Exploring Vaccine Hesitancy and Uptake during COVID-19: A Review of PM's Mann Ki Baat Dialogue.

Public health programmes are interlinked and intertwined with communication, advocacy and social mobilisation for their success. The unprecedented situation created by COVID-19 brought a medical emergency all over the world, the like of which was probably not seen after the Spanish Flu outbreak, a century ago. First there seemed no solution in sight when tens of thousands of people lost their lives to the coronavirus in various countries, but when the vaccine arrived, there were, in general, doubts about its efficacy and safety. Indian scenario was not any different. When the government launched the vaccine in a campaign mode in January 2021, it was also battling with misperceptions and vaccine hesitancy. Prime Minister Narendra Modi took it upon himself to address the issue through his various addresses to the nation and his signature programme Mann ki Baat (MKB) on the radio. This review paper examines the empirical research on MKB coverage of the COVID-19 pandemic, the media multiplier impact of the MKB, people's voices through their engagement with various social media platforms, and what is the impact on vaccine uptake.

Polymeric immunoglobulin receptor and galectin-3-binding protein are raised in biliary atresia: Reveals a proteomic-based study.

To identify and evaluate differentially expressed plasma proteins in biliary atresia (BA), we performed plasma proteome profiling using liquid chromatography with tandem mass spectrometry (LC-MS/MS) in 20 patients with BA and 10 control children. Serological assays validated the most significant and highly upregulated proteins in a cohort of 45 patients and 15 controls. Bioinformatics tools were used for functional classification and protein-protein interactions of differentially expressed proteins (DEPs). Of 405 proteins detected in patients and 360 in controls, 242 proteins, each with ≥2 unique peptides (total of 3230 peptides), were common in both groups. Compared to controls, 90 proteins in patients were differentially expressed and were dysregulated. Twenty-five were significantly upregulated with polymeric immunoglobulin receptor (PIgR), galectin-3-binding protein (Gal-3BP), complement C2, the most prominent, and 15 had low expression. The bioinformatic analysis revealed functional interaction between DEPs and their role in an inflammatory immune response. Enzyme immunoassay for PIgR and Gal-3BP in patients' plasma showed their levels raised significantly (p = 0.0021 and p = 0.0369, respectively). The PIgR and Gal-3BP are novel proteins upregulated in BA and may be tested further for their utility as potential circulating disease biomarker(s). SIGNIFICANCE: The study shows that plasma PIgR and GAL-3BP levels are significantly raised in infants with BA within the first 3 months of life. If tested in a larger cohort, these proteins may be found to have their diagnostic potential and utility as disease biomarkers. The study also provides valuable information on the involvement of several DEPs in innate immune response, chronic inflammation, and fibrosis. This strengthens the hypothesis that the immune-mediated inflammatory processes are responsible for the progressive nature of BA.

Examining the Impact of Key Factors on COVID-19 Vaccination Coverage in India: A PLS-SEM Approach.

During the coronavirus disease 2019 (COVID-19) pandemic, numerous factors determined the performance of COVID-19 vaccination coverage. The purpose of this study is to examine the influence of factors such as government stewardship, planning and implementation, and community participation on COVID-19 vaccination coverage. This study applied partial least square structured equation modeling (PLS-SEM) by analyzing 187 responses from the stakeholders involved in vaccination programs in four select states of India. This study empirically validates a framework for improving vaccination coverage by confirming the significant impact of planning and implementation on vaccination coverage followed by government stewardship and community participation. Additionally, this study highlights the individual impact of each factor on vaccination coverage. Based on the findings, strategic recommendations were proposed that can be utilized for formulating policy-level actions to facilitate the vaccination program.

An Equity Analysis of Zero-Dose Children in India Using the National Family Health Survey Data: Status, Challenges, and Next Steps.

