Withaferin A Protects against Primary and Recurrent Tuberculosis by Modulating Mycobacterium-Specific Host Immune Responses.

The fate of Mycobacterium tuberculosis infection is governed by immune signaling pathways that can either eliminate the pathogen or result in tuberculosis (TB). Anti-TB therapy (ATT) is extensive and is efficacious only against active, drug-sensitive strains of M. tuberculosis. Due to severe side effects, ATT often causes impairment of host immunity, making it imperative to use novel immunotherapeutics for better clinical outcomes. In this study, we have explored the immunomodulatory potential of withaferin A (WA) as an immunotherapeutic against TB. Here, we demonstrate that WA can constrain intracellular drug-sensitive and -resistant strains of M. tuberculosis by augmenting host immune responses. We also established the potential of WA treatment in conjunction with isoniazid. We show that WA directs the host macrophages toward defensive M1 polarization and enhances TH1 and TH17 immune responses against M. tuberculosis infection. The reduced bacterial burden upon T cell adoptive transfer further corroborated the augmented T cell responses. Interestingly, WA stimulated the generation of T cell memory populations by instigating STAT signaling, thereby reducing the rate of TB recurrence due to reactivation and reinfection. We substantiate the prospects of WA as a potent adjunct immunomodulator that enriches protective memory cells by prompting STAT signaling and improves host defense against M. tuberculosis. IMPORTANCE Despite being extensive, conventional antituberculosis therapy (ATT) is barely proficient in providing sterile immunity to tuberculosis (TB). Failure to constrain the escalating global TB burden due to the emergence of drug-resistant bacterial strains and immune dampening effects of ATT necessitates adjunct immunotherapeutics for better clinical outcomes. We evaluated the prospects of withaferin A (WA), an active constituent of Withania somnifera, as an adjunct immunomodulator against diverse M. tuberculosis strains. WA efficiently restricts the progression of TB by stimulating antimycobacterial host responses, protective immune signaling, and activation of diverse immune cell populations. Protective effects of WA can be attributed to the enrichment of memory T cells by induction of STAT signaling, thereby enhancing resistance to reinfections and reactivation of disease. We ascertained the immunotherapeutic potential of WA in boosting host immune responses against M. tuberculosis.

Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis.

Stimulation of naïve T cells during primary infection or vaccination drives the differentiation and expansion of effector and memory T cells that mediate immediate and long-term protection. Despite self-reliant rescue from infection, BCG vaccination, and treatment, long-term memory is rarely established against Mycobacterium tuberculosis (M.tb) resulting in recurrent tuberculosis (TB). Here, we show that berberine (BBR) enhances innate defense mechanisms against M.tb and stimulates the differentiation of Th1/Th17 specific effector memory (TEM), central memory (TCM), and tissue-resident memory (TRM) responses leading to enhanced host protection against drug-sensitive and drug-resistant TB. Through whole proteome analysis of human PBMCs derived from PPD+ healthy individuals, we identify BBR modulated NOTCH3/PTEN/AKT/FOXO1 pathway as the central mechanism of elevated TEM and TRM responses in the human CD4+ T cells. Moreover, BBR-induced glycolysis resulted in enhanced effector functions leading to superior Th1/Th17 responses in human and murine T cells. This regulation of T cell memory by BBR remarkably enhanced the BCG-induced anti-tubercular immunity and lowered the rate of TB recurrence due to relapse and re-infection. These results thus suggest tuning immunological memory as a feasible approach to augment host resistance against TB and unveil BBR as a potential adjunct immunotherapeutic and immunoprophylactic against TB.

Consensus guidelines on management of oral potentially malignant disorders.

Oral cancer is usually preceded by oral potentially malignant disorders (OPMDs) and early detection can downstage the disease. The majority of OPMDs are asymptomatic in early stages and can be detected on routine oral examination. Though only a proportion of OPMDs may transform to oral squamous cell carcinoma (OSCC), they may serve as a surrogate clinical lesion to identify individuals at risk of developing OSCC. Currently, there is a scarcity of scientific evidence on specific interventions and management of OPMDs and there is no consensus regarding their management. A consensus meeting with a panel of experts was convened to frame guidelines for clinical practices and recommendations for management strategies for OPMDs. A review of literature from medical databases was conducted to provide the best possible evidence and provide recommendations in management of OPMDs.

