In silico structural and mechanical insights into bedaquiline resistance associated with high-grade non-synonymous mutations in atpE, mmpR5, and pepQ.

Clinical resistance against bedaquiline (BDQ) remains intractable to anti-tuberculosis therapies since its introduction to the market over a decade ago. Herein, we investigated the structural and mechanical aspects of BDQ resistance in AtpE, MmpR5, and PepQ. The known target-specific resistant single non-synonymous mutations were refined to high-grade candidates. Thus, 7 (AtpE), 5 (MmpR5), and 1 (PepQ) single nucleotide polymorphisms (SNPs) and one insertion frameshift mutation in MmpR5 were recreated at the molecular level, and these phenotypic models were then directed to stringent dynamics to define time-scaled changes. The AtpE variants destabilized the structure; mainly, L59V, E61D, and I66M were detrimental to the complex fitness, while L74V and L114P boosted the BDQ binding to MmpR5. The first three and last two alterations gave rise to loss- and gain-of-function to AtpE and MmpR5, respectively. Hence, these five mutants are functionally relevant and therapeutically targetable hotspots of BDQ resistance. There were no noticeable changes in PepQ data analysis. The present study revealed that MmpR5 mutations confer BDQ resistance, whereas AtpE and PepQ SNPs display low susceptibility. These results were tallied with the published findings, which testified to the pursued method's reliability and accuracy. We hope these data and inferences could be helpful for the futuristic design of novel TB drugs.Communicated by Ramaswamy H. Sarma.

A study of insulin resistance and its clinico-metabolic associations among apparently healthy individuals attending a tertiary care hospital.

BACKGROUND: Insulin resistance (IR), as a result of unhealthy life-styles and westernization, most likely contributes to the increased incidence of metabolic abnormalities and consequently, the development of metabolic syndrome (MS). AIM: The present study was undertaken to determine the magnitude of IR and associated clinico-metabolic risk factors among the out-patients of a tertiary care hospital in Bihar, India. SUBJECTS AND METHODS: Anthropometric profile, lipid profile, fasting blood glucose, C-reactive protein (CRP) and C-peptide of 112 individuals were measured using the standard procedures. IR was assessed using the homeostasis model (Homeostatic model assessment [HOMA]-IR). RESULTS: The mean IR was 1.5 (1.0). Individuals with MS, higher body mass index and CRP ≥6 mg/l had higher IR. Linear regression showed, among the components of MS, waist circumference had the highest contribution toward IR. The optimal cut-off value to detect IR by HOMA2-IR was 1.35. CONCLUSION: IR was found to have a strong association with various clinico-metabolic risk factors.

Diversity in transcripts and translational pattern of stress proteins in marine extremophiles.

Extremophiles occur in a diverse range of habitats, from the frigid waters of Antarctic to the superheated plumes of hydrothermal vents. Their in-depth study could provide important insights into the biochemical, ecological and evolutionary aspects of marine microbes. The cellular machinery of such extreme-lovers could be highly flexible to cope with such harsh environments. Extreme conditions of temperature, pressure, salinity, pH, oxidative stress, radiation, etc., above the physiological tolerance level can disrupt the natural conformation of proteins in the cell. The induction of stress proteins (heat/cold shock proteins/salt stress proteins/pressure-induced proteins) plays a vital role in the acclimatization of extremophiles. The present review focuses on the in vitro studies conducted on the transcripts and translational pattern of stress proteins in extremophiles. Though some proteins are unique, a commonality in stress resistance mechanism has been observed, for example, the universal occurrence of HSP60, 70 and the expression of metabolic and DNA repair proteins. The review highlights that among all the stressful conditions, salt/osmotic stress evokes the expression of highest number of transcripts/proteins while psychrophilic condition the least.

The effect of antigen presenting cells on the cytokine profiles of stable and reactional lepromatous leprosy patients.

