Ex vivo and in vivo evidence that cigarette smoke-exposed T regulatory cells impair host immunity against Mycobacterium tuberculosis.

Introduction: A strong epidemiologic link exists between cigarette smoke (CS) exposure and susceptibility to tuberculosis (TB). Macrophage and murine studies showed that CS and nicotine impair host-protective immune cells against Mycobacterium tuberculosis (MTB) infection. While CS and nicotine may activate T regulatory cells (Tregs), little is known about how CS may affect these immunosuppressive cells with MTB infection. Methods: We investigated whether CS-exposed Tregs could exacerbate MTB infection in co-culture with human macrophages and in recipient mice that underwent adoptive transfer of Tregs from donor CS-exposed mice. Results: We found that exposure of primary human Tregs to CS extract impaired the ability of unexposed human macrophages to control an MTB infection by inhibiting phagosome-lysosome fusion and autophagosome formation. Neutralizing CTLA-4 on the CS extract-exposed Tregs abrogated the impaired control of MTB infection in the macrophage and Treg co-cultures. In Foxp3+GFP+DTR+ (Thy1.2) mice depleted of endogenous Tregs, adoptive transfer of Tregs from donor CS-exposed B6.PL(Thy1.1) mice with subsequent MTB infection of the Thy1.2 mice resulted in a greater burden of MTB in the lungs and spleens than those that received Tregs from air-exposed mice. Mice that received Tregs from donor CS-exposed mice and infected with MTB had modest but significantly reduced numbers of interleukin-12-positive dendritic cells and interferon-gamma-positive CD4+ T cells in the lungs, and an increased number of total programmed cell death protein-1 (PD-1) positive CD4+ T cells in both the lungs and spleens. Discussion: Previous studies demonstrated that CS impairs macrophages and host-protective T effector cells in controlling MTB infection. We now show that CS-exposed Tregs can also impair control of MTB in co-culture with macrophages and in a murine model.

The EJC disassembly factor PYM is an intrinsically disordered protein and forms a fuzzy complex with RNA.

The discovery of several functional interactions where one or even both partners remain disordered has demonstrated that specific interactions do not necessarily require well-defined intermolecular interfaces. Here we describe a fuzzy protein-RNA complex formed by the intrinsically unfolded protein PYM and RNA. PYM is a cytosolic protein, which has been reported to bind the exon junction complex (EJC). In the process of oskar mRNA localization in Drosophila melanogaster, removal of the first intron and deposition of the EJC are essential, while PYM is required to recycle the EJC components after localization has been accomplished. Here we demonstrate that the first 160 amino acids of PYM (PYM1-160) are intrinsically disordered. PYM1-160 binds RNA independently of its nucleotide sequence, forming a fuzzy protein-RNA complex that is incompatible with PYM's function as an EJC recycling factor. We propose that the role of RNA binding consists in down-regulating PYM activity by blocking the EJC interaction surface of PYM until localization has been accomplished. We suggest that the largely unstructured character of PYM may act to enable binding to a variety of diverse interaction partners, such as multiple RNA sequences and the EJC proteins Y14 and Mago.

Characterization of Immune Cells From the Lungs of Patients With Chronic Non-Tuberculous Mycobacteria or Pseudomonas aeruginosa Infection.

Little is known of the lung cellular immunophenotypes in patients with non-tuberculous mycobacterial lung disease (NTM-LD). Flow-cytometric analyses for the major myeloid and lymphoid cell subsets were performed in less- and more-diseased areas of surgically resected lungs from six patients with NTM-LD and two with Pseudomonas aeruginosa lung disease (PsA-LD). Lymphocytes, comprised mainly of NK cells, CD4+ and CD8+ T cells, and B cells, accounted for ~60% of all leukocytes, with greater prevalence of T and B cells in more-diseased areas. In contrast, fewer neutrophils were found with decreased number in more-diseased areas. Compared to NTM-LD, lung tissues from patients with PsA-LD demonstrated relatively lower numbers of T and B lymphocytes but similar numbers of NK cells. While this study demonstrated a large influx of lymphocytes into the lungs of patients with chronic NTM-LD, further analyses of their phenotypes are necessary to determine the significance of these findings.

