Cigarette Smoking as a Risk Factor for Tuberculosis in Adults: Epidemiology and Aspects of Disease Pathogenesis.

It has been noted by the World Health Organisation that cases of tuberculosis in 2022 globally numbered 10.6 million, resulting in 1.3 million deaths, such that TB is one of the infectious diseases causing the greatest morbidity and mortality worldwide. Since as early as 1918, there has been an ongoing debate as to the relationship between cigarette smoking and TB. However, numerous epidemiological studies, as well as meta-analyses, have indicated that both active and passive smoking are independent risk factors for TB infection, development of reactivation TB, progression of primary TB, increased severity of cavitary disease, and death from TB, among several other considerations. With this considerable body of evidence confirming the association between smoking and TB, it is not surprising that TB control programmes represent a key potential preventative intervention. In addition to coverage of the epidemiology of TB and its compelling causative link with smoking, the current review is also focused on evidence derived from clinical- and laboratory-based studies of disease pathogenesis, most prominently the protective anti-mycobacterial mechanisms of the alveolar macrophage, the primary intracellular refuge of M. tuberculosis. This section of the review is followed by an overview of the major strategies utilised by the pathogen to subvert these antimicrobial mechanisms in the airway, which are intensified by the suppressive effects of smoke inhalation on alveolar macrophage function. Finally, consideration is given to a somewhat under-explored, pro-infective activity of cigarette smoking, namely augmentation of antibiotic resistance due to direct effects of smoke per se on the pathogen. These include biofilm formation, induction of cellular efflux pumps, which eliminate both smoke-derived toxicants and antibiotics, as well as gene modifications that underpin antibiotic resistance.

Pro-Inflammatory Interactions of Dolutegravir with Human Neutrophils in an In Vitro Study.

There is increasing awareness of an association between the uptake of the HIV integrase inhibitor, dolutegravir, in first-line antiretroviral regimens with unusual weight gain and development of the metabolic syndrome, particularly in African women. Although seemingly unexplored, the development of systemic inflammation linked to the putative pro-inflammatory activity of dolutegravir represents a plausible pathophysiological mechanism of this unusual weight gain. This possibility was explored in the current study undertaken to investigate the effects of dolutegravir (2.5−20 µg/mL) on several pro-inflammatory activities of neutrophils isolated from the blood of healthy, adult humans. These activities included the generation of reactive oxygen species (ROS), degranulation (elastase release) and alterations in the concentrations of cytosolic Ca2+ using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of neutrophils to dolutegravir alone resulted in the abrupt, dose-related, and significant (p < 0.0039−p < 0.0022) generation of ROS that was attenuated by the inclusion of the Ca2+-chelating agent, EGTA, or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI), phospholipase C (U733122), myeloperoxidase (sodium azide) and phosphoinositol-3-kinase (wortmannin). In addition, exposure to dolutegravir augmented the release of elastase by stimulus-activated neutrophils. These pro-inflammatory effects of dolutegravir on neutrophils were associated with significant, rapid, and sustained increases in the concentrations of cytosolic Ca2+ that appeared to originate from the extracellular compartment, seemingly consistent with an ionophore-like property of dolutegravir. These findings are preliminary and necessitate verification in the clinical setting of HIV infection. Nevertheless, given the complex link between inflammation and obesity, these pro-inflammatory interactions of dolutegravir with neutrophils may contribute to unexplained weight gain, possibly via the development of insulin resistance.

Assessment of the efficacy of clofazimine alone and in combination with primary agents against Mycobacterium tuberculosis in vitro.