In view of varied reports on the Th1/Th2 paradigm in leprosy, we used a novel real time (RT) fluorogenic reverse transcriptase based PCR (RT-PCR) to measure cytokine expression in peripheral blood cells from lepromatous leprosy patients with stable disease and those suffering from erythema nodosum leprosum (ENL/Type II) reactions. To evaluate the role of accessory cells in Th cell differentiation, co-expression of Th cytokines interferon gamma (IFNgamma) and interleukin (IL) 4 and regulatory cytokines IL 10 and IL 12 was compared in antigen stimulated peripheral blood mononuclear cells (PBMC), cultures containing T cells reconstituted with autologous monocytes (MO) and cultures containing T cells reconstituted with autologous dendritic cells (DC). 7/8 stable lepromatous leprosy patients showed co-expression of both IFNgamma and IL 4, suggesting a Th0 or a combination of Th1 + Th2 subsets in PBMC. The RT-PCR demonstrated that stable lepromatous patients and patients in ENL had significantly higher levels of IFNgamma mRNA molecules compared to IL 4. In fact, 5/8 ENL patients had undetectable levels of IL 4 mRNA, with a skewing of the cytokine response towards a Th1-like profile. Consistent with this. IL 12p40 mRNA molecules were significantly higher in the PBMC of ENL patients compared to stable lepromatous patients (P < 0.01). Reconstitution of purified T cells with autologous DC and MO from the stable lepromatous group resulted in down regulation of IL 4 (P < 0.03 for DC and P < 0.02 for MO) and IL 10 (P < 0. 01 for DC and P < 0.02 for MO), and a consequent skewing towards a Th1 profile similar to that seen in ENL patients. The fact that accessory cells could alter the cytokine profile in the reconstituted cultures suggests that they may play a role in determining Th subset differentiation in chronic diseases, and may influence the immunological stability of such diseases.

Dysregulation of IL-4 expression in lepromatous leprosy patients with and without erythema nodosum leprosum.

In order to increase our understanding of the immunological basis of erythema nodosum leprosum (ENL), we studied Th-like cytokine profiles in 130 leprosy patients, employing both the conventional and a novel, real-time, fluorogenic reverse transcriptase-based PCR (RT-PCR). The concomitant expression of both Th-like cytokines, interferon-gamma and IL-4, and the regulatory cytokines, IL-10 and IL-12, was studied in the peripheral blood cells of leprosy patients with and without ENL. In the conventional RT-PCR, varied cytokine profiles were observed in individual patients of all clinical types. Fifty-three percent of lepromatous patients without ENL and 59% of tuberculoid leprosy patients showed co-expression of IFN gamma and IL-4, indicating a non-polarized Th 0 pattern. Of the 36 patients with ENL, 58% demonstrated a polarized Th 1 pattern, with only 30% expressing both cytokines. Semiquantitative RT-PCR indicated a lower expression of IL-4 compared to that of IFN gamma in the lepromatous patients without ENL; the difference was even greater among those with ENL. The sensitive, real-time PCR confirmed the down-regulation of IL-4 and IL-10, with absence of IL-4 in half of the patients, resulting in skewing of the cytokine response toward a Th 1-like profile.

Lepromatous leprosy patients show T helper 1-like cytokine profile with differential expression of interleukin-10 during type 1 and 2 reactions.

Some leprosy patients suffer from clinical episodes associated with tissue damage which are designated as Type 1 (reversal reaction) when localized to the lesions and Type 2 (erythema nodosum leprosum, ENL) when accompanied by systemic involvement. We had reported earlier that stable, non-reaction lepromatous leprosy subjects show T helper 2 (Th2)- and Th0- but not Th1-like responses in the peripheral blood. To further understand the development of Th-like responses during disease, 32 lepromatous patients undergoing reactions were studied using cytokine-specific reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in peripheral blood and some skin biopsies. Of interest was the evidence of a Th1-like response with presence of interferon-gamma (IFN-gamma) and absence of interleukin-4 (IL-4) mRNA in the peripheral blood mononuclear cells (PBMC) of 85 and 64% of Type 1 and 2 reaction patients, respectively, and in all reaction sites. Whereas a Th0- was seen in some, a Th2-like response was absent. IL-12p40 mRNA was seen in 21/25 ENL and all Type 1 reaction subjects irrespective of the Th phenotype. IL-12p40 and IFN-gamma were detectable in unstimulated PBMC suggesting an in vivo priming during reactions. IL-10 was mainly associated with adherent cells and showed a differential expression in the two reactions. It was present in the PBMC of ENL but not in reversal reaction patients. Moreover, it was not detectable in the skin lesions of either type of reactions. A Th1-like cytokine profile was associated with immunopathology and persisted up to 6-7 months after the onset of reactions.