Electrocatalytic oxidation of aromatic amine (4-aminobiphenyl): Kinetics and transformation products with mechanistic approach.

Electrocatalytic oxidation (EO) of carcinogenic 4-aminobiphenyl (4-ABP) aromatic amine was performed using Ti-RuO2 anodes. Current (I), pH, electrolysis time (t), and 4-ABP initial concentration (Co ) were selected as EO parameters, and their effects on %4-ABP removal (R1 ) and energy consumed (R2 ) were studied. Experimental design, parameters optimization, and their interaction with responses R1 and R2 were performed using response surface methodology. At optimized parameters, %TOC removal and 4-BP mineralization current efficiency (%MCE) were assessed to evaluate the potential of Ti/RuO2 anodes towards 4-ABP mineralization. Simultaneous TOC and 4-ABP degradation kinetics were also studied to evaluate the competition in 4-ABP mineralization and degradation. Further, UPLC-Q-TOF-MS analysis was performed to identify the 4-ABP transformation products during the EO, and a mechanism describing the EO transformation was proposed. At optimum parameters (I = 1.2 A; pH = 4.0; t = 30 min; Co = 30 ppm), responses were found to be R1 = 60.25%; R2 = 2.49 kWh/g of 4-ABP removed. %TOC removal and %MCE were 52.4% and 34.2%, respectively. PRACTITIONER POINTS: 4-Aminobiphenyl electro-oxidation (EO) was explored using Ti/RuO2 anode. Achieved 34.2% mineralization current efficiency, 52.4% TOC and 61.3% TKN removal. Three electro-oxidation transformation products of 4-ABP were detected. 4-Aminobiphenyl was found degrading at ≈1.6 times higher rate than TOC A plausible EO transformation pathway and mechanism was proposed.

Therapeutic efficacy of antimalarial drugs targeting DosRS signaling in Mycobacterium abscessus.

A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis, is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS. OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.

Apramycin Overcomes the Inherent Lack of Antimicrobial Bactericidal Activity in Mycobacterium abscessus.

Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's inherent resistance to clinically available antimicrobials. The low bactericidal potency of currently available treatment regimens is of concern and testifies to the poor therapeutic outcomes for pulmonary M. abscessus infections. Mechanistically, we demonstrate here that the acetyltransferase Eis2 is responsible for the lack of bactericidal activity of amikacin, the standard aminoglycoside used in combination treatment. In contrast, the aminoglycoside apramycin, with a distinct structure, is not modified by any of the pathogen's innate aminoglycoside resistance mechanisms and is not affected by the multidrug resistance regulator WhiB7. As a consequence, apramycin uniquely shows potent bactericidal activity against M. abscessus. This favorable feature of apramycin is reflected in a mouse model of pulmonary M. abscessus infection, which demonstrates superior activity, compared with amikacin. These findings encourage the development of apramycin for the treatment of M. abscessus infections and suggest that M. abscessus eradication in pulmonary disease may be within therapeutic reach.

Etiological Profiles of Patients With Thrombocytopenia in Central India: A Tertiary Centre Study.

