Trends in science: successes and responsible research conduct.

Science, particularly in life sciences and biotechnologies, is continuing to make remarkable progress in the past decade. This has been possible due to the governments and people recognizing that scientific discoveries bring development and prosperity to the nation. The new trend in research is to collaborate across disciplines with large teams of participants across the globe. This has brought success but has led to varying standards in ethics and responsible conduct which require harmonization. Recent discoveries point to a need for new approaches to ethics. The rise in cases of misconduct and retraction of research papers from high-profile individuals has been a cause for concern. It is encouraging that many countries that have detected misconduct in research have instituted strong steps to correct the situation. This brief review discusses the recent developments of interest to me, the issues of global research, ethics and responsible conduct.

The value proposition of the Global Health Security Index.

Infectious disease outbreaks pose major threats to human health and security. Countries with robust capacities for preventing, detecting and responding to outbreaks can avert many of the social, political, economic and health system costs of such crises. The Global Health Security Index (GHS Index)-the first comprehensive assessment and benchmarking of health security and related capabilities across 195 countries-recently found that no country is sufficiently prepared for epidemics or pandemics. The GHS Index can help health security stakeholders identify areas of weakness, as well as opportunities to collaborate across sectors, collectively strengthen health systems and achieve shared public health goals. Some scholars have recently offered constructive critiques of the GHS Index's approach to scoring and ranking countries; its weighting of select indicators; its emphasis on transparency; its focus on biosecurity and biosafety capacities; and divergence between select country scores and corresponding COVID-19-associated caseloads, morbidity, and mortality. Here, we (1) describe the practical value of the GHS Index; (2) present potential use cases to help policymakers and practitioners maximise the utility of the tool; (3) discuss the importance of scoring and ranking; (4) describe the robust methodology underpinning country scores and ranks; (5) highlight the GHS Index's emphasis on transparency and (6) articulate caveats for users wishing to use GHS Index data in health security research, policymaking and practice.

Leprosy Reactions Show Increased Th17 Cell Activity and Reduced FOXP3+ Tregs with Concomitant Decrease in TGF-ß and Increase in IL-6.

BACKGROUND: 50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4+ subsets of Th17 cells and CD25+FOXP3+ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions. METHODOLOGY AND PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3+ Treg cells as well as reduction in TGF-ß and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients. CONCLUSIONS: Patients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-ß and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.

Immunology of leprosy and diagnostic challenges.

Leprosy, caused by noncultivable Mycobacterium leprae (ML), has varied manifestations, which are associated with the host immune responses. The dermal involvement is accompanied by peripheral nerve damage, which leads to sensory motor loss and deformities. Both innate and acquired immune responses are involved. The main cell to be compromised is the CD4 + T helper cell, which shows antigen specific unresponsiveness to only ML and not to other common antigens in the bacilliferous generalized lepromatous form of the disease. In contrast, the paucibacillary localized tuberculoid form shows appropriate T cell functions and poor antibody response. The dichotomy between T cell functions and antibodies are discussed against the current information on cytokines, Th subsets, and regulatory T cells. During lepromatous reactions, there is a temporary, heightened T cell immunity, even in lepromatous subjects. The dermal lesions confirm many features observed with peripheral blood mononuclear cells and give additional information on local immune responses. Nerve damage involves both immune and nonimmune mechanisms. Leprosy is a model disease for understanding host immune responses to intracellular bacilli. There are challenges in diagnosing early leprosy. In spite of intensive efforts by many groups, consensus on a universal test suitable for endemic areas is awaited.

Immunology fo leprosy and diagnostic challenges

Leprosy, caused by noncultivable Mycobacterium leprae (ML), has varied manifestations, which are associated with the host immune responses. The dermal involvement is accompanied by peripheral nerve damage, which leads to sensory motor loss and deformities. Both innate and acquired immune responses are involved. The main cell to be compromised is the CD4 + T helper cell, which shows antigen specific unresponsiveness to only ML and not to other common antigens in the bacilliferous generalized lepromatous form of the disease. In contrast, the paucibacillary localized tuberculoid form shows appropriate T cell functions and poor antibody response. The dichotomy between T cell functions and antibodies are discussed against the current information on cytokines, Th subsets, and regulatory T cells. During lepromatous reactions, there is a temporary, heightened T cell immunity, even in lepromatous subjects. The dermal lesions confirm many features observed with peripheral blood mononuclear cells and give additional information on local immune responses. Nerve damage involves both immune and nonimmune mechanisms. Leprosy is a model disease for understanding host immune responses to intracellular bacilli. There are challenges in diagnosing early leprosy. In spite of intensive efforts by many groups, consensus on a universal test suitable for endemic areas is awaited.