Modification of delivery system enhances MHC nonrestricted immunogenicity of V3 loop region of HIV-1 gp120.

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2b, H-2d, H-2k and H-2s, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P < 0.05 to P < 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.

Splenic and intestinal lymphocyte proliferation response in mice fed milk or yogurt and challenged with Salmonella typhimurium.

Two groups of 4-5 week old DBA/2J Nii mice were put on either a yogurt-based (n = 33) or a milk-based (n = 32) diet for a period of 4 weeks. At the end of the feeding trial one sub group of mice each from the two dietary groups was sacrificed for assessment of immune response. The remaining mice were challenged intragastrically with 2 x 10(10) live Salmonella typhimurium organisms and continued on their respective diets for 8 days after which they were also sacrificed. The immune response was measured by tritiated thymidine uptake by splenic or intestinal lymphocytes in response to the mitogens concanavalin A (Con A), Phytohaemaggutinin (PHA), and Lipopolysaccharide from Escherichia coli (LPS). Serum Immunoglobulin A levels were also estimated. Feed efficiency, measured as weight gain per unit energy intake, was significantly higher for the yogurt diet than for the milk diet. The mitogenic response of splenic and intestinal lymphocytes in the two groups of unchallenged mice was not different. In the Salmonella-challenged mice the stimulation index (SI) of splenic lymphocytes from yogurt-fed mice (mean +/- SD) was significantly higher (P = 0.001) in response to Con A (24.71 +/- 3.40) than that of milk-fed mice (15.85 +/- 2.09). Further, in these mice the SI of intestinal lymphocytes from yogurt-fed mice was higher than that of milk-fed mice in response to Con A (7.35 +/- 0.61 vs 5.65 +/- 0.78, P = 0.016) and LPS (9.04 +/- 0.93 vs 6.15 +/- 1.32, P = 0.016). Serum IgA levels in Salmonella-challenged mice were significantly higher 8 days after the challenge in the yogurt-fed group than in the milk-fed group (P < 0.001). The experiments indicate an improvement in local gastrointestinal as well as systemic immunity on a yogurt diet as compared to a milk diet.

Expression and functional characterisation of the clpC gene of Mycobacterium leprae: ClpC protein elicits human antibody response.

This paper reports the expression of a previously described gene [Nath and Laal, Nucleic Acids Res. 18 (1990) 4935], currently identified as the clpC gene of Mycobacterium leprae, using an in vitro rabbit reticulocyte lysate-coupled transcription/translation system. The produced protein moved as a 95-kDa band on SDS-PAGE. An additional band of 79 kDa was seen which may have resulted from a GTG codon downstream to the initiating ATG in the clpC sequence. A threefold increase in synthesis of the 95-kDa protein was achieved by altering the translation codon context sequence of the ATG start codon. The ClpC (caseinolytic protease C) amino acid sequence, which contained two nucleotide-binding sites, exhibited in vitro ATP binding. Of functional significance was its immunoreactivity in human subjects with mycobacterial infection. Leprosy and tuberculosis patients with active disease had antibodies which recognised ClpC in dot ELISA.

Monocyte derived IL 10 and PGE2 are associated with the absence of Th 1 cells and in vitro T cell suppression in lepromatous leprosy.

Our previous studies had shown that the clinicopathological spectrum in leprosy was associated with discrete T cell subsets in circulation, with tuberculoid patients having antigen-induced Th 1, whereas lepromatous leprosy patients with antigen-specific T cell anergy possessed Th 2 cells. The present study shows that infected monocytes from lepromatous but not tuberculoid leprosy patients released soluble factors (MoF(s)) containing IL-10 and PGE2 which inhibited M. leprae induced in vitro lymphoproliferation of previously sensitised healthy or tuberculoid leprosy subjects. A strong negative correlation was observed between adherent cell derived IL-10 and IL-2 at the level of both the product and cytokine mRNA. Moreover, anti-IL-10 antibodies and indomethacin partially reversed the suppressor effects of MoF(s). Taken together these studies indicate that infected monocytes contribute to the development of T cell anergy by releasing factors that affect regulatory cytokines and T cell subset differentiation in lepromatous leprosy.