Introduction Platelet-related disease may result from an abnormal platelet count, namely thrombocytopenia or thrombocythemia, or altered platelet function, and thus is associated with bleeding or with thrombotic manifestation. Thrombocytopenia is defined as a subnormal number of platelets i.e. less than 1,50,000/µL in the peripheral blood. It can lead to inadequate clot formation and increased risk of bleeding and is a common indication for bone marrow aspiration and biopsy. Methodology The study was a hospital-based prospective observational study from January 2019 to June 2020. All cases of thrombocytopenia which were diagnosed first on haematology analyser (platelet counts <150,000/µL) and confirmed subsequently by peripheral smear with/without bleeding manifestations due to thrombocytopenia were taken up for the study. The aims and objectives of this study were to find out the epidemiological spectrum and prevalence of thrombocytopenia according to age and sex along with the correlation of haematological and bone marrow findings of such patients. Result This study comprised 100 cases of thrombocytopenia, with acute leukaemia accounting for the majority (28/100), followed by dimorphic anaemia (15/100), megaloblastic anaemia (11/100), hypocellular marrow, infection, and other conditions. All cases (100%) displayed the clinical symptom of widespread weakness and pallor, which was followed by fatiguability (72%) and dyspnoea (48%). Many thrombocytopenic individuals also had lymphadenopathy and hepatomegaly, the last two least common appearances. Lymphadenopathy and hepatomegaly which were the last two least common presentations were present in a significant number of thrombocytopenic patients. Conclusion The study of bone marrow is helpful in the diagnosis of thrombocytopenia cases. Bone marrow examination is a simple, safe outpatient procedure and yields an impressive amount of diagnostically valuable data in a wide variety of disorders of thrombocytopenia. An evaluation of the patient's bone marrow unquestionably aids in the early diagnosis and treatment of their ailment.

Increased Virulence of Outer Membrane Porin Mutants of Mycobacterium abscessus.

Chronic pulmonary infections caused by non-tuberculous mycobacteria of the Mycobacterium abscessus complex (MABSC) are emerging as a global health problem and pose a threat to susceptible individuals with structural lung disease such as cystic fibrosis. The molecular mechanisms underlying the pathogenicity and intrinsic resistance of MABSC to antibiotics remain largely unknown. The involvement of Msp-type porins in the virulence and biocide resistance of some rapidly growing non-tuberculous mycobacteria and the finding of deletions and rearrangements in the porin genes of serially collected MABSC isolates from cystic fibrosis patients prompted us to investigate the contribution of these major surface proteins to MABSC infection. Inactivation by allelic replacement of the each of the two Msp-type porin genes of M. abscessus subsp. massiliense CIP108297, mmpA and mmpB, led to a marked increase in the virulence and pathogenicity of both mutants in murine macrophages and infected mice. Neither of the mutants were found to be significantly more resistant to antibiotics. These results suggest that adaptation to the host environment rather than antibiotic pressure is the key driver of the emergence of porin mutants during infection.

Stepwise pathogenic evolution of Mycobacterium abscessus.

Although almost all mycobacterial species are saprophytic environmental organisms, a few, such as Mycobacterium tuberculosis, have evolved to cause transmissible human infection. By analyzing the recent emergence and spread of the environmental organism M. abscessus through the global cystic fibrosis population, we have defined key, generalizable steps involved in the pathogenic evolution of mycobacteria. We show that epigenetic modifiers, acquired through horizontal gene transfer, cause saltational increases in the pathogenic potential of specific environmental clones. Allopatric parallel evolution during chronic lung infection then promotes rapid increases in virulence through mutations in a discrete gene network; these mutations enhance growth within macrophages but impair fomite survival. As a consequence, we observe constrained pathogenic evolution while person-to-person transmission remains indirect, but postulate accelerated pathogenic adaptation once direct transmission is possible, as observed for M. tuberculosis Our findings indicate how key interventions, such as early treatment and cross-infection control, might restrict the spread of existing mycobacterial pathogens and prevent new, emergent ones.

The double-edged sword of Tregs in M tuberculosis, M avium, and M absessus infection.

Immunity against different Mycobacteria species targeting the lung requires distinctly different pulmonary immune responses for bacterial clearance. Many parameters of acquired and regulatory immune responses differ quantitatively and qualitatively from immunity during infection with Mycobacteria species. Nontuberculosis Mycobacteria species (NTM) Mycobacterium avium- (M avium), Mycobacterium abscessus-(M abscessus), and the Mycobacteria species Mycobacterium tuberculosis-(Mtb). Herein, we discuss the potential implications of acquired and regulatory immune responses in the context of animal and human studies, as well as future directions for efforts to treat Mycobacteria diseases.

Non-Tuberculous Mycobacteria Interference with BCG-Current Controversies and Future Directions.