Bile multi-omics analysis classifies lipid species and microbial peptides predictive of carcinoma of gallbladder.

BACKGROUND AND AIMS: Histopathological examination is the gold standard for detection of gallstone (GS) or gallbladder carcinoma (CAGB). Bile concentrated in the gallbladder (GB) is expected to recapitulate metagenomics and molecular changes associated with development of CAGB. APPROACH AND RESULTS: Bile samples were screened for lipidomics and metaproteome (metagenomics) signatures capable of early detection of cancer in GB anomalies. Analysis of the training cohort (n = 87) showed that metastability of bile was reduced in CAGB (p < 0.05). CAGB bile showed significant alteration of lipidome and microbiome as indicated by multivariate partial least squares regression analysis and alpha-diversity and beta-diversity indexes (p < 0.05). Significant reduction of lipid species and increase in bacterial taxa were found to be associated with patients with CAGB, CAGB with GS, and GS (p < 0.05, log fold change >1.5). A multimodular correlation network created using weighted lipid/metaproteomic correlation network analysis showed striking associations between lipid and metaproteomic modules and functionality. CAGB-linked metaproteomic modules/functionality directly correlated with lipid modules, species, clinical parameters, and bile acid profile (p < 0.05). Increased bacterial taxa (Leptospira, Salmonella enterica, Mycoplasma gallisepticum) and their functionality showed a direct correlation with lipid classes such as lysophosphatidylinositol, ceramide 1-phosphate, and lysophosphatidylethanolamine and development of CAGB (r2  > 0.85). Lipid/metaproteomic signature-based probability of detection for CAGB was > 90%, whereas that for GS was > 80% (p < 0.05). Validation of eight lipid species using four machine learning algorithms in two separate cohorts (n = 38; bile [test cohort 1] and paired plasma [test cohort 2]) showed accuracy (99%) and sensitivity/specificity (>98%) for CAGB detection. CONCLUSIONS: Bile samples of patients with CAGB showed significant reduction in lipid species and increase in bacterial taxa. Our study identifies a core set of bile lipidome and metaproteome signatures which may offer universal utility for early diagnosis of CAGB.

History Cancer Care in Kerala.

The history of cancer care of Kerala spread back to colonial era, where the first hospitals with cancer care facilities were started and run by the London Missionary Society. Later, many government hospitals started cancer care and establishment of the Regional Cancer Centre in 1981 elevated the status of Kerala in the cancer care map of India. The history of modern cancer care in Kerala dates back to the nineteenth century. In Travancore, where the capital of Kerala was later located, modern medicine was first made available in 1811 to the royal family followed by their officials, prisoners and the general public respectively.

Protocol for global proteome, virome, and metaproteome profiling of respiratory specimen (VTM) in COVID-19 patient by LC-MS/MS-based analysis.

In this protocol, we describe global proteome profiling for the respiratory specimen of COVID-19 patients, patients suspected with COVID-19, and H1N1 patients. In this protocol, details for identifying host, viral, or bacterial proteome (Meta-proteome) are provided. Major steps of the protocol include virus inactivation, protein quantification and digestion, desalting of peptides, high-resolution mass spectrometry (HRMS)-based analysis, and downstream bioinformatics analysis. For complete details on the use and execution of this profile, please refer to Maras et al. (2021).

An updated review on activated PI3 kinase delta syndrome (APDS).

Activated Phosphoinositide 3-kinase δ syndrome (APDS) is a newly recognised primary immunodeficiency disease. It has currently been a hot topic of clinical research and new data are emerging regarding its pathogenesis, clinical manifestations and treatment. Patients with APDS syndrome have significant autoimmune manifestations and lymphoproliferation. It is important to differentiate APDS from the usual polygenic CVID in view of the availability of targeted therapy like mTOR inhibitors such as Rapamycin and selective PI3Kδ inhibitors. We provide a comprehensive review on this interesting disorder focusing light on its etiology, genetic research and emerging therapy.