Background While immunization programs across the world have made considerable progress, children and communities continue to be beyond the reach of healthcare services. Globally, they are now referred to as zero-dose (ZD) children (those who have not received a single dose of diphtheria, pertussis, and tetanus-containing vaccine). Pre-COVID-19 pandemic analyses suggest that nearly 50% of vaccine-preventable deaths occur among ZD children. Two-thirds of these children live in extremely poor households suffering from multiple deprivations including lack of access to reproductive health services, water, and sanitation. Hence, ZD children have now been prioritized as a key cohort for identification and integration with the health systems as we build back from the pandemic. Methodology Extracting data from the last two National Family Health Survey (NFHS) rounds (NFHS 4, 2015-2016 and NFHS 5, 2019-2021), this study aims to ascertain the status of ZD children aged 12-23 months in India, the challenges, and the necessary action agenda going forward. Data were analyzed for equity determinants such as gender, place of residence, religion, birth order, caste, and mother's schooling. Key determinants included the change in ZD prevalence at the national, state, and district levels; variations across equity parameters and states with maximum improvements; and disparity across these indicators. A correlation analysis was also conducted to understand the nature of the association between ZD prevalence and critical maternal and child health indicators. Results The overall ZD prevalence between the two rounds was reduced by 4.1% (10.5-6.4%). A total of 26 states in the country reported a ZD prevalence of <10% in NFHS 5 compared to 18 in NFHS 4. In total, 324 districts reported a ZD prevalence of <5%, and 145 districts reported a prevalence of >10%. The equity parameters reflected a slow-footed reduction among ZD for girl children, across urban geographies, firstborn children, mothers with 12 or more years of schooling, and children in families with the highest wealth quintiles. A negative correlation accentuated between the two NFHS rounds was established between first-trimester registration, four or more antenatal visits, institutional deliveries, and ZD prevalence. Conclusions The findings point toward sustained improvement across key equity parameters, however, challenges do exist. Moreover, the impact of the pandemic on immunization programs across the globe and in India is bound to halt and reverse the progress and potentiate further inequities. It is thus imperative that continued and augmented efforts are continued to identify, integrate, and immunize ZD children, families, and communities.

Interleukin-35 Mitigates ox-LDL-Induced Proatherogenic Effects via Modulating miRNAs Associated with Coronary Artery Disease (CAD).

PURPOSE: Recent emergence of miRNAs as important regulators of processes involving lesion formation and regression has highlighted miRNAs as potent therapeutic targets for the treatment of atherosclerosis. Few studies have reported the atheroprotective role of IL-35, a novel immunosuppressive and anti-inflammatory cytokine; however, miRNA-dependent regulation underlying the anti-atherosclerotic potential of IL-35 remains elusive. METHODS: THP-1 macrophages were incubated with human recombinant IL-35 (rIL-35) either in the presence or absence of ox-LDL. qRT-PCR was conducted to validate the expression levels of previously identified miRNAs including miR-197-5p, miR-4442, miR-324-3p, miR-6879-5p, and miR-6069 that were differentially expressed in peripheral blood mononuclear cells of coronary artery disease (CAD) patients vs. controls. Additionally, bioinformatic analysis was performed to predict miRNA-associated targets and their corresponding functional significance in CAD. RESULTS: Exogenous IL-35 significantly decreased the average area of ox-LDL-stimulated macrophages, indicating the inhibitory effect of IL-35 on lipid-laden foam cell formation. Furthermore, rIL-35 treatment alleviated the ox-LDL-mediated atherogenic effects by modulating the expression levels of aforementioned CAD-associated miRNAs in the cultured macrophages. Moreover, functional enrichment analysis of these miRNA-related targets revealed their role in the molecular processes affecting different stages of atheroslerotic plaque development, such as macrophage polarization, T cell suppression, lipoprotein metabolism, foam cell formation, and iNOS-mediated inflammation. CONCLUSION: Our observations uncover the novel role of IL-35 as an epigenetic modifier as it influences the expression level of miRNAs implicated in the pathogenesis of atherosclerosis. Thus, IL-35 cytokine therapy-mediated miRNA targeting could be an effective therapeutic strategy against the development of early atheromas in asymptomatic high-risk CAD patients.

Mission Indradhanush and Intensified Mission Indradhanush-Success Story of India's Universal Immunization Program and the Role of Mann Ki Baat in Bridging the Immunization Gap.

Child immunization is crucial for reducing the morbidity and mortality associated with vaccine-preventable diseases (VPDs). The program grew over the years, however, progress towards full immunization coverage (FIC) remained slow, with only 44% of children fully immunized in 1992-1993, and 62% in 2015-2016, as reported in the National Family Health Survey. To address this challenge, Government of India launched Routine Immunization intensification drive- Mission Indradhanush (MI) in 2014, with the aim of achieving 90% FIC. The success of MI led to the launch of Intensified Mission Indradhanush (IMI) in 2017, with more intensive planning, monitoring, review, and inter-sectoral partnerships.