Increase in TGF-ß secreting CD4⁺CD25⁺ FOXP3⁺ T regulatory cells in anergic lepromatous leprosy patients.

BACKGROUND: Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25(+)FOXP3(+) regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples. METHODOLOGY/PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3(+), TGF-ß in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4(+)CD25(+)FOXP3(+) T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-ß which was further confirmed by increase in TGF-ß in culture supernatants (p<0.003). Furthermore, TGF-ß in iTreg cells was associated with phosphorylation of STAT5A. TGF-ß was seen in CD25(+) cells of the CD4(+) but not that of CD8(+) T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients. CONCLUSIONS/SIGNIFICANCE: Our results indicate that FOXP3(+) iTregs with TGF-ß may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.

CD4+ Th17 cells discriminate clinical types and constitute a third subset of non Th1, Non Th2 T cells in human leprosy.

BACKGROUND: Patients with localized tuberculoid and generalized lepromatous leprosy show respectively Th1 and Th2 cytokine profile. Additionally, other patients in both types of leprosy also show a non discriminating Th0 cytokine profile with both interferon-γ and IL-4. The present study investigated the role of Th17 cells which appear to be a distinct subtype of Th subtypes in 19 tuberculoid and 18 lepromatous leprosy patients. Five healthy subjects with long term exposure to infection and 4 skin biopsies from healthy subjects undergoing cosmetic surgery were used as controls. METHODOLOGY/PRINCIPLE FINDINGS: An array of Th17 related primers for cytokines, chemokines and transcription factors was used in real time reverse transcribed PCR to evaluate gene expression, ELISA for cytokine secretion in the supernatants of antigen stimulated PBMC cultures and flow cytometry for establishing the phenotype of the IL-17, IL-21 producing cells. CONCLUSIONS/SIGNIFICANCE: IL-17 isoforms showed significantly higher expression and release in supernatants of antigen stimulated PBMC cultures and dermal lesions of healthy contacts and tuberculoid leprosy as compared to lepromatous leprosy (p<0.003). This was further confirmed by Th17 associated transcription factor RORC, cytokines IL-21, IL-22, and IL-23, chemokines MMP13, CCL20, CCL22. Of interest was the association of IL-23R and not IL-6R with IL-17(+) cells. The Th17 cells were CD4(+) CCR6(+) confirming their effector cell lineage. Polarized Th1 cytokines were seen in 3/7 tuberculoid and Th2 cytokines in 5/10 lepromatous leprosy patients. Of importance was the higher association of Th17 pathway factors with the non-polarized Th0 types as compared to the polarized Th1 and Th2 (p<0.01). Our study draws attention to a third type of effector Th cell that may play a role in leprosy.

Lsr2 of Mycobacterium leprae and its synthetic peptides elicit restitution of T cell responses in erythema nodosum leprosum and reversal reactions in patients with lepromatous leprosy.

The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742-752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.

Genetic evidence of TAP1 gene variant as a susceptibility factor in Indian leprosy patients.

The heterodimeric transporter associated with antigen processing (TAP) gene loci is known to play a vital role in immune surveillance. We investigated a possible association of gene polymorphisms both in TAP1 and TAP2 in a cohort of clinically classified leprosy patients (n=222) and in ethnically matched controls (n=223). The TAP1 and TAP2 genes were genotyped for four single nucleotide polymorphisms TAP1 (rs1057141 Iso333Val and rs1135216 Asp637Gly) and TAP2 (rs2228396 Ala565Thr and rs241447 Ala665Thr) by direct sequencing and ARMS-PCR. The minor allele of TAP1 637G contributes to an increased risk to leprosy compared to controls (OR: 1.68, 95% CI 1.2-2.36, P=0.0057). An increased risk for the variant minor allele of the TAP1 637G to multibacillary (BL+LL) or paucibacillary (BT+TT) infections was also observed [multibacillary vs. controls (OR: 1.56, 95% CI 1.07-2.28, P=0.054); paucibacillary vs. controls (OR: 1.92, 95% CI 1.21-3.01, P=0.013)]. In the dominant model, the genotypes of the TAP1 rs1135216AG+GG additionally contributed to an increased risk. Overall our findings demonstrate that the TAP1 gene variant (rs1135216 Asp637Gly) influences the susceptibility to clinically classified leprosy patients in Indian population.