Flow Cytometry for Diagnosis of Primary Immune Deficiencies-A Tertiary Center Experience From North India.

Flow cytometry has emerged as a useful technology that has facilitated our understanding of the human immune system. Primary immune deficiency disorders (PIDDs) are a heterogeneous group of inherited disorders affecting the immune system. More than 350 genes causing various PIDDs have been identified. While the initial suspicion and recognition of PIDDs is clinical, laboratory tools such as flow cytometry and genetic sequencing are essential for confirmation and categorization. Genetic sequencing, however, are prohibitively expensive and not readily available in resource constrained settings. Flow cytometry remains a simple, yet powerful, tool for multi-parametric analysis of cells. While it is confirmatory of diagnosis in certain conditions, in others it helps in narrowing the list of putative genes to be analyzed. The utility of flow cytometry in diagnosis of PIDDs can be divided into four major categories: (a) Enumeration of lymphocyte subsets in peripheral blood. (b) Detection of intracellular signaling molecules, transcription factors, and cytokines. (c) Functional assessment of adaptive and innate immune cells (e.g., T cell function in severe combined immune deficiency and natural killer cell function in familial hemophagocytic lymphohistiocytosis). (d) Evaluation of normal biological processes (e.g., class switching in B cells by B cell immunophenotyping). This review focuses on use of flow cytometry in disease-specific diagnosis of PIDDs in the context of a developing country.

Genetics of COPA syndrome.

Inborn errors of immunity usually not only result in immunodeficiency but may also manifest as immune dysregulation in the form of autoinflammation, autoimmunity, or sometimes malignancy. One of the most recently discovered monogenic disorder of immune dysregulation is COPA syndrome. COPA syndrome is an inherited autoimmune disorder caused by mutations in COPA gene. COPA gene encodes for α subunit of the COP1 protein, which is involved in the reverse vesicular protein transport from Golgi apparatus to the endoplasmic reticulum (ER). The inheritance pattern of COPA syndrome is autosomal dominant, and the patients typically present with interstitial lung disease with pulmonary hemorrhage and subsequently develop arthritis. Immunological features involve autoantibody formation, elevated expression of IL-1ß and IL-6, and increase in the number of Th17 cells. Molecular pathophysiology of COPA syndrome is not clearly understood. However, it is known that accumulation of unfolded proteins in ER leads to ER stress, which is an indirect result of aberrant vesicular transport of proteins from Golgi apparatus to ER and defective cellular autophagy. ER stress induces inflammation and is responsible for pathogenesis of a large number of chronic inflammatory diseases. Unfolded protein response process responds to improperly folded proteins and defends against stress in ER to ensure the fidelity of the protein folding. It maintains the expression of stress-response genes and causes initiation of inflammatory signaling pathways essential for the innate immunity. Mutation in COPA gene associated with defective protein sorting to ER has unearthed a new primary immunodeficiency disease with a unique clinical phenotype. This review highlights the clinical and molecular aspects of COPA syndrome.

Clinical and molecular features of X-linked hyper IgM syndrome - An experience from North India.

X-linked hyper IgM Syndrome (XLHIGM), the most frequent form of the Hyper IgM syndromes is a primary immune deficiency resulting from a mutation in the CD40 ligand gene (CD40LG). We analyzed the clinical and laboratory features of ten patients with XLHIGM, who were diagnosed at a tertiary care hospital in North India. Most common infections were sinopulmonary infections (80%) and diarrhea (50%). Sclerosing cholangitis and necrotising fasciitis were noted in one patient each. Three novel mutations in CD40LG (c.429_429 delA, p. G144DfsX5; c.500 G > A, p.G167E and c.156 G > C, p.K52 N) were detected. In addition, we found one missense mutation, two splice site mutations and two large deletions, which have been previously reported. Four (4) patients had expired at the time of analysis. We report the first series of XLHIGM from North India where we have documented unique features such as pulmonary alveolar proteinosis and infections with Mycobacterium sp.