Progress Toward Measles and Rubella Elimination - India, 2005-2021.

In 2019, India, along with other countries in the World Health Organization (WHO) South-East Asia Region,* adopted the goal of measles and rubella elimination by 2023,† a revision of the previous goal of measles elimination and control of rubella and congenital rubella syndrome (CRS) by 2020§ (1-3). During 2017-2021, India adopted a national strategic plan for measles and rubella elimination (4), introduced rubella-containing vaccine (RCV) into the routine immunization program, launched a nationwide measles-rubella supplementary immunization activity (SIA) catch-up campaign, transitioned from outbreak-based surveillance to case-based acute fever and rash surveillance, and more than doubled the number of laboratories in the measles-rubella network, from 13 to 27. Strategies included 1) achieving and maintaining high population immunity with at least 95% vaccination coverage by providing 2 doses of measles- and rubella-containing vaccines; 2) ensuring a sensitive and timely case-based measles, rubella and CRS surveillance system; 3) maintaining an accredited measles and rubella laboratory network; 4) ensuring adequate outbreak preparedness and rapid response to measles and rubella outbreaks; and 5) strengthening support and linkages to achieve these strategies, including planning and progress monitoring, advocacy, social mobilization and communication, identification and utilization of synergistic linkages of integrated program efforts, research, and development. This report describes India's progress toward the elimination of measles and rubella during 2005-2021, with a focus on the years 2017-2021.¶ During 2005-2021, coverage with the first dose of a measles-containing vaccine (MCV) administered through routine immunization increased 31%, from 68% to 89%. During 2011-2021, coverage with a second MCV dose (MCV2) increased by 204%, from 27% to 82%. During 2017-2021, coverage with a first dose of RCV (RCV1) increased almost 14-fold, from 6% to 89%. More than 324 million children received a measles- and rubella-containing vaccine (MRCV) during measles-rubella SIAs completed in 34 (94%) of 36 states and union territories (states) during 2017-2019. During 2017-2021, annual measles incidence decreased 62%, from 10.4 to 4.0 cases per 1 million population, and rubella incidence decreased 48%, from 2.3 to 1.2 cases per 1 million population. India has made substantial progress toward measles and rubella elimination; however, urgent and intensified efforts are required to achieve measles and rubella elimination by 2023.

Return on investment of the electronic vaccine intelligence network in India.

The electronic vaccine intelligence network (eVIN) was introduced by India's Ministry of Health and Family Welfare in 12 states and was implemented by the United Nations Development Programme through the Gavi health system strengthening support during 2014-17 to replace the traditional paper-based cold-chain management system with an electronic vaccine logistics management system. An economic assessment was conducted as part of the overall assessment of eVIN. The objective of the economic assessment was to conduct a return on investment analysis of eVIN implementation. Return on investment was defined as the ratio of total benefits (savings) from eVIN to total investment in eVIN. All costs were calculated in 2020 prices and reported in Indian rupees (1 US dollar = INR 74.132). A one-rupee investment in eVIN led to a return of INR 0.52 for traditional vaccines. The highest cost savings from eVIN was from better vaccine stock management. When same percentage of savings from the new vaccines were incorporated into the analysis, one-rupee investment in eVIN led to a return of INR 1.41. In the future, when only recurrent costs will exist, the return from eVIN will be even higher: a one-rupee investment in eVIN will yield a return of INR 2.93. The assessment of eVIN showed promising results in streamlining the vaccine flow network and ensuring equity in vaccine stock management along with good return on investment; hence, there was a rapid expansion of eVIN in all 731 districts across 36 states and union territories in the country.

Comparison of two dose escalation strategies of methotrexate in active rheumatoid arthritis: a multicentre, parallel group, randomised controlled trial.

