upregulation of profibrotic macrophages (Mϕ) and subsequent development of cardiac fibrosis in CD. We used in vitro and in vivo models of T. cruzi infection and chemical and genetic inhibition of Parp1 to examine the molecular mechanisms by which Mϕ might augment profibrotic events in CD. Cultured (RAW 264.7 and THP-1) Mϕ infected with T. cruzi and primary cardiac and splenic Mϕ of chronically infected mice exhibited a significant increase in the expression, activity, and release of metalloproteinases (MMP2, MMP9, and MMP12) and the cytokine transforming growth factor ß (TGF-ß). Mϕ release of MMPs and TGF-ß signaled the cardiac fibroblast to myofibroblast differentiation, as evidenced by a shift from S100A4 to alpha smooth muscle actin (α-SMA) expression. Incubation of infected Mϕ with MMP2 and MMP9 inhibitors resulted in 60 to 74% decline in TGF-ß release, and MMP9 and PARP1 inhibitors resulted in 57 to 70% decline in Mϕ TGF-ß-driven cardiac fibroblast differentiation. Likewise, histological studies showed a 12- to 16-fold increase in myocardial expression of CD68 (Mϕ marker) and its colocalization with MMP9/TGF-ß, galectin-3, and vimentin in wild-type mice with CD. In comparison, chronically infected Parp1-/- mice exhibited a >50% decline in myocardial levels of Mϕ and associated fibrosis markers. Further study showed that PARP1 synergized with c-Fos and JunB AP-1 family members for transcriptional activation of profibrotic response after T. cruzi infection. We conclude that PARP1 inhibition offers a potential therapy for controlling the T. cruzi-driven fibroblast differentiation in CD through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-ß pathway.IMPORTANCE Cardiomyopathy is the most important clinical manifestation of T. cruzi-driven CD. Recent studies have suggested the detrimental role of the matrix metalloproteinases MMP2 and MMP9 in extracellular matrix (ECM) degradation during cardiac remodeling in T. cruzi infection. Peripheral TGF-ß levels are increased in clinically symptomatic CD patients over those in clinically asymptomatic seropositive individuals. We provide the first evidence that during T. cruzi infection, Mϕ release of MMP2 and MMP9 plays an active role in activation of TGF-ß signaling of ECM remodeling and cardiac fibroblast-to-myofibroblast differentiation. We also determined that PARP1 signals c-Fos- and JunB-mediated AP-1 transcriptional activation of profibrotic gene expression and demonstrated the significance of PARP1 inhibition in controlling chronic fibrosis in Chagas disease. Our study provides a promising therapeutic approach for controlling T. cruzi-driven fibroblast differentiation in CD by PARP1 inhibitors through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-ß pathway.

Mitochondrial Regulation of Macrophage Response Against Pathogens.

Innate immune cells play the first line of defense against pathogens. Phagocytosis or invasion by pathogens can affect mitochondrial metabolism in macrophages by diverse mechanisms and shape the macrophage response (proinflammatory vs. immunomodulatory) against pathogens. Besides ß-nicotinamide adenine dinucleotide 2'-phosphate, reduced (NADPH) oxidase, mitochondrial electron transport chain complexes release superoxide for direct killing of the pathogen. Mitochondria that are injured are removed by mitophagy, and this process can be critical for regulating macrophage activation. For example, impaired mitophagy can result in cytosolic leakage of mitochondrial DNA (mtDNA) that can lead to activation of cGAS-STING signaling pathway of macrophage proinflammatory response. In this review, we will discuss how metabolism, mtDNA, mitophagy, and cGAS-STING pathway shape the macrophage response to infectious agents.