SummaryESAT6 has recently been demonstrated to cause haemolysis and macrophage lysis. Our studies demonstrate that ESAT6 causes cytolysis of type 1 and type 2 pneumocytes. Both types of pneumocytes express membrane laminin, and ESAT6 exhibits dosedependent binding to both cell types and to purified human laminin. While minimal ESAT6 was detected on the surface of Mycobacterium tuberculosis grown in vitro, exogenously provided ESAT6 specifically associated with the bacterial cell surface, and the bacterium-associated ESAT6 retained its cytolytic ability. esat6 transcripts were upregulated~4-to~13-fold in bacteria replicating in type 1 cells, and~3-to~5 fold in type 2 cells. In vivo, laminin is primarily concentrated at the basolateral surface of pneumocytes where they rest on the basement membrane, which is composed primarily of laminin and collagen. The upregulation of esat6 transcripts in bacteria replicating in pneumocytes, the specific association of ESAT6 with the bacterial surface, the binding of ESAT6 to laminin and the lysis of pneumocytes by free and bacterium-associated ESAT6 together suggest a scenario wherein Mycobacterium tuberculosis replicating in pneumocytes may utilize surface ESAT6 to anchor onto the basolateral lamininexpressing surface of the pneumocytes, and damage the cells and the basement membrane to directly disseminate through the alveolar wall.

Context:Prevalence of hypothyroidism is 2–4% in women in the reproductive age group. Hypothyroidism can affect fertility due to anovulatory cycles, luteal phase defects, hyperprolactinemia, and sex hormone imbalance.Aims and Objectives:To study the prevalence of clinical/sub-clinical hypothyroidism in infertile women and the response of treatment for hypothyroidism on infertility.Materials and Methods:A total of 394 infertile women visiting the infertility clinic for the first time were investigated for thyroid stimulating hormone (TSH) and prolactin (PRL). Infertile women with hypothyroidism alone or with associated hyperprolactinemia were given treatment for hypothyroidism with thyroxine 25–150 μg.Results:Of 394 infertile women, 23.9% were hypothyroid (TSH > 4.2 μIU/ml). After treatment for hypothyroidism, 76.6% of infertile women conceived within 6 weeks to 1 year. Infertile women with both hypothyroidism and hyperprolactinemia also responded to treatment and their PRL levels returned to normal.Conclusion:Measurement of TSH and PRL should be done at early stage of infertility check up rather than straight away going for more costly tests or invasive procedures. Simple, oral hypothyroidism treatment for 3 months to 1 year can be of great benefit to conceive in otherwise asymptomatic infertile women.

The aim of the study was to investigate the activity of human neutrophil peptide (HNP)-1 to kill Mycobacterium tuberculosis H37Rv in vitro and ex vivo in the murine macrophage cell line J744A.1 on the basis of colony forming units. Macromolecular biosynthesis was studied by monitoring the incorporation of radioactive precursors into different macromolecules. The binding and localization studies were carried out with radioiodinated HNP-1 whereas the cytotoxicity of HNP-1 to macrophages was determined by trypan blue exclusion assay. A concentration dependent inhibition in the growth of M. tuberculosis H37Rv was observed in the presence of HNP-1. The minimum inhibitory concentration and median inhibitory concentration of HNP-1 were found to be 2.5 microg x mL(-1) and 0.8 microg x mL(-1). Treatment of both in vitro grown and phagocytosed mycobacterial cells with HNP-1 resulted in generalized inhibition in the macromolecular biosynthesis with maximum inhibition in deoxyribonucleic acid and lipid biosynthesis. HNP-1 exhibited equilibrium binding with respect to time and two-thirds of bound radioactivity was shown to be present inside the macrophages. Approximately 50% and 98% killing of intracellular mycobacteria was observed after 3 days of treatment with 5 microg x mL(-1) and 40 microg x mL(-1) of HNP-1, respectively. HNP-1 exhibited low cytotoxicity towards the macrophage cell line at the bactericidal concentration to mycobacteria. From the results of this study, it is concluded that human neutrophil peptide-1 possesses potent bactericidal activity against virulent mycobacteria in vitro as well as mycobacteria replicating within macrophages.

The therapeutic efficacy of human neutrophil peptide 1 (HNP-1) against experimental tuberculosis in mice on the basis of numbers of CFU has been examined. Mice infected with 1.5 ؋ 10 4 CFU of Mycobacterium tuberculosis H 37 Rv and treated with different doses of HNP-1 injected subcutaneously exhibited significant clearance of bacilli from lungs, livers, and spleens. There were time-and dose-dependent decreases in the bacillary load in lungs, livers, and spleens of the HNP-1-treated animals compared to that in controls (untreated animals). These observations strongly suggest the therapeutic activity of HNP-1 against tuberculosis.The recent resurgence in the incidence of tuberculosis and its association with human immunodeficiency virus infection and AIDS warrants the development of new therapeutic strategies for the effective control of tuberculosis. Despite the outbreaks of multidrug-resistant strains of Mycobacterium tuberculosis and calls for new drug development, truly novel compounds, which would significantly improve treatment, continue to elude us. Antibiotic peptides from higher eukaryotes have gained considerable attention as an alternative to conventional antibiotics owing to their potent antimicrobial activities in vitro (for a recent review see reference 2). The defensins are a family of small antimicrobial peptides having six highly conserved cysteine residues, resulting in three disulfide linkages. Human neutrophil peptide 1 (HNP-1) is one of the four types of defensins present in the azurophilic granules of polymorphonuclear neutrophils. HNP-1 has been demonstrated to exhibit potent in vitro bactericidal activity against a wide variety of microbes, including mycobacterial species (5). Recently, we have reported the ability of HNP-1 to kill M. tuberculosis in vitro as well as ex vivo (6, 7). To date, no study has been carried out to investigate the therapeutic efficacy of HNP-1 against M. tuberculosis infections. In the present study, the in vivo therapeutic potential of HNP-1 against experimental tuberculosis in a mouse model was investigated.Six-to eight-week-old pathogen-free Laca (inbred) strain mice were infected intravenously with 1.5 ϫ 10 4 CFU of M. tuberculosis H 37 Rv per mouse. After 15 days of challenge, establishment of infection was confirmed by Ziehl-Neelsen staining of whole-tissue homogenates of lungs, livers, and spleens from four animals. Chemically synthesized HNP-1 with the same primary structure and the same disulfide linkages (i.e., between Cys-2 and Cys-30, Cys-4 and Cys-19, and Cys-9 and Cys-29) as native HNP-1 (obtained from Peptide Institute Inc., Osaka, Japan) was used in this study. It was dissolved in 0.01% acetic acid and stored as a stock solution of 100 g/ml at Ϫ20°C (dissolved peptide was used within 3 weeks, as the aqueous solution was stable for a few weeks only). To investigate the therapeutic potential of HNP-1, infected animals were divided into three groups (at least four animals in each group) and were injected subcutaneously with two different doses of HNP-1...