Long term effect of visual screening on oral cancer incidence and mortality in a randomized trial in Kerala, India.

OBJECTIVES: We studied oral cancer incidence and mortality and the impact of compliance to repeat screening rounds during a 15-year follow-up in a cluster-randomized controlled trial in Trivandrum district, Kerala, India. METHODS: Healthy individuals aged 35 and above in seven clusters randomized to the intervention arm received four rounds of oral visual inspection by trained health workers at 3-year intervals, and those in six clusters randomized to the control arm received routine care during 1996-2005 and one round of visual screening during 2006-2009. Screen-positive persons were referred for diagnosis and treatment. Oral cancer incidence and mortality were compared between the study arms by intention to treat analysis. RESULTS: Of the 96,517 eligible subjects in the intervention arm, 25,144 (26.1%) had one, 22,382 (23.2%) had two, 22,008 (22.8%) had three and 19,288 (20.0%) had four rounds of screening. Of the 95,356 eligible subjects in the control group 43,992 (46.1%) received one round of screening. Although the 12% reduction in oral cancer mortality in all individuals did not reach statistical significance, there was a 24% reduction in oral cancer mortality (95% CI 3-40%) in users of tobacco and/or alcohol in the intervention arm after 4-rounds of screening; there was 38% reduction in oral cancer incidence (95% CI 8-59%) and 81% reduction in oral cancer mortality (95% CI 69-89%) in tobacco and/or alcohol users adhering to four screening rounds. CONCLUSION: Sustained reduction in oral cancer mortality during the 15-year follow-up, with larger reductions in those adhering to repeated screening rounds support the introduction of population-based screening programs targeting users of smoking or chewing tobacco or alcohol or both in high-incidence countries.

Cost-effectiveness of oral cancer screening: results from a cluster randomized controlled trial in India.

OBJECTIVE: To evaluate oral cancer screening by visual inspection. METHODS: A cluster randomized controlled trial was initiated in Trivandrum district, Kerala, India. Of 13 population clusters, seven were randomly allocated to three rounds of screening between 1996 and 2004, while standard care was provided in six (control arm). An activity-based approach was employed to calculate costs associated with various components of the screening trial. Information on the resources used and on clinical events in each trial arm was derived from trial databases. Total costs for each cluster were estimated in 2004 United States dollars (US$). The incremental cost per life-year saved was calculated for all eligible individuals and for high-risk individuals (i.e. tobacco or alcohol users). FINDINGS: The proportion of oral cancers detected at an early stage (i.e. stage I or II) was higher in the intervention arm than the control arm (42% versus 24%, respectively). The incremental cost per life-year saved was US$ 835 for all individuals eligible for screening and US$ 156 for high-risk individuals. Oral cancer screening by visual inspection was performed for under US$ 6 per person. CONCLUSION: The most cost-effective approach to oral cancer screening by visual inspection is to offer it to the high-risk population. Targeted screening of this group will ensure that screening can be offered at a reasonable cost in a limited-resource setting.

Effect of screening on oral cancer mortality in Kerala, India: a cluster-randomised controlled trial.

BACKGROUND: Oral cancer is common in men from developing countries, and is increased by tobacco and alcohol use. We aimed to assess the effect of visual screening on oral cancer mortality in a cluster-randomised controlled trial in India. METHODS: Of the 13 clusters chosen for the study, seven were randomised to three rounds of oral visual inspection by trained health workers at 3-year intervals and six to a control group during 1996-2004, in Trivandrum district, Kerala, India. Healthy participants aged 35 years and older were eligible for the study. Screen-positive people were referred for clinical examination by doctors, biopsy, and treatment. Outcome measures were survival, case fatality, and oral cancer mortality. Oral cancer mortality in the study groups was analysed and compared by use of cluster analysis. Analysis was by intention to treat. FINDINGS: Of the 96,517 eligible participants in the intervention group, 87,655 (91%) were screened at least once, 53,312 (55%) twice, and 29,102 (30%) three times. Of the 5145 individuals who screened positive, 3218 (63%) complied with referral. 95,356 eligible participants in the control group received standard care. 205 oral cancer cases and 77 oral cancer deaths were recorded in the intervention group compared with 158 cases and 87 deaths in the control group (mortality rate ratio 0.79 [95% CI 0.51-1.22]). 70 oral cancer deaths took place in users of tobacco or alcohol, or both, in the intervention group, compared with 85 in controls (0.66 [0.45-0.95]). The mortality rate ratio was 0.57 (0.35-0.93) in male tobacco or alcohol users and 0.78 (0.43-1.42) in female users. INTERPRETATION: : Oral visual screening can reduce mortality in high-risk individuals and has the potential of preventing at least 37,000 oral cancer deaths worldwide.