OBJECTIVES: The chemotherapeutic regimens of patients with drug-susceptible (DS)- tuberculosis (TB) comprise four primary anti-TB drugs: rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and pyrazinamide (PZA), administered for six-to-nine months. These drug regimens target the various microbial populations that include actively replicating (AR), slow-replicating (SR) and non-replicating (NR) organisms. Clofazimine (CFZ) has showed benefit in shortening DS-TB treatment in vivo from six to four months when used in combination with this regimen in murine models of experimental infection. However, its antimicrobial efficacy when used in combination with the primary drugs against the various microbial populations of Mycobacterium tuberculosis has not been demonstrated. METHODS: In the current in vitro study, the inhibitory and bactericidal activities of CFZ in combination with the primary anti-TB drugs, RMP, INH and EMB against the AR and SR organisms in planktonic and biofilm-forming cultures, respectively, were evaluated by fractional inhibitory concentration index (FICI) and fractional bactericidal concentration index (FBCI) determinations, using the Loewe Additivity Model. RESULTS: In planktonic cultures, CFZ demonstrated synergistic growth inhibitory activity in combination with RMP and INH individually and collectively. With respect to bactericidal activity, CFZ exhibited synergistic activity only in a two-drug combination with RMP. However, in biofilm-forming cultures, all CFZ-containing anti-TB drug combinations exhibited synergistic inhibitory and bactericidal effects, particularly in combination with RIF and INH. CONCLUSION: Clofazimine exhibited synergistic effects in combination with primary anti-TB drugs against both planktonic and biofilm-forming cultures, showing potential benefit in augmenting treatment outcome when used during standard TB chemotherapy.

Role of the kdpDE Regulatory Operon of Mycobacterium tuberculosis in Modulating Bacterial Growth in vitro.

Bacteria use K+-uptake transporters differentially for adaptation in varying growth conditions. In Mycobacterium tuberculosis, two K+-uptake systems, the Trk comprising the CeoB and CeoC proteins and the Kdp consisting of the two-component system (TCS), KdpDE and KdpFABC, have been characterized, but their selective utilization during bacterial growth has not been completely explored. In the current study, the roles of the M. tuberculosis KdpDE regulatory system alone and in association with the Trk transporters in bacterial growth were investigated by evaluating the growth of M. tuberculosis KdpDE-deletion and KdpDE/Trk (KT)-double knockout mutant strains in planktonic culture under standard growth conditions. The KT-double knockout mutant strain was first constructed using homologous recombination procedures and was evaluated together with the KdpDE-deletion mutant and the wild-type (WT) strains with respect to their rates of growth, K+-uptake efficiencies, and K+-transporter gene expression during planktonic growth. During growth at optimal K+ concentrations and pH levels, selective deletion of the TCS KdpDE (KdpDE-deletion mutant) led to attenuation of bacterial growth and an increase in bacterial K+-uptake efficiency, as well as dysregulated expression of the kdpFABC and trk genes. Deletion of both the KdpDE and the Trk systems (KT-double knockout) also led to severely attenuated bacterial growth, as well as an increase in bacterial K+-uptake efficiency. These results demonstrate that the KdpDE regulatory system plays a key role during bacterial growth by regulating K+ uptake via modulation of the expression and activities of both the KdpFABC and Trk systems and is important for bacterial growth possibly by preventing cytoplasmic K+ overload.

Effects of Cigarette Smoke Condensate on Growth and Biofilm Formation by Mycobacterium tuberculosis.

MATERIALS AND METHODS: The planktonic and biofilm-forming cultures were prepared in Middlebrook 7H9 and Sauton broth media, respectively, using Mtb strain, H37Rv. The effects of CSC at concentrations of 0.05-3.12 mg/L on growth, biofilm formation and structure were evaluated using microplate Alamar Blue assay, spectrophotometric procedure and scanning electron microscopy (SEM), respectively. Involvement of reactive oxygen species in CSC-mediated biofilm formation was investigated by including catalase in biofilm-forming cultures. RESULTS: CSC did not affect the growth of planktonic bacteria, but rather led to a statistically significant increase in biofilm formation at concentrations of 0.4-3.12 mg/L, as well as in the viability of biofilm-forming bacteria at CSC concentrations of 0.2-1.56 mg/L. SEM confirmed an agglomerated biofilm matrix and irregular bacterial morphology in CSC-treated biofilms. Inclusion of catalase caused significant attenuation of CSC-mediated augmentation of biofilm formation by Mtb, implying involvement of oxidative stress. These findings demonstrate that exposure of Mtb to CSC resulted in increased biofilm formation that appeared to be mediated, at least in part, by oxidative stress, while no effect on planktonic cultures was observed. CONCLUSION: Smoking-related augmentation of biofilm formation by Mtb may contribute to persistence of the pathogen, predisposing to disease reactivation and counteracting the efficacy of antimicrobial chemotherapy.