OBJECTIVES: There are no head-to-head trials of different dose escalation strategies of methotrexate (MTX) in RA. We compared the efficacy, safety and tolerability of 'usual' (5 mg every 4 weeks) versus 'fast' (5 mg every 2 weeks) escalation of oral MTX. METHODS: This multicentre, open-label (assessor blinded) RCT included patients 18-55 years of age having active RA with disease duration <5 years, and not on DMARDs. Patients were randomized 1:1 into usual or fast escalation groups, both groups starting MTX at 15 mg/week till a maximum of 25 mg/week. Primary outcome was EULAR good response at 16 weeks, secondary outcomes were ΔDAS28 and adverse effects (AE). Analyses were intention-to-treat. RESULTS: 178 patients with mean DAS28-CRP of 5.4(1.1) were randomized to usual (n=89) or fast escalation groups (n=89). At 16 weeks, there was no difference in good EULAR response in the usual (28.1%) or fast escalation (22.5%) groups (p=0.8). There was no difference in mean ΔDAS28-CRP at 8 weeks (-0.9, -0.8, p=0.72) or 16 weeks (-1.3, -1.3, p=0.98). Even at 24 weeks (extended follow-up), responses were similar. There were no inter-group differences in ΔHAQ, or MTX-polyglutamates 1-3 levels at 8 or 16 weeks. Gastrointestinal AE were higher in the fast escalation group over initial 8 weeks (27%, 40%, p=0.048), but not over 16 weeks. There was no difference in cytopenias, transaminitis, or drug discontinuation/dose reduction between the groups. No serious AE were seen. CONCLUSION: A faster MTX escalation strategy in RA was not more efficacious over 16-24 weeks, and did not significantly increase AE, except higher gastrointestinal AE initially. TRIAL REGISTRATION NUMBER: CTRI/2018/12/016549.

Correlation between soluble receptor for advanced glycation end products levels and coronary artery disease in postmenopausal nondiabetic women.

BACKGROUND: The established cardiovascular risk factors cannot explain the overall risk of coronary artery disease (CAD), especially in women. Therefore, there is a growing need for the assessment of novel biomarkers to identify women at risk. The receptor for advanced glycation end products (RAGE) and its interaction with the advanced glycation end product (AGE) ligand have been associated with atherogenesis. The soluble fraction of RAGE (sRAGE) antagonizes RAGE signaling and exerts an antiatherogenic effect. AIM: The study aim was to explore the association between plasma levels of sRAGE and CAD in nondiabetic postmenopausal women. METHODS: This case-control study included 110 nondiabetic postmenopausal women who were enrolled in two groups. Group I included 55 angiographically proven CAD subjects with > 50% stenosis in at least one of the major coronary arteries and Group II included 55 healthy control women who did not have CAD or had < 50% stenosis of the coronary arteries. Stenosis was confirmed by invasive angiography. Plasma sRAGE was determined by an enzyme-linked immunosorbent assay. RESULTS: We observed significantly lower plasma sRAGE concentrations in subjects with CAD vs healthy controls (P < 0.05). Univariate and multivariate logistic regression analysis also revealed a significant correlation between plasma sRAGE levels and CAD (P = 0.01). Multivariate odds ratios for CAD revealed that subjects with sRAGE concentrations below 225 pg/mL (lowest quartile) had a 6-fold increase in CAD prevalence independent of other risk factors. CONCLUSION: Our findings indicated that low sRAGE levels were independently associated with CAD in nondiabetic postmenopausal women. Risk assessment of CAD in postmenopausal women can be improved by including sRAGE along with other risk factors.

Uric acid as a predictor of endothelial dysfunction in patients with metabolic syndrome.

OBJECTIVE: We conducted a study to examine the association of endothelial dysfunction and oxidative stress with uric acid levels in patients of metabolic syndrome. METHODS: One hundred and two patients of Metabolic Syndrome (International Diabetes Federation definition) were included in the study. Anthropometric measurements, serum uric acid levels, fasting blood sugar levels and lipid levels, as well as malondialdehyde and reactive nitrogen intermediates were measured after an 8-hour fasting period. Flow mediated vasodilation (FMD) of the brachial artery was measured and endothelial dysfunction was defined as an increase in diameter < 10% post compression. RESULTS: A total of 102 patients were included in the study. Mean uric acid level was 5.49 ± 1.61 mg%. A total of 59 patients in the study had endothelial dysfunction, defined by an abnormal FMD. Patients with an abnormal FMD had higher levels of serum uric acid which was statistically significant (p value = 0.010). Serum RNI and MDA levels were negatively correlated with uric acid, but did not reach statistical significance. Patients with an abnormal FMD had a lower RNI level, but this did not reach statistical significance. Serum MDA levels were significantly higher in patients with an abnormal FMD (p value = 0.038). CONCLUSION: Uric acid was significantly associated with endothelial dysfunction in patients with metabolic syndrome in our study. It was inversely correlated with serum RNI and MDA levels, but this did not reach statistical significance.

