Location of Intra- and Extracellular M. tuberculosis Populations in Lungs of Mice and Guinea Pigs during Disease Progression and after Drug Treatment

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development

Optimization and Lead Selection of Benzothiazole Amide Analogs Toward a Novel Antimycobacterial Agent

Mycobacteria remain an important problem worldwide, especially drug resistant human pathogens. Novel therapeutics are urgently needed to tackle both drug-resistant tuberculosis (TB) and difficult-to-treat infections with nontuberculous mycobacteria (NTM). Benzothiazole adamantyl amide had previously emerged as a high throughput screening hit against M. tuberculosis (Mtb) and was subsequently found to be active against NTM as well. For lead optimization, we applied an iterative process of design, synthesis and screening of several 100 analogs to improve antibacterial potency as well as physicochemical and pharmacological properties to ultimately achieve efficacy. Replacement of the adamantyl group with cyclohexyl derivatives, including bicyclic moieties, resulted in advanced lead compounds that showed excellent potency and a mycobacteria-specific spectrum of activity. MIC values ranged from 0.03 to 0.12 μg/mL against M. abscessus (Mabs) and other rapid- growing NTM, 1–2 μg/mL against M. avium complex (MAC), and 0.12–0.5 μg/mL against Mtb. No pre-existing resistance was found in a collection of n = 54 clinical isolates of rapid-growing NTM. Unlike many antibacterial agents commonly used to treat mycobacterial infections, benzothiazole amides demonstrated bactericidal effects against both Mtb and Mabs. Metabolic labeling provided evidence that the compounds affect the transfer of mycolic acids to their cell envelope acceptors in mycobacteria. Mapping of resistance mutations pointed to the trehalose monomycolate transporter (MmpL3) as the most likely target. In vivo efficacy and tolerability of a benzothiazole amide was demonstrated in a mouse model of chronic NTM lung infection with Mabs. Once daily dosing over 4 weeks by intrapulmonary microspray administration as 5% corn oil/saline emulsion achieved statistically significant CFU reductions compared to vehicle control and non-inferiority compared to azithromycin. The benzothiazole amides hold promise for development of a novel therapeutic agent with broad antimycobacterial activity, though further work is needed to develop drug formulations for direct intrapulmonary delivery via aerosol

Increased Virulence of an Epidemic Strain of Mycobacterium massiliense in Mice

Chronic pulmonary disease and skin/soft tissue infections due to non-tuberculous mycobacteria (NTM) of the Mycobacterium chelonae-abscessus-massiliense group is an emerging health problem worldwide. Moreover, the cure rate for the infections this group causes is low despite aggressive treatment. Post-surgical outbreaks that reached epidemic proportions in Brazil recently were caused by M. massiliense isolates resistant to high-level disinfection with glutaraldehyde (GTA). Understanding the differences in the virulence and host immune responses induced by NTM differing in their sensitivity to disinfectants, and therefore their relative threat of causing outbreaks in hospitals, is an important issue.We compared the replication and survival inside macrophages of a GTA-susceptible reference Mycobacterium massiliense clinical isolate CIP 108297 and an epidemic strain from Brazil, CRM-0019, and characterized the immune responses of IFNγ knockout mice exposed to a high dose aerosol with these two isolates. CRM-0019 replicated more efficiently than CIP 108297 inside mouse bone marrow macrophages. Moreover, the animals infected with CRM-0019 showed a progressive lung infection characterized by a delayed influx of CD4+ and CD8+ T cells, culminating in extensive lung consolidation and demonstrated increased numbers of pulmonary CD4+ Foxp3+ regulatory T cells compared to those infected with the reference strain. Immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of NTM disease by preventing the induction of specific protective immune responses.These results provide the first direct evidence of the increased virulence in macrophages and mice and pathogenicity in vivo of the Brazilian epidemic isolate and the first observation that NTM infections can be associated with variable levels of regulatory T cells which may impact on their virulence and ability to persist in the host

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Comparative Studies Evaluating Mouse Models Used for Efficacy Testing of Experimental Drugs against Mycobacterium tuberculosis▿