The global tuberculosis (TB) epidemic caused by the bacterial pathogen Mycobacterium tuberculosis (M.tb) continues unabated. The Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination is widely utilized worldwide to protect against infection with M.tb. BCG vaccine protection against TB has had widely varying results for reasons that are not well understood. BCG vaccine interference by non-tuberculosis (NTM) mycobacterial species has been implicated as the potential cause of reduced BCG vaccine efficacy against M.tb. Ongoing efforts to develop new vaccines for TB requires a thorough understanding of the effect of NTM exposure on BCG vaccine efficacy, which may ultimately be a critical determinant of success. We reviewed the conflicting reports on whether NTM interferes with the BCG vaccine, potential explanations to help resolve the controversy, and strategies for developing better animal models. Further studies are needed to longitudinally track the effects of NTM exposure on BCG vaccine-induced host-protective anti-TB immunity.

Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility.

Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen.

Animal Models of Mycobacteria Infection.

This manuscript describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species. A wide variety of animal models have been used to test new vaccines, drugs, and the impact of cigarette exposure. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Aerosol infection of mice with mycobacteria Basic Protocol 2: Aerosol infection of guinea pig with mycobacteria using a Madison chamber Alternate Protocol 1: Cigarette exposure prior to infection of mice with mycobacteria Alternate Protocol 2: Intravenous infection of mice with mycobacteria Basic Protocol 3: Necropsy methods for animals experimentally infected with mycobacteria Basic Protocol 4: Following the course of infection Basic Protocol 5: Measuring the animal immune response to infection Support Protocol: Cultivation of mycobacteria for use in animal experiments.

Ca2+-binding protein from Entamoeba histolytica (EhCaBP6) is a novel GTPase.

GTPases are molecular switches, which regulate a variety of cellular processes such as cell polarity, gene transcription, microtubule dynamics, cell-cycle etc. In this paper, we characterize a Ca2+-binding protein from Entamoeba histolytica (EhCaBP6) as a novel GTPase. We locate the active site for GTP hydrolysis within the C-terminal domain of EhCaBP6, although it requires full length protein for its complete range of activity. Using NMR studies, we observe that GTP binding induces conformational change in EhCaBP6. The identification of this novel and unusual Ca2+-dependent GTPase is important to elucidate the unconventional cell cycle of E. histolytica.

Factors affecting adherence to treatment in children living with HIV.

BACKGROUND: With the successful introduction of ART, the life expectancy of children living with HIV (CLHIV) is substantially increased. However, strict compliance is a necessary step to begin with as noncompliance is again associated with its own demerits of incomplete suppression and decreasing the overall survival of the patients. AIMS: The aim of this study was to measure factors associated with adherence to ART among CLHIV. MATERIALS AND METHODS: This is a 1-year follow-up study conducted from November 2012 to December 2013. A total of 171 children between 18 months and 15 years living with HIV and on ART attending the two ART centers of Delhi were enrolled in the study. Adherence and factors affecting adherence were obtained from the study participants using a semi-structured interview schedule. STATISTICAL ANALYSIS USED: Collected data were transformed into variables and analyzed into SPSS. All observations were in terms of mean, median, standard deviations, percentages, and proportions. Tests of significance such as Chi-square test and t-test were applied wherever required. RESULTS: In nearly 89% of the study participants, adherence to ART was ≥95% at the end of the study. The most common reason for nonadherence was forgetfulness (59%), and 57% of the parents/caretakers were facing monetary problems. Long distance, greater duration to reach center, and unavailability of the parents/caretakers were also quoted as problems. CONCLUSION: Adherence is genesis to successful treatment outcome and is strongly associated with availability of support by their parents/caretakers. Professional help along with guidance and encouragement is required not only at the caretaker level but also to the family as a whole.

Clofazimine inhalation suspension for the aerosol treatment of pulmonary nontuberculous mycobacterial infections.