Hypoxia-induced mitochondrial reactive oxygen species (mtROS) differentially regulates smooth muscle cell (SMC) proliferation of pulmonary and systemic vasculature.

BACKGROUND: Vascular remodeling plays a pivotal role in regulation of hypoxia-mediated pulmonary and systemic hypertension via the phenotypic modulation of smooth muscle cells (SMCs) of pulmonary and systemic arteries, respectively. Mitochondria serve as putative oxygen (O2) sensors, and consequently, adaptations to hypoxia are mediated via HIF (hypoxia-inducible factors) activation, which impinges on mitochondrial function by suppressing the mitochondrial activity. Therefore, we explored the implication of hypoxia-mediated mitochondrial stress in pulmonary and systemic arterial remodeling. METHODS: The hypoxic (10% O2) effect on human pulmonary artery and aortic SMCs was examined in vitro by cell viability assay, proliferation index, autophagy, and comet assays. Mitochondrial ROS (mtROS), membrane potential (MMP), and mitochondrial morphology were assessed using mitochondrial-selective fluorescent probes. Further, the cell cycle distribution was analyzed by flow cytometry using propidium iodide staining. RESULTS: Our data indicate no significant alterations in cell viability and active proliferation of hypoxic PASMCs; however, an excessive rise in mtROS production and disrupted MMP, accompanied by enhanced DNA damage and reduced autophagy was observed, highlighting the 'apoptosis resistance' phenotype in these cells. Conversely, in hypoxia-treated hASMCs, a modest rise in mtROS levels was associated with reduced DNA damage; followed by upregulated autophagy; increased S-phase DNA content and cell viability, depicting the cytoprotective effect of hypoxia-induced autophagy against mitochondrial damage in hASMCs. CONCLUSION: Our findings suggest that differential impact of mtROS on proliferative capacity may contribute to the variable hypoxic responses in pulmonary and systemic vasculature. Therefore, targeting mtROS may serve as an effective therapeutic strategy to prevent hypoxia-induced hypertension.

Uric acid as a predictor of endothelial dysfunction in patients with metabolic syndrome

ABSTRACT Objective: We conducted a study to examine the association of endothelial dysfunction and oxidative stress with uric acid levels in patients of metabolic syndrome. Subjects and methods: One hundred and two patients of Metabolic Syndrome (International Diabetes Federation definition) were included in the study. Anthropometric measurements, serum uric acid levels, fasting blood sugar levels and lipid levels, as well as malondialdehyde and reactive nitrogen intermediates were measured after an 8-hour fasting period. Flow mediated vasodilation (FMD) of the brachial artery was measured and endothelial dysfunction was defined as an increase in diameter < 10% post compression. Results: A total of 102 patients were included in the study. Mean uric acid level was 5.49 ± 1.61 mg%. A total of 59 patients in the study had endothelial dysfunction, defined by an abnormal FMD. Patients with an abnormal FMD had higher levels of serum uric acid which was statistically significant (p value = 0.010). Serum RNI and MDA levels were negatively correlated with uric acid, but did not reach statistical significance. Patients with an abnormal FMD had a lower RNI level, but this did not reach statistical significance. Serum MDA levels were significantly higher in patients with an abnormal FMD (p value = 0.038). Conclusions: Uric acid was significantly associated with endothelial dysfunction in patients with metabolic syndrome in our study. It was inversely correlated with serum RNI and MDA levels, but this did not reach statistical significance.

Molecular insights of cigarette smoke condensate-activated NLRP3 inflammasome in THP-1 cells in a stage-specific atherogenesis.