Is targeting dysregulation in apoptosis splice variants in Mycobacterium tuberculosis (MTB) host interactions and splicing factors resulting in immune evasion by MTB strategies a possibility?

Mycobacterium tuberculosis (Mtb), is one of the foremost organisms causing mortality in humans, and has been for most of human history. When faced with an infection the human immune system is ordinarily very competent in killing both extracellular and intracellular bacilli. However, Mtb is able to evade the host immune system and is even able to establish a persistent infectious reservoir by "hiding" in the immune cells of the host. While the mechanisms by which the bacteria accomplishes this are not fully understood, it is known that the bacterium can subvert cellular processes in cells such as macrophages that prevent the lysis of the bacteria or the cell undergoing apoptosis. They are also able to interfere with immune cell signalling. One of the greatest effects that Mtb has is too alter the transcriptome of the macrophage. An easy way for the bacterium to accomplish this is to alter the alternative splicing patterns of the host. This can lead to a large change in the population of different protein isoforms, some of which have very different functions when compared to the original protein. At the same time the long history of Mtb infecting humans have led to specific immune reactions that occur in the host immune system in order to fight the infection. Many of these specific reactions involve new isoforms of host defence proteins. In this way the human host can use alternate splicing to create new isoforms of immune- related proteins that are more effective in defending against Mtb.

Submission for Special Issue: The Role of Platelet Activation in the Pathophysiology of HIV, Tuberculosis, and Pneumococcal Disease. Bedaquiline Suppresses ADP-Mediated Activation of Human Platelets In Vitro via Interference With Phosphatidylinositol 3-Kinase.

Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 µg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.

Clofazimine, but Not Isoniazid or Rifampicin, Augments Platelet Activation in vitro.

Although the inclusion of the cationic amphiphilic, anti-mycobacterial agent, clofazimine, in the chemotherapeutic regimens of patients with multidrug-resistant tuberculosis (TB) has contributed to improved outcomes, concerns remain about the cardiotoxic potential of this agent. Accordingly, the current study was undertaken with the primary objective of investigating the effects of clofazimine, on the reactivity of human platelets in vitro, a seemingly unexplored, mechanism of cardiotoxicity. Platelet-rich plasma (PRP) prepared from the blood of healthy, adult humans was treated with clofazimine (0.625-10 mg/L), or the primary anti-TB agents, isoniazid and rifampicin (at final concentrations of 5 and 10 mg/L), followed by addition of either adenosine 5'-diphosphate (ADP) or thrombin and measurement of platelet activation according to the magnitude of expression of CD62P (P-selectin), as well as the CD62P-mediated formation of heterotypic neutrophil:platelet (NP) aggregates, using flow cytometry. Clofazimine, but neither isoniazid nor rifampicin, caused dose-related potentiation of both ADP- and thrombin-activated expression of CD62P by platelets, achieving statistical significance at threshold concentrations of 0.625 and 2.5 mg/L, respectively, as well as significant formation of N:P aggregates. These stimulatory effects of clofazimine on platelet activation were partly attenuated by pre-treatment of PRP with the membrane-stabilizing agent, α-tocopherol, possibly consistent with a membrane-disruptive mechanism. In conclusion, clofazimine, at concentrations within the therapeutic range, augments platelet activation in vitro, probably by a mechanism linked to membrane destabilization. If operative in vivo, these pro-thrombotic activities of clofazimine may predispose for development of microvascular occlusion, exacerbating an already existing high risk for development of TB-associated cardiovascular disease.