Betel quid without tobacco as a risk factor for oral precancers.

The IARC monographs recently classified chewing betel quid without tobacco as a human carcinogen. Several studies in Taiwan have reported that betel quid without tobacco may increase the risk of oral precancers such as oral leukoplakia and oral submucous fibrosis. However in India, since most betel quid chewers prefer to add tobacco to the quid, the independent effect of betel quid on the risk of oral precancers is difficult to assess and has not yet been fully explored. We conducted a large case-control study in Kerala, India, including 927 oral leukoplakia cases, 170 oral submucous fibrosis cases, 100 erythroplakia cases, 115 multiple oral precancer cases and 47,773 controls. The focus of this reanalysis is on the minority of individuals who chewed betel quid without tobacco. Among nonsmokers and nondrinkers, chewing betel quid without tobacco conferred ORs of 22.2 (95%CI = 11.3, 43.7) for oral leukoplakia, 56.2 (95%CI = 21.8, 144.8) for oral submucous fibrosis, 29.0 (95%CI = 5.63, 149.5) for erythroplakia and 28.3 (95%CI = 6.88, 116.7) for multiple oral precancers, after adjustment for age, sex, education and BMI. Dose-response relationships were observed for both the frequency and duration of betel quid chewing without tobacco on the risk of oral precancers. In conclusion, our study supports the hypothesis that chewing betel quid without tobacco elevates the risks of various oral precancers.

Genomic instability in oral squamous cell carcinoma: relationship to betel-quid chewing.

Genomic instability in repeated DNA sequences is exhibited by a variety of cancer types, including oral squamous cell carcinoma. Exposure to carcinogenic compounds may further increase the instability. We have used Inter-Simple Sequence Repeat (Inter-SSR) PCR methodology to detect genetic alterations in 37 oral cancer patients who had chewed betel-quid. Thirty-eight percent of DNA from tumors had genomic alterations in the sequences flanked by (CA)(8) and (GT)(8) repeats. Patients with tumor DNAs harboring genomic alterations had a two-fold higher consumption of betel-quid than patients without alterations in tumor DNA. Matched normal and tumor DNAs were also screened for microsatellite instability where four patients (10.8%) showed alterations in at least one microsatellite marker but there was no relationship between this phenotype and betel-quid chewing. These data indicate that exposure to carcinogens present in the betel-quid may contribute to genomic instability detected by inter-SSR PCR in a subset of oral tumors.

Risk factors for multiple oral premalignant lesions.