BACKGROUND: Smoking induces excessive inflammation which is associated with all the stages of atherosclerosis. Earlier, we reported Nod-like receptor protein 3 (NLRP3) inflammasome activation as a pro-atherosclerotic property of cigarette smoking. In the present study, we aimed to explore the underlying detailed upstream mechanism and the cellular status of putative downstream molecules of cigarette smoke condensate (CSC)-activated NLRP3 inflammasome in atherosclerotic disease. METHODS AND RESULTS: THP-1 monocytes, macrophages and foam cells represent crucial stages of atherogenesis as initiation, progression and development. To determine the upstream molecular regulators of smoking-induced NLRP3 inflammasome in atherogenesis, Myeloid differentiation primary response 88/Nuclear Factor kappa-light-chain-enhancer of activated B cells (MyD88/NF-κB) and Suppressor of cytokine signaling 3/Signal transduction and activator of transcription 3 (SOCS3/STAT3) pathways were elucidated. Stage-specific THP-1 cells were treated with MyD88 and SOCS3/STAT3 inhibitors. The results showed that MyD88 inhibition markedly attenuated the expression of NLRP3 markers (NLRP3, caspase-1, Interleukin (IL)-1ß and IL-18), IL-6, SOCS3 and NF-κB. Moreover, the secretory levels of pro-cytokines were also significantly reduced in culture media. In contrast, no changes were observed with SOCS3/STAT3 inhibitor. Further, ac-vyad-cmk, an inflammasome inhibitor was used to explore the downstream targets of CSC-activated NLRP3 inflammasome in atherosclerotic process. The transcriptional profiling of 25 atherosclerotic markers was carried out using ExProfile™ Custom Gene qPCR Arrays. CSC exposure upregulated the expression of 17 genes and downregulated 4 genes in a stage-specific manner. Inhibitory experiments showed aberrant changes in CSC-regulated genes. Altogether, 15 molecules were common in all three stages. CONCLUSION: The findings may suggest that MyD88/NF-κB pathway is an upstream regulator of NLRP3 inflammasome underlying smoking-induced atherosclerosis. Notably, 15 atherosclerotic molecules associated with endothelial dysfunction, scavenger receptors, cholesterol esterification and matrix-metalloproteins were found downstream to CSC-activated NLRP3 inflammasome.

Activation of NLRP3 inflammasome assembly is associated with smoking status of patients with coronary artery disease.

OBJECTIVE: Cigarette smoke is considered as a sterile inflammatory stimulus which triggers an innate immune response, accountable for vascular events. Previously, we reported smoking-induced NLRP3 inflammasome activation in the pathogenesis of atherosclerosis through caspase-1 activation and secretion of pro-cytokines (interleukin (IL)-1ß and IL-18) in vitro and in vivo. Therefore, the present study aimed to reconnoitre the association of cigarette smoking and NLRP3 inflammasome activation ex vivo in human subjects with coronary atherosclerosis. METHODS AND RESULTS: In order to establish and validate the association between smoking status and NLRP3 inflammasome ex vivo, mononuclear cells were isolated from smokers with angiographically-proven coronary artery disease (CAD); non-smokers with CAD; smokers without CAD, and healthy non-smokers (controls) (n = 20 each). The transcriptional and translational expression of NLRP3 inflammasome markers i.e. NLRP3, pro-caspase-1, caspase-1, pro-IL-1ß, IL-1ß, pro-IL-18 and IL-18 was significantly increased (2 to 7-fold) in smokers with CAD vs non-smokers with CAD; and smokers without CAD vs non-smoker controls. In addition, the oxidative stress, an upstream mediator of NLRP3 inflammasome was evaluated and found to be significantly augmented in smokers vs non-smokers (with and without CAD respectively). Further, the levels of serum cotinine, oxidative stress markers (8-isoprostane and 8-oxo-2́'-deoxyguanosine), caspase-1 and pro-cytokines (IL-1ß and IL-18) were also higher in smokers vs non-smokers. Moreover, the levels of pro-cytokines were positively correlated with caspase-1 and serum cotinine, corroborating the secretion of cytokines in a caspase-1-dependent manner. CONCLUSION: Our data may imply NLRP3 inflammasome as a mediator of the pro-atherosclerotic property of cigarette smoking in atherosclerotic patients.

Exposure of cigarette smoke condensate activates NLRP3 inflammasome in vitro and in vivo: A connotation of innate immunity and atherosclerosis.

