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The benzothiazole amide CRS0393 demonstrated excellent in vitro activity against nontuberculous mycobacteria (NTM), including M. abscessus isolates from cystic fibrosis (CF) patients, with minimum inhibitory concentrations (MICs) of ≤0.03-0.5 μg/mL. The essential transport protein MmpL3 was confirmed as the target via analysis of spontaneous resistant mutants and further biological profiling. In mouse pharmacokinetic studies, intratracheal instillation of a single dose of CRS0393 resulted in high concentrations of drug in epithelial lining fluid (ELF) and lung tissue, which remained above the M. abscessus MIC for at least 9 hours post-dose. This exposure resulted in a penetration ratio of 261 for ELF and 54 for lung tissue relative to plasma. CRS0393 showed good oral bioavailability, particularly when formulated in kolliphor oil, with a lung-to-plasma penetration ratio ranging from 0.5 to 4. CRS0393 demonstrated concentration-dependent reduction of intracellular M. abscessus in a THP-1 macrophage infection model. CRS0393 was well tolerated following intranasal administration (8 mg/kg) or oral dosing (25 mg/kg) once daily for 28 days in dexamethasone-treated C3HeB/FeJ mice. Efficacy against M. abscessus strain 103 was achieved via the intranasal route, while oral dosing will need further optimization. CRS0393 holds promise for development as a novel agent with broad antimycobacterial activity.

Tuberculosis (TB) remains a major cause of morbidity and mortality, particularly in low- and middle-income countries where access to health care workers, cold-chain storage, and sterile water sources may be limited. Inhaled drug delivery is a promising alternative to systemic delivery of antimycobacterial drugs, as it enables rapid achievement of high infection-site drug concentrations. The off-patent drug clofazimine (CFZ) may be particularly suitable for this route, given its known systemic toxicities. In this study, micronized CFZ particles produced by air jet milling were assessed for shelf-stability, pharmacokinetics, and anti-TB efficacy by the oral and pulmonary routes in BALB/c mice. Intratracheal instillation of micronized CFZ particles produced several-fold higher lung concentrations after a single 30 mg/kg dose compared to delivery via oral gavage, and faster onset of bactericidal activity was observed in lungs of mice with chronic Mycobacterium tuberculosis infection compared to the oral route. Both infection status and administration route affected the multidose pharmacokinetics (PK) of micronized CFZ. Increased lung and spleen accumulation of the drug after pulmonary administration was noted in infected mice compared to naive mice, while the opposite trend was noted in the oral dosing groups. The infection-dependent PK of inhaled micronized CFZ may point to a role of macrophage trafficking in drug distribution, given the intracellular-targeting nature of the formulation. Lastly, air jet milled CFZ exhibited robustness to storage-induced chemical degradation and changes in aerosol performance, thereby indicating the suitability of the formulation for treatment of TB in regions with limited cold chain supply.

MenJ, a flavoprotein oxidoreductase, is responsible for the saturation of the β-isoprene unit of mycobacterial menaquinone, resulting in the conversion of menaquinone with nine isoprene units (MK-9) to menaquinone with nine isoprene units where the double bond in the second unit is reduced [MK-9(II-H2)]. The hydrogenation of MK-9 increases the efficiency of the mycobacterial electron transport system, whereas the deletion of MenJ results in decreased survival of the bacteria inside J774A.1 macrophage-like cells but is not required for growth in culture. Thus, it was suggested that MenJ may represent a contextual drug target in M. tuberculosis, that is, a drug target that is valid only in the context of an infected macrophage. However, it was unclear if the conversion of MK-9 to MK-9(II-H2) or the MenJ protein itself was responsible for bacterial survival. In order to resolve this issue, a plasmid expressing folded, full-length, inactive MenJ was engineered. Primary sequence analysis data revealed that MenJ shares conserved FAD binding, NADH binding, and catalytic and C-terminal motifs with archaeal geranylgeranyl reductases. A MenJ mutant deficient in any one of these motifs is devoid of reductase activity. Therefore, point mutations of highly conserved amino acids in the conserved motifs were generated and the recombinant proteins were monitored for conformational changes by circular dichroism and oxidoreductase activity. The mutational analysis indicates that amino acids tryptophan 215 (W215