Cytokine profile of circulating T cells of leprosy patients reflects both indiscriminate and polarized T-helper subsets: T-helper phenotype is stable and uninfluenced by related antigens of Mycobacterium leprae.

Cytokine profiles of circulating mononuclear cells were studied with the aim of delineating T-cell subsets in leprosy patients with active disease. Using reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokine mRNA and enzyme-linked immunoassay (ELISA) for the secreted products, interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied. Three antigens, native Mycobacterium leprae, a recombinant antigen LSR/A15 of M. leprae and peptide 624 spanning 58-77 amino acids of the latter, were used to induce cytokine expression and release. Half of the subjects, irrespective of the clinical type or antigen used, showed a mixed T-helper type 0 (Th0)-like cytokine pattern, with evidence of the concomitant presence of IFN-gamma and IL-4. The remainder showed a polarized pattern based on the type of leprosy. Lepromatous patients with disseminated disease had Th2-type cytokines, with IL-4 but not IFN-gamma. In contrast, tuberculoid leprosy patients with localized disease showed a Th1-like profile, with the presence of IFN-gamma but not IL-4. Of interest was the stability of the Th phenotype for M. leprae-related antigens. Both the recombinant and the peptide antigens induced the same phenotype as the natural M. leprae bacillus in all except four of 45 leprosy patients.

Polytuftsin: its possible effects and mechanism during macrophage activation.

Polytuftsin (PT) a 35-40 repeat unit of tuftsin (TKPR), when administered as a conjugate with the malarial peptide, ring-infected erythrocyte surface antigen (RESA), enhanced antigen-induced lymphoproliferation and antibody levels in mice as compared to RESA alone. This enhancement was unrelated to the H-2 background of the animals. The present study was undertaken with a view to understanding the mechanism(s) responsible for this immune enhancement. Peritoneal adherent cells (PAC) from H-2b and H-2d mice were incubated with RESA alone, PT-conjugated RESA, a physical mixture of RESA + PT and PT alone. They were subsequently evaluated for I-A expression using monoclonal antibodies and flow cytometry as well as cell-ELISA. Significant increase in I-A expression on PAC was observed in all 4 groups as compared to untreated cells. Whereas cells treated with PT-conjugated RESA showed highly significant increase in I-A (P < 0.001), the other groups showed moderate increase (P < 0.05). This enhancement was attributable to increase in the number of I-A-positive cells rather than I-A molecules per cell. Moreover, IL-1 release, as assayed by bioassay, was significantly higher in cells treated with conjugated RESA as compared to cells treated with RESA or PT alone (P < 0.05). Thus, it would appear that PT-conjugated RESA peptide of the malarial antigen selectively enhances major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APC) and may therefore improve immune functions by stimulating better antigen presentation and proliferation of T cells.

Clinical utility of LSR/A15 gene for Mycobacterium leprae detection in leprosy tissues using the polymerase chain reaction.

Skin biopsy and slit-skin smears from 46 leprosy patients and 4 nonleprosy patients were tested for the presence of Mycobacterium leprae by the polymerase chain reaction (PCR) using primers based on the sequence of the LSR/15 kD gene. The PCR was found to be specific and sensitive, with a detection level of 10 and 100 bacilli. PCR using skin biopsies gave a higher detection rate than did slit-skin smears, probably due to the higher density of bacilli in a 4-mm punch biopsy. Dot blot hybridization with radioactive probes was 10-fold more sensitive than the ethidium bromide staining. Eight patients who did not show acid-fast bacilli in tissues by the conventional methods were shown to have PCR-amplified M. leprae DNA. False-negative results were obtained in 3 cases even though formal evidence for tissue inhibitors was absent.