OBJECTIVE: Smoking is known to have detrimental effects on cardiovascular system. However, the potential molecular basis of smoking-induced atherosclerosis remains unclear. NLRP3 inflammasome is implicated in perpetuation of inflammatory response in atherosclerosis. Therefore, we aimed to explore the cytotoxic effects of cigarette smoke condensate (CSC) on the activation of NLRP3 inflammasome in vitro and in vivo. METHODS: For in vitro study, the pro-atherogenic effects of CSC were evaluated in THP-1 monocytes with different dose concentrations (0.1, 1, 5, 10 and 20 µg/ml) for varied time periods (6, 12, 24 and 48 h). For in vivo study, 30 male C57BL/6J mice were employed. 6 mice were sacrificed for baseline investigations. 24 mice were randomly divided into four groups: Group-I:Control mice, Group-II:CSC model, Group-III:High-fat diet(HFD) model, and Group-IV:HFD + CSC model for 14 weeks (n = 6/group). The group-II and IV mice were injected with 720 µg CSC/20 g body weight intraperitoneally (6 days/week). RESULTS: In vitro, higher dosage of CSC (20 µg/ml) was toxic to cells as significant decline in cell viability and proliferation was observed. Furthermore, the mRNA expression of NLRP3 inflammasome and its pro-cytokine levels were significantly augmented on CSC exposure in a dose-dependent manner but impeded in time-dependent manner. In vivo, CSC and HFD independently augmented the expression of NLRP3 inflammasome (~4-10 fold-change) along with pro-cytokine levels in Group-II and III vs Group-I mice whereas, HFD + CSC treatment demonstrated synergistic effects in Group-IV. CONCLUSION: Our data suggest that CSC activates NLRP3 inflammasome in vitro and in vivo and collectively with HFD has synergistic effects in vivo that may promote atherosclerosis.

Exposure of cigarette smoke condensate activates NLRP3 inflammasome in THP-1 cells in a stage-specific manner: An underlying role of innate immunity in atherosclerosis.

BACKGROUND: Smoking is known to affect all the phases of atherosclerosis, thus is considered as an independent and major risk factor. The underlying mechanism responsible for the atherogenic effects of smoking is still uncertain and a major concern. Recent evidence implicates NLRP3 inflammasome, an innate immunity component in the pathogenesis of atherosclerosis. Therefore, we hypothesized that NLRP3 inflammasome may be an associated pathway between smoking and atherosclerosis. METHODS AND RESULTS: Differentiation in monocytes, macrophages and foam cells are the key stages in atherosclerotic plaque development, best mimicked by THP-1 cells. Therefore, to determine whether cigarette smoke condensate (CSC) could induce differentiation of THP-1 monocytes into macrophages, morphological changes and the expression levels of the inflammatory surface markers, i.e. CD11b, CD14 and CD36 were analyzed. The results showed that CD14 and CD36 levels were significantly increased in CSC-treated THP-1 monocytes. Further, we investigated the effect of CSC exposure on the status of NLRP3 inflammasome markers, i.e. NLRP3, pro-caspase-1, caspase-1, pro-IL-18, pro-IL-1ß, IL-1ß and IL-18 in a stage-specific manner. For this, THP-1 monocytes, PMA-differentiated macrophages and oxidized-low density lipoprotein (ox-LDL)-induced macrophage foam cells were exposed to 10 µg/ml of CSC for 6 h. CSC exposure significantly upregulated the expression of NLRP3 inflammasome in CSC-treated cells at both transcriptional and translational levels. Moreover, downstream pro-cytokines, i.e. IL-1ß and IL-18 levels were also significantly increased in culture supernatants of CSC-exposed cells. CONCLUSION: These observations suggest that CSC exposure may activate NLRP3 inflammasome in a stage-specific manner and may promote initiation and progression of atherosclerosis.

Metformin upregulates mitophagy in patients with T2DM: A randomized placebo-controlled study.

Impaired mitochondrial autophagy (mitophagy) and NLRP3 inflammasome activation have been incriminated in the pathogenesis of T2DM. Metformin besides being an insulin sensitizer also induces autophagy; however, its effect on mitophagy and NLRP3 activation in patients with T2DM still remains elusive. Forty-five drug-naïve T2DM patients with HbA1C 7%-9% (53-75 mmol/mol) were randomly assigned to receive either metformin, voglibose, or placebo for 3 months, and were also recommended for lifestyle intervention programme (n = 15 each). Mitochondrial oxidative stress (MOS) parameters, qPCR and immunoblotting of mitophagy-related markers (PINK1, PARKIN, MFN2, NIX, LC3-II, LAMP2), p-AMPKα (T172), and NLRP3 proteins, as well as transmission electron microscopy (TEM) for assessing mitochondrial morphology were performed in the mononuclear cells of study patients. Both metformin and voglibose showed a similar efficacy towards the reduction in HbA1c and MOS indices. However, multivariate ANCOVA divulged that mRNA and protein expression of mitophagy markers, NLRP3 and p-AMPKα (T172), were significantly increased only with metformin therapy. Moreover, PINK1 expression displayed a significant positive association with HOMA-ß indices, and TEM studies further confirmed reduced distortions in mitochondrial morphology in the metformin group only. Our observations underscore that metformin upregulates mitophagy and subsequently ameliorates the altered mitochondrial morphology and function, independent of its glucose-lowering effect. Further, restoration of normal mitochondrial phenotype may improve cellular function, including ß-cells, which may prevent further worsening of hyperglycaemia in patients with T2DM.