Oral leukoplakia, oral submucous fibrosis and erythroplakia are 3 major types of oral premalignant lesions. Multiple oral premalignant lesions may possibly develop due to field cancerization, where carcinogenic exposures can cause simultaneous genetic defects to the upper aerodigestive tract epithelium, putting the epithelium at high risk for development of premalignant lesions at different stages of carcinogenesis. There have been no epidemiological studies on risk or protective factors of the disease. A case-control study was conducted with data from the baseline screening of a randomized oral cancer screening trial in Kerala, India. A total of 115 subjects with multiple oral premalignant lesions (8-10% of oral premalignant lesions in our case series) were included: 64 subjects with oral leukoplakia and oral submucous fibrosis, 19 subjects with oral leukoplakia and erythroplakia, 22 subjects with oral submucous fibrosis and erythroplakia and 10 subjects with all 3 lesions. Individuals without oral lesions were considered controls (n=47,773). The odds ratio (OR) for ever tobacco chewers was 37.8 (95% confidence interval (CI)=16.2-88.1) when adjusted for age, sex, education, BMI, smoking, drinking and fruit/vegetable intake. Dose-response relationships were seen for the frequency (p<0.0001) and duration of tobacco chewing (p<0.0001) with the risk of multiple oral premalignant lesions. Whereas alcohol drinking may possibly be a risk factor for multiple oral premalignant lesions, smoking was not associated with the risk of multiple oral premalignant lesions (OR=0.9, 95%CI=0.5-1.7). The results suggest that tobacco chewing was the most important risk factor for multiple oral premalignant lesions and may be a major source of field cancerization on the oral epithelium in the Indian population.

Interim results from a cluster randomized controlled oral cancer screening trial in Kerala, India.

A cluster randomized controlled oral cancer screening trial is on-going in the Trivandrum district, India, to evaluate the efficacy of screening in reducing oral cancer mortality. Subjects, aged 35 years and above, in 13 clusters in the Trivandrum district, India, were randomized to the intervention (screening) group (7 clusters, 78969 subjects) to receive three rounds of screening by oral visual inspection by trained health workers at 3-year intervals or to a control group (6 clusters, 74739 subjects). Two rounds of screening were completed between 1995 and 2002 during which 69896 (88.5%) subjects were screened at least once, and 59.7% of the 4408 screen-positive subjects were further investigated. In the intervention group, 344404 person-years were accrued and 329326 person-years were in the control group. In the intervention group, 149 incident oral cancer cases and 65 deaths from oral cancer were observed, and 106 incident cases and 62 deaths from oral cancer were observed in the control group. The programme sensitivity for detection of oral precancerous lesions and cancer was 81.5% and the programme specificity was 84.8%; the programme positive predictive value was 39.6%. In the intervention group 37.6% of the cases were in stages I-II, as opposed to 18.9% in the control group. The 3 year survival rate was 57.5% in the intervention and 38.8% in the control group (P<0.05). The age standardized oral cancer mortality rates were 21.2/100000 person-years in the intervention and 21.3/100000 in the control group. After completing two rounds of screening, oral cancer mortality rates were similar in both study groups.

Body mass index, tobacco chewing, alcohol drinking and the risk of oral submucous fibrosis in Kerala, India.

OBJECTIVE: While chewing areca nut is considered a risk factor for oral submucous fibrosis, the effects of cigarette smoking, alcohol drinking, and body mass index (BMI) have not been examined; nor are they well established. In this study we investigated the association between BMI, smoking, drinking, and the risk of oral submucous fibrosis. METHODS: We conducted a case-control study within the framework of an ongoing randomized oral cancer screening trial in Kerala, India. Trained health workers conducted interviews with structured questionnaires and oral visual inspections to diagnose oral premalignant lesions. A total of 170 oral submucous fibrosis cases (139 women and 31 men) and 47,773 controls were identified. The odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression in SAS. RESULTS: The adjusted OR for ever-tobacco chewing was 44.1 (95% CI = 22.0-88.2). An inverse dose-response relationship was seen between BMI and the risk of oral submucous fibrosis when both genders were combined (p for trend = 0.0010), with an OR of 0.5 (95% CI = 0.3-0.9) for the highest BMI quartile compared to the lowest. Alcohol drinking may possibly be associated with the risk of oral submucous fibrosis; the adjusted OR for ever drinking was 2.1 (95% CI = 1.0-4.4). Cigarette smoking did not appear to be a risk factor for women or for men. Both smoking and drinking were rare habits among women. CONCLUSION: This study suggested, for the first time, that BMI was inversely associated with the risk of oral submucous fibrosis for both genders when potential confounding factors were adjusted. Our results indicated that alcohol drinking might be a moderate risk factor and confirmed the previous observation that chewing tobacco was a strong risk factor for oral submucous fibrosis.