Facial appearance reveals immunity in African men.

Facial appearance is thought to indicate immunity in humans, but very few studies have tested this relationship directly. The aim of this study was to test the relationship between direct measures of immunity, perceived facial health and attractiveness, and facial cues in African men. We show that men with a stronger cytokine response are considered significantly more attractive and healthy. Men with more masculine, heavier facial features (i.e. muscular appearance) have a significantly higher cytokine response and appear significantly healthier and more attractive, while men with a yellower, lighter, "carotenoid" skin colour, have a marginally higher immune response and are also considered significantly more healthy and attractive. In contrast, more symmetrical, skinnier looking men appeared more attractive and healthier, but did not have a stronger cytokine response. These findings also shed new light on the "androgen-mediated" traits proposed by the immunocompetence handicap hypothesis (ICHH) and we propose that facial muscularity serves as a better estimate of an "androgen-mediated" trait than facial masculinity. Finally, we build on previous evidence to show that men's facial features do indeed reveal aspects of immunity, even better than more traditional measures of health, such as body mass index (BMI).

Mechanisms of action and therapeutic efficacies of the lipophilic antimycobacterial agents clofazimine and bedaquiline.

Drug-resistant (DR)-TB is the major challenge confronting the global TB control programme, necessitating treatment with second-line anti-TB drugs, often with limited therapeutic efficacy. This scenario has resulted in the inclusion of Group 5 antibiotics in various therapeutic regimens, two of which promise to impact significantly on the outcome of the therapy of DR-TB. These are the 're-purposed' riminophenazine, clofazimine, and the recently approved diarylquinoline, bedaquiline. Although they differ structurally, both of these lipophilic agents possess cationic amphiphilic properties that enable them to target and inactivate essential ion transporters in the outer membrane of Mycobacterium tuberculosis. In the case of bedaquiline, the primary target is the key respiratory chain enzyme F1/F0-ATPase, whereas clofazimine is less selective, apparently inhibiting several targets, which may underpin the extremely low level of resistance to this agent. This review is focused on similarities and differences between clofazimine and bedaquiline, specifically in respect of molecular mechanisms of antimycobacterial action, targeting of quiescent and metabolically active organisms, therapeutic efficacy in the clinical setting of DR-TB, resistance mechanisms, pharmacodynamics, pharmacokinetics and adverse events.

Expression of the Genes Encoding the Trk and Kdp Potassium Transport Systems of Mycobacterium tuberculosis during Growth In Vitro.

Two potassium (K(+))-uptake systems, Trk and Kdp, are operative in Mycobacterium tuberculosis (Mtb), but the environmental factors triggering their expression have not been determined. The current study has evaluated the expression of these genes in the Mtb wild-type and a trk-gene knockout strain at various stages of logarithmic growth in relation to extracellular K(+) concentrations and pH. In both strains, mRNA levels of the K(+)-uptake encoding genes were relatively low compared to those of the housekeeping gene, sigA, at the early- and mid-log phases, increasing during late-log. Increased gene expression coincided with decreased K(+) uptake in the context of a drop in extracellular pH and sustained high extracellular K(+) concentrations. In an additional series of experiments, the pH of the growth medium was manipulated by the addition of 1N HCl/NaOH. Decreasing the pH resulted in reductions in both membrane potential and K(+) uptake in the setting of significant induction of genes encoding both K(+) transporters. These observations are consistent with induction of the genes encoding the active K(+) transporters of Mtb as a strategy to compensate for loss of membrane potential-driven uptake of K(+) at low extracellular pH. Induction of these genes may promote survival in the acidic environments of the intracellular vacuole and granuloma.

Effects of clofazimine on planktonic and biofilm growth of Mycobacterium tuberculosis and Mycobacterium smegmatis.