Metformin promotes mitophagy in mononuclear cells: a potential in vitro model for unraveling metformin's mechanism of action.

Increased oxidative stress in patients with type 2 diabetes mellitus (T2DM) results in abnormalities in cell repair processes, such as mitophagy, which compromises mitochondrial function and contributes to insulin resistance and ß cell failure. Metformin, widely recommended in the management of T2DM, exerts its pleiotropic effects via 5'-AMP-activated protein kinase (AMPK); however, its effect on mitophagy remains elusive. Recent evidence demonstrates that peripheral blood mononuclear cells (PBMCs) express insulin receptors and the human organic cation transporter protein, and they are extensively being used as a surrogate for examining mitochondrial function in T2DM. Metformin treatment increased the formation of acidic vesicles and mitophagosomes, upregulated mitophagy markers, and enhanced mitophagic flux, as indicated by increased LC3-II expression and reduced p62 protein levels. In addition, pretreatment with compound C (an AMPK inhibitor) significantly decreased the expression of mitophagy markers in metformin-treated cells, indicating that metformin induces mitophagy via the AMPK pathway. In conclusion, metformin-induced mitophagy may improve cellular function, including in ß cells, by restoring normal mitochondrial phenotype, which may prove beneficial in patients with T2DM and other mitochondrial-related diseases. Moreover, PBMCs may be used as a novel diagnostic biomarker for identifying mitochondrial disorders.

Clinico-genetic model to predict methotrexate intolerance in rheumatoid arthritis.

INTRODUCTION: Methotrexate is the gold-standard DMARD in rheumatoid arthritis but is often associated with "mild" adverse effects like intolerance or laboratory abnormalities. Although non-life threatening, they are responsible for drug discontinuation in 17-50%. There is limited data on clinical and genetic markers that predict their occurrence. METHODS: This prospective study enrolled patients with active rheumatoid arthritis. They were started on methotrexate at a weekly dose of 15 mg, escalated gradually to reach 25 mg which was continued till the end of the study. Intolerance (symptomatic adverse effects) was ascertained by a questionnaire at 4, 8, 16, and 24 weeks. Laboratory testing for occurrence of cytopenia and/or transaminitis was done at the same study visits. Seven SNPs in four genes involved in methotrexate handling were genotyped using real-time polymerase chain reaction. RESULTS: This study included 110 patients with rheumatoid arthritis who received methotrexate for 24 weeks; the final mean weekly methotrexate dose was 22.0 ± 4.0 mg. Methotrexate intolerance occurred in 40 (37%), common being nausea (and vomiting) in 29 and anxiety (and dizziness) in 9. It was associated with lower BMI at baseline (21.5 ± 3.7, 23.8 ± 4.6 kg/m2, p = 0.01). FPGS rs10106 was significantly associated with intolerance with an allelic odds ratio (95% CI) of 2.02 (1.14-3.57) and the recessive genetic model (AA+AG versus GG) with an odds ratio of 3.8 (95% CI 1.5-9.6, p = 0.004). A model including both BMI and FPGS rs10106 could modestly predict methotrexate intolerance with an accuracy of 66.3%. CONCLUSIONS: A clinical-genetic model including BMI and SNP FPGS 10101 was found to have a modest prediction ability for methotrexate intolerance.Key Points• Methotrexate intolerance (symptomatic adverse effects) was common and occurred in 37% patients over 6 months.• SNP FPGS rs10106 and low body mass index were associated with methotrexate intolerance.• Clinico-genetic model had a modest ability of 66% for predicting intolerance.