We report the role of human neutrophil peptide (HNP)-1 as an adjunct to antituberculosis (anti-TB) drugs. The combination of HNP-1, isoniazid, and rifampicin was evaluated against Mycobacterium tuberculosis H(37)Rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (MICs) of these agents. In vitro results revealed >1-log unit reductions even when HNP-1 and anti-TB drugs were used at 1/16 MICs. This combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 MICs of HNP-1 and drugs. HNP-1 used in conjunction with anti-TB drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. The effective therapeutic dosage of drugs could be reduced to half by supplementing HNP-1 in the therapeutic schedule. These results clearly suggest that HNP-1 can be used as adjunct chemotherapy with conventional drugs against TB.

Pulmonary tuberculosis, the disease caused by Mycobacterium tuberculosis, still retains a top rank among the deadliest communicable diseases. Sputum expectorated during the disease continues to be a primary diagnostic specimen and also serves as a reservoir of bacteria. The expression pattern of mycobacteria in sputum will lead to an insight into bacterial adaptation at the most highly transmissible stage of infection and can also help in identifying newer diagnostic as well as drug targets. Thus, in the present study, a whole genome microarray of Mycobacterium tuberculosis was used to elucidate the transcriptional profile of mycobacteria in the sputum samples of smear positive pulmonary tuberculosis patients. Overall, the mycobacteria in sputum appeared to be in a low energy and low replicative state as compared to in vitro grown log phase M. tb with downregulation of genes involved in ATP synthesis, aerobic respiration and translational machinery. Simultaneously, downregulation was also seen in the genes involved in secretion machinery of mycobacteria along with the downregulation of genes involved in the synthesis of phthiocerol dimycocerosate and phenol glycolipids. In contrast, the majority of the genes which showed an upregulation in sputum mycobacteria were of unknown function. Further identification of these genes may provide new insights into the mycobacterial behavior during this phase of infection and may help in deciphering candidates for development of better diagnostic and drug candidates.

BackgroundCharacteristics of laboratory findings of COVID-19 patients are of great significance for diagnosis and treatment. Studies that have analysed the variations in hepatic profile in correlation with the inflammatory markers in SARS-CoV-2 are limited.MethodsWe retrospectively analysed liver function tests and inflammatory markers of 170 admitted patients with confirmed COVID-19 in the tertiary care centre, Post Graduate Institute of Medical Education and Research (PGIMER), India, using Roche Cobas Autoanalyzer.ResultsNumber of patients with normal liver enzyme levels were 63 (41.5%), while with raised levels of any of the liver enzymes were 89 (58.5%), out of which 43 (48.31%) had liver injury which manifested as increased severity in terms of intensive care unit (ICU) requirement (p=0.0005). Significantly raised levels of liver enzymes and liver injury were observed with age (p<0.0001) and in males (p=0.004). Significantly decreased levels of albumin and total proteins and increased levels of total bilirubin (p<0.0001) were seen in patients with abnormal liver enzyme levels and liver injury as compared to patients with normal levels. Significant increase in the levels of alanine transaminase and gamma-glutamyl transferase was seen on the 7th day, CRP and ferritin (p<0.0001) peaks were observed on 2nd and 3rd day respectively. A significant positive correlation was found between the levels of these inflammatory markers and liver function parameters.ConclusionsMore than half of patients admitted to the hospital with SARS-CoV-2 infection had an abnormal liver function which was found to be associated with raised levels of inflammatory markers. Significantly higher proportions of patients with abnormal liver function were elderly and males and were at higher risk of progressing to severe disease.

Cholesterol-mediated mycobacteria entry into and survival within macrophages has added a new dimension to Tuberculosis research. The molecular mechanism through which cholesterol initiates this process is still poorly understood. The present study addressed to resolve this mechanism revealed that Mycobacterium tuberculosis possesses cholesterol-specific Receptor 'Ck'-like molecule responsible for mycobacterial entry into macrophages. Further human Receptor-Ck was found to regulate transcriptional expression of a gene that codes for Tryptophan-Aspartate containing coat (TACO) protein responsible for survival of mycobacteria within cells. Based upon these results, we propose that interaction of Receptor-Ck with cholesterol-rich membrane domains helps to create a 'Synaptic-junction' between mycobacteria and macrophage resulting in signalling events that are responsible for mycobacterium entry into and survival within macrophages.