BACKGROUND: Nontuberculous mycobacteria are recognized as a concern for cystic fibrosis (CF) patients due to increasing disease prevalence and the potential for detrimental effects on pulmonary function and mortality. Current standard of care involves prolonged systemic antibiotics, which often leads to severe side effects and poor treatment outcomes. In this study, we investigated the tolerability and efficacy of a novel inhaled therapeutic in various mouse models of NTM disease. METHODS: We developed clofazimine inhalation suspension (CIS), a novel formulation of clofazimine developed for inhaled administration. To determine the efficacy, minimum inhibitory concentrations were evaluated in vitro, and tolerability of CIS was determined in naïve mouse models over various durations. After establishing tolerability, CIS efficacy was tested in in vivo infection models of both Mycobacterium avium and M. abscessus. Lung and plasma clofazimine levels after chronic treatments were evaluated. RESULTS: Clofazimine inhalation suspension demonstrated antimycobacterial activity in vitro, with MIC values between 0.125 and 2 µg/ml for M. avium complex and M. abscessus. Administration into naïve mice showed that CIS was well tolerated at doses up to 28 mg/kg over 28 consecutive treatments. In vivo, CIS was shown to significantly improve bacterial elimination from the lungs of both acute and chronic NTM-infected mouse models compared to negative controls and oral clofazimine administration. Clofazimine concentrations in lung tissue were approximately four times higher than the concentrations achieved by oral dosing. CONCLUSION: Clofazimine inhalation suspension is a well tolerated and effective novel therapeutic candidate for the treatment of NTM infections in mouse models.

Mycobacterium avium Infection in a C3HeB/FeJ Mouse Model.

Infections caused by Mycobacterium avium complex (MAC) species are increasing worldwide, resulting in a serious public health problem. Patients with MAC lung disease face an arduous journey of a prolonged multidrug regimen that is often poorly tolerated and associated with relatively poor outcome. Identification of new animal models that demonstrate a similar pulmonary pathology as humans infected with MAC has the potential to significantly advance our understanding of nontuberculosis mycobacteria (NTM) pathogenesis as well as provide a tractable model for screening candidate compounds for therapy. One new mouse model is the C3HeB/FeJ which is similar to MAC patients in that these mice can form foci of necrosis in granulomas. In this study, we evaluated the ability of C3HeB/FeJ mice exposure to an aerosol infection of a rough strain of MAC 2285 to produce a progressive infection resulting in small necrotic foci during granuloma formation. C3HeB/FeJ mice were infected with MAC and demonstrated a progressive lung infection resulting in an increase in bacterial burden peaking around day 40, developed micronecrosis in granulomas and was associated with increased influx of CD4+ Th1, Th17, and Treg lymphocytes into the lungs. However, during chronic infection around day 50, the bacterial burden plateaued and was associated with the reduced influx of CD4+ Th1, Th17 cells, and increased numbers of Treg lymphocytes and necrotic foci during granuloma formation. These results suggest the C3HeB/FeJ MAC infection mouse model will be an important model to evaluate immune pathogenesis and compound efficacy.

Single-site phosphorylation within the His-tag sequence attached to a recombinant protein.

We report the observation of single-site phosphorylation in a His-tag sequence N-terminally attached to a recombinant protein (UVI31+) in vitro. This modification was detected at position 23 at a serine residue of the His-tag sequence encoded by the vector pET28a. Furthermore, the phosphorylated tag sequence was found to be dephosphorylated by the action of alkaline phosphatases. The functional activity and dynamics of the protein carrying the His-tag sequence were unchanged after phosphorylation. The possibility of phosphorylation within the N-terminal His-tag demonstrates that care has to be taken upon analysis of post-translational modifications via mass spectrometry for recombinant protein expression strategies.

Microscopic study of zinc nanoparticles synthesised using thermosetting polymer.

The present study reports the novel synthesis of Zinc nanoparticles (Zn NPs) by thermal decomposition method and its characterisation by Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), and X-ray Diffraction Measurements (XRD). Synthesis of Zn NPs was achieved by using thermosetting polymer and zinc salts as precursor. Zn NPs were obtained on calcination at 850 °C for 30 min. SEM study reveals that synthesized nanoparticles are spherical in shape. XRD analysis shows that the Zn NPs formed are low crystalline in nature.