Lsr2 peptides of Mycobacterium leprae show hierarchical responses in lymphoproliferative assays, with selective recognition by patients with anergic lepromatous leprosy.

Lsr2 protein of Mycobacterium leprae was shown earlier to elicit B and T cell responses in leprosy patients (20, 28). Lymphoproliferation to M. leprae and Lsr2 antigens was observed in >70% of tuberculoid (T) patients and in 16 and 34% of lepromatous (L) patients, respectively. We focused on the M. leprae nonresponders in the lepromatous group using 22 synthetic Lsr2 peptides (end-to-end peptides A to F and overlapping peptides p1 to p16) in in vitro T cell responses. A total of 125 leprosy and 13 tuberculosis patients and 19 healthy controls from the area of endemicity (here, healthy controls, or HC) were investigated. The highest responses were observed (67 to 100%) in HC for all peptides except p1 to p3, and the lowest was observed in tuberculosis patients. Significant differences in lymphoproliferation were observed in T, L, and HC groups (analysis of variance [ANOVA], P = 0.000 to 0.015) for all end-to-end peptides except B and for p5 and p7 to p10. Hierarchical recognition between lepromatous and tuberculoid leprosy was noted for p8 (P < 0.05) and between the HC and L groups for p7 to p10, p15, and p16 (P < 0.005 to P < 0.02). Significant lymphoproliferation was observed to peptides A to F and p1 to p9, p11, p12, p15, p16 (P = 0.000 to 0.001) with 40% responding to peptides C and p16 in L patients. Lepromatous patients also showed significantly higher levels of a gamma interferon (IFN-γ) response to peptide C than to other peptides (P < 0.05). Major histocompatibility complex (MHC) class II bias for peptide recognition was not observed. These studies indicate that Lsr2 has multiple T cell epitopes that induce in vitro T cell responses in the highly infective lepromatous leprosy patients.

Transmission of dapsone-resistant leprosy detected by molecular epidemiological approaches.

Drug resistance surveillance identified six untreated leprosy patients in the Philippines with Mycobacterium leprae folP1 mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked by M. leprae variable-number tandem repeat (VNTR) strain types. In India, folP1 mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target gene rpoB. These findings indicate that dapsone resistance is being transmitted.

Increased CXC ligand 10 levels and gene expression in type 1 leprosy reactions.

Type 1 reaction (T1R) is a systemic inflammatory syndrome causing substantial morbidity in leprosy. T1R results from spontaneously enhanced cellular immunity in borderline types of leprosy, but there are no established laboratory markers for the reaction. Preliminary studies have identified elevated circulating CXC ligand 10 (CXCL10) during T1R. Correlation of CXCL10 with clinical T1R was studied in repeated serum specimens obtained before, during, and after T1R. CXCL10 gene expression was assessed in biopsy specimens taken before and during T1R, and sections were stained for the cytokine using monoclonal antibodies. Sequential serum specimens revealed elevation of circulating CXCL10 associated with episodes of T1R (P = 0.0001) but no evidence of an earlier, predictive change in the level of the chemokine. Reverse transcriptase (RT)-PCR revealed elevated expression of CXCL10 transcripts during T1R, but not in patients who did not have T1R. No significant correlation between CXCL10 and gamma interferon (IFN-γ) mRNA levels was observed. Immunohistochemical staining of the skin biopsy specimens suggested an overall increase in CXCL10 but did not identify a particular strongly staining population of leukocytes. Increased CXCL10 in lesions and serum is characteristic of T1R. CXCL10 measurement offers new possibilities for laboratory diagnosis and monitoring of T1R. Studies of the regulation of CXCL10 may provide insight into the mechanisms of T1R and identify potential new drug targets for treatment.

Evidence-based National Vaccine Policy.

India has over a century old tradition of development and production of vaccines. The Government rightly adopted self-sufficiency in vaccine production and self-reliance in vaccine technology as its policy objectives in 1986. However, in the absence of a full-fledged vaccine policy, there have been concerns related to demand and supply, manufacture vs. import, role of public and private sectors, choice of vaccines, new and combination vaccines, universal vs. selective vaccination, routine immunization vs. special drives, cost-benefit aspects, regulatory issues, logistics etc. The need for a comprehensive and evidence based vaccine policy that enables informed decisions on all these aspects from the public health point of view brought together doctors, scientists, policy analysts, lawyers and civil society representatives to formulate this policy paper for the consideration of the Government. This paper evolved out of the first ever ICMR-NISTADS national brainstorming workshop on vaccine policy held during 4-5 June, 2009 in New Delhi, and subsequent discussions over email for several weeks, before being adopted unanimously in the present form.

VNTR typing of Mycobacterium leprae in South Indian leprosy patients.

OBJECTIVES: To study the suitability, stability and diversity of short tandem repeat (STR) genomic markers to elicit strain variation in the Mycobacterium leprae isolates within leprosy patients from Andhra Pradesh and Tamil Nadu states in South India. MATERIALS AND METHODS: Slit skin smear (SSS) samples were collected from lesions and various body sites of newly diagnosed leprosy patients. The SSSs from each patient were pooled, except in the case of five patients. Total DNA was extracted from SSS samples. M. leprae STRs were amplified from the DNA either by multiplex PCR (MP) or single PCR methods. The number of repeats for each STR locus (the STR allele) was obtained either by fragment length analysis (FLA) or by DNA sequencing of the PCR amplicons. RESULTS AND CONCLUSION: Multiplex PCR minimised the use of DNA and reagents, and together with FLA, was time and cost effective for STR strain typing. After examination of the isolates of South Indian origin at 13 STR loci, it was determined that the alleles for (AC)8b, (GGT)5, 6-3a (rpoT), 21-3, 27-5, and 23-3 were conserved in two study populations. In a family from Andhra Pradesh, the M. leprae STR patterns in two patients were identical in 16 of 18 loci which indicate a common source of infection. Fourteen of 15 STR loci showed no intra-patient variation in the five patients tested in Tamil Nadu. Altogether, these studies indicate the suitability of STR strain typing for assessing short-range transmission chains.

High-resolution sonography: a new technique to detect nerve damage in leprosy.

BACKGROUND: Leprosy is the most common treatable peripheral nerve disorder worldwide with periods of acute neuritis leading to functional impairment of limbs, ulcer formation and stigmatizing deformities. Since the hallmarks of leprosy are nerve enlargement and inflammation, we used high-resolution sonography (US) and color Doppler (CD) imaging to demonstrate nerve enlargement and inflammation. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] We performed bilateral US of the ulnar (UN), median (MN), lateral popliteal (LP) and posterior tibial (PT) nerves in 20 leprosy patients and compared this with the clinical findings in these patients and with the sonographic findings in 30 healthy Indian controls. The nerves were significantly thicker in the leprosy patients as compared to healthy controls (p<0.0001 for each nerve). The two patients without nerve enlargements did not have a type 1 or type 2 reaction or signs of neuritis. The kappa for clinical palpation and nerve enlargement by sonography was 0.30 for all examined nerves (0.32 for UN, 0.41 for PN and 0.13 for LP). Increased neural vascularity by CD imaging was present in 39 of 152 examined nerves (26%). Increased vascularity was observed in multiple nerves in 6 of 12 patients with type 1 reaction and in 3 of 4 patients with type 2 reaction. Significant correlation was observed between clinical parameters of grade of thickening, sensory loss and muscle weakness and US abnormalities of nerve echotexture, endoneural flow and cross-sectional area (p<0.001). CONCLUSIONS/SIGNIFICANCE: We conclude that clinical examination of enlarged nerves in leprosy patients is subjective and inaccurate, whereas sonography provides an objective measure of nerve damage by showing increased vascularity, distorted echotexture and enlargement. This damage is sonographically more extensive and includes more nerves than clinically expected.

Lower touch sensibility in the extremities of healthy Indians: further deterioration with age.

Touch sensibility testing is a cost-effective, psychophysical measure of peripheral nerve function and impairment. However, there is limited information regarding the natural variability in touch sensibility across different populations and different age groups. We studied 568 healthy Indian volunteers without any clinical evidence of peripheral nerve disease. Touch sensibility was evaluated bilaterally in palms, feet, and heels, using Semmes-Weinstein monofilaments, with target forces ranging from 0.008 to 300 g. No differences were observed between the right and the left limbs. The lowest target force detected ranged from 0.4 to 2 g in the palms and 1.4 to 15 g in the feet. These values showed further increase with age. Women compared with men had higher sensibility in the palms in most age groups. Touch sensibility thresholds recorded in a large group of Indians were higher than that reported in other populations. These findings have clinical implications for the diagnosis of early nerve impairment in the elderly and in disease states drawing attention to geographic variations in touch sensation.