Mycobacteria form lipid-rich biofilms that restrict the efficacy of antimicrobial chemotherapy, possibly necessitating the use of lipophilic antibiotics. In the current study, the activity of one such agent, clofazimine, against Mycobacterium tuberculosis and Mycobacterium smegmatis planktonic cells and biofilms was investigated. Minimum inhibitory concentrations (MICs) of clofazimine were determined for planktonic cultures, whilst minimum bactericidal concentrations (MBCs) were determined for planktonic, biofilm-producing and biofilm-encased organisms using standard bacteriological procedures. The effects of clofazimine on biofilm formation and the stability of pre-formed biofilm were measured using a crystal violet-based spectrophotometric procedure. In the case of M. smegmatis, clofazimine was found to be active against planktonic phase (MICs and MBCs of 2.5mg/L and >20mg/L, respectively) and biofilm-producing organisms (MBC of 2.5mg/L); clofazimine demonstrated greater activity against M. tuberculosis, corresponding values of 0.06, 5 and 0.3mg/L. Although clofazimine inhibited biofilm production both by M. tuberculosis and M. smegmatis (P<0.05 at ≥0.07mg/L and ≥0.3mg/L, respectively) and appeared to reduce the pre-formed M. tuberculosis biofilm, addition of antimicrobial agent to pre-existing biofilm matrices failed to kill biofilm-encased organisms. In conclusion, clofazimine is more effective against M. tuberculosis than against M. smegmatis, exhibiting bactericidal activity both for actively growing and slowly replicating bacilli but not for non-replicating organisms of both species.

Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro.

Manganese (as Mn(2+)), a superoxide dismutase mimetic, catalyzes the formation of the relatively stable membrane-permeable reactive oxygen species (ROS) hydrogen peroxide (H2O2), a mediator of intracellular redox signaling in immune and inflammatory cells. The goal of this study was to investigate the potential for Mn(2+), via its pro-oxidative properties, to activate production of pro-inflammatory cytokines/chemokines IL-1ß, IL-6, IL-8, IFNγ, TNFα, and G-CSF by human monocyte-derived macrophages in vitro. For these studies, the cells were isolated from peripheral blood mononuclear leukocytes and matured to generate a population of large CD14/CD16 co-expressing cells. The monocyte-derived macrophages were then exposed to bacterial lipopolysaccharide (LPS, 1 µg/ml) or MnCl2 (25-100 µM)-alone or in combination-for 24 h at 37 °C, after which cell-free supernatants were analyzed using a multiplex cytokine assay procedure. Exposure of the cells to LPS caused modest statistically insignificant increases in cytokine production; MnCl2 caused dose-related increases in production of all six cytokines (achieving statistical significance of p < 0.0171- < 0.0005 for IL-1ß, IL-6, IL-8, IFNγ, and TNFα). In the case of LPS and MnCl2 combinations, the observed increases in production of IL-1ß, IL-6, IL-8, IFNγ, and G-CSF were greater than those seen with cells exposed to the individual agents. The Mn(2+)-mediated induction of cytokine production was associated with increased production of H2O2 and completely attenuated by inclusion of the H2O2-scavenger dithiothreitol, and partially by inhibitors of NF-κB and p38MAP kinase. The findings from the studies here help to further characterize the pro-inflammatory mechanisms that may underpin clinical disorders associated with excess exposure to Mn(2+), particularly those disorders seen in the central nervous and respiratory systems.

Clofazimine: current status and future prospects.

Clofazimine, a lipophilic riminophenazine antibiotic, possesses both antimycobacterial and anti-inflammatory activities. However, its efficacy has been demonstrated only in the treatment of leprosy, not in human tuberculosis, despite the fact that this agent is impressively active in vitro against multidrug-resistant strains of Mycobacterium tuberculosis. Recent insights into novel targets and mechanisms of antimicrobial and anti-inflammatory activity coupled with the acquisition of innovative drug delivery technologies have, however, rekindled interest in clofazimine as a potential therapy for multidrug- and extensively multidrug-resistant tuberculosis in particular, as well as several autoimmune diseases. The primary objective of this review is to critically evaluate these recent developments and to assess their potential impact on improving the therapeutic efficacy and versatility of clofazimine.