Critical residues of the Mycobacterium leprae LSR recombinant protein discriminate clinical activity in erythema nodosum leprosum reactions.

We reported earlier (S. Singh, N. P. Shanker Narayan, P. J. Jenner, G. Ramu, M. J. Colston, H. K. Prasad, and I. Nath, Infect. Immun. 62:86-90, 1994) that polyclonal antibodies directed against selective sequences in the Mycobacterium leprae recombinant protein designated LSR were present in lepromatous leprosy patients undergoing erythema nodosum leprosum (ENL) reactions (type 2 reactions). In this study using peptides with single-residue deletions from positions 6 to 24, we define three distinct regions, GVTY, NAA, and RGD, which were important for antibody recognition and for the discrimination of clinically silent and active ENL reactions. Antibodies against NAA were found only in patients undergoing active reactions. This is in contrast to the results for the RGD motif, which was recognized in all ENL patients, irrespective of the clinical status. Though GVTY was recognized in both groups of patients, its recognition was masked by the flanking glutamic acid. These findings point towards a specific molecular recognition pattern that emerges when a lepromatous leprosy patient undergoes immune perturbations leading to ENL reactions. Moreover, the fine specificity of immunological recognition changes during the natural evolution of the host-parasite interaction.

Sera of leprosy patients with type 2 reactions recognize selective sequences in Mycobacterium leprae recombinant LSR protein.

Type 2 reactions (erythema nodosum leprosum [ENL]) are episodic, reactional states causing significant morbidity in lepromatous leprosy patients. With a view to defining the immunological differences between the stable and reactional forms of lepromatous leprosy, we determined antibody responses to LSR, a recombinant protein of Mycobacterium leprae previously described by us (S. Laal, Y.D. Sharma, H.K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. S. Mishra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991), as well as to 10- to 15-mer overlapping peptides synthesized on the basis of the LSR amino acid sequence. We report here the selective recognition of B cell epitopes by sera from patients with ENL as compared with a control group with nonreactional lepromatous leprosy. Peptides 2 and 3, with the sequences GVTYEIDLTNKNAA and IDLTNKNAAKLRGD, respectively, were recognized by > 95% of sera from patients with active ENL. Peptide 3 in addition showed reactivity with sera taken from 91.6% of lepromatous leprosy patients who were apparently stable but who were recorded to have had ENL several weeks before or after the sample collection. The core sequence IDLTNKNAA common to both these peptides may be a major target of humoral responses in ENL. In addition, the RGD motif at the C terminus appeared to influence the antigenicity of peptide 3 in enzyme-linked immunosorbent assay. It would appear that humoral responses during ENL are directed to selective antigenic determinants of the leprosy bacillus. The use of such serological markers to identify lepromatous leprosy patients with a high risk for developing ENL would be of clinical and predictive value.

Effect of recombinant interferon gamma administration on lesional monocytes/macrophages in lepromatous leprosy patients.

Hydrogen peroxide (H2O2) and superoxide anion (O2-) were estimated in lesional cells from 10 lepromatous leprosy patients injected intralesionally with recombinant interferon-gamma (rIFN-gamma). Clinically similar contralateral lesions injected with excipient served as controls. Lesional esterase-positive cells (suggestive of monocytes/macrophages) from rIFN-gamma-injected sites of many subjects showed net increments in the H2O2 and O2 levels compared to controls. When these cells were exposed to Mycobacterium leprae in vitro, there was a down-regulation of O2- in 4 of 5 subjects. Such inhibition was not observed in rIFN-gamma-injected sites. From the present studies it was not possible to determine whether the above effects of rIFN-gamma were primarily on lesional mature macrophages or on newly migrated young monocytes. Erythema and induration were observed at the cytokine-injected site but not at the control site between 24 and 72 hr. A monthly slit-skin smear examination of the former site showed a bacterial index (BI) reduction compared to the controls in 4 of 10 patients, this reduction occurring as early as 4 to 8 weeks. Histopathology of the biopsies of 6 of 10 subjects between 9 and 10 months showed a further BI decrease attributable to rIFN-gamma and not to the subsequently instituted chemotherapy.