Methodologies for preclinical animal model testing of drugs against Mycobacterium tuberculosis vary from laboratory to laboratory; however, it is unknown if these variations result in different outcomes. Thus, a series of head-to-head comparisons of drug regimens in three commonly used mouse models (intravenous, a low-dose aerosol, and a high-dose aerosol infection model) and in two strains of mice are reported here. Treatment with standard tuberculosis (TB) drugs resulted in similar efficacies in two mouse species after a low-dose aerosol infection. When comparing the three different infection models, the efficacies in mice of rifampin and pyrazinamide were similar when administered with either isoniazid or moxifloxacin. Relapse studies revealed that the standard drug regimen showed a significantly higher relapse rate than the moxifloxacin-containing regimen. In fact, 4 months of the moxifloxacin-containing combination regimen showed similar relapse rates as 6 months of the standard regimen. The intravenous model showed slower bactericidal killing kinetics with the combination regimens tested and a higher relapse of infection than either aerosol infection models. All three models showed similar outcomes for in vivo efficacy and relapse of infection for the drug combinations tested, regardless of the mouse infection model used. Efficacy data for the drug combinations used also showed similar results, regardless of the formulation used for rifampin or timing of the drugs administered in combination. In all three infection models, the dual combination of rifampin and pyrazinamide was less sterilizing than the standard three-drug regimen, and therefore the results do not support the previously reported antagonism between standard TB agents

Location of <i>M. tuberculosis</i> bacilli in lungs from <i>M. tuberculosis</i> infected guinea pigs, throughout infection and after 6 weeks of INH or TMC207 drug treatment.

<p>(AR) auramine-rhodamine, hematoxylin QS and DAPI; (H&E) hematoxylin and eosin. (A, B) Low (A) and high (B) magnifications of an early primary granuloma in the lungs of <i>M. tuberculosis</i> infected guinea pigs 4 weeks post-aerosol infection. Primary granulomas are distinguished from secondary lesions by the presence of necrosis (N) (H&E staining, 100× and 1000× magnifications). (C) A secondary lesion in the lungs of an <i>M. tuberculosis</i> infected guinea pig 4 weeks post-aerosol infection. Secondary lesions are distinguished from primary granulomas by the lack of central necrosis in the former (H&E staining, 100× magnification). (D, E) Low (D) and high (E) magnifications of a primary granuloma from the lungs of an <i>M. tuberculosis</i> infected guinea pig 4 weeks after aerosol infection. The majority of AR+ bacilli are extracellular within the necrotic core (c) (AR, 100× and 200× magnifications). (F) High magnification of area demarcated in figure 5E (square). Extracellular AR+ bacilli in the necrotic core of primary granulomas exist as single cells or are situated in clusters (AR, 1000× magnification). (G, H) Confocal micrographs of a necrotic granuloma showing an acellular necrotic core (c) with extracellular AR+ bacilli (red) (AR and DAPI, 200× and 630× magnifications). (I) A primary granuloma from the lungs of an <i>M. tuberculosis</i> infected guinea pig 10 weeks after aerosol infection showing advanced calcification and calcification of the necrotic core (c) and the acellular rim (R) surrounding the core (H&E staining, 100× magnification). (J) Fluorescent image of a primary lung granuloma from an <i>M. tuberculosis</i> infected guinea pig 10 weeks after aerosol infection. Extracellular AR+ bacilli are present within the necrotic core (c) and the acellular rim (R) surrounding the necrotic regions (AR, 200× magnification). (K) Cropped image from figure 5I (square) showing extracellular AR+ stained bacilli in the acellular rim. (L) Fluorescent image of a necrotic primary lung granuloma from an <i>M. tuberculosis</i> infected guinea pig treated for 6 weeks with INH. Extra-cellular AR+ bacilli are primarily within the core (c) of the partially calcified lytic necrosis, and to a lesser extent within the acellular, uncalcified rim (R) (AR, 200× and 400× magnifications). (M) A low magnification of the remnant of a primary lung granuloma in an <i>M. tuberculosis</i> infected guinea pig treated for 6 weeks with TMC207. The primary granuloma shows a caseous necrotic core (c) surrounded by inflammatory cells (H&E staining, 100× magnification). (N) Fluorescent image of the caseous necrotic core (c) in the primary lung granuloma shown in figure 5M taken from a serial tissue section. The few extra-cellular AR+ bacilli remaining after TMC207 treatment are primarily located within the central core of caseous necrosis (AR, 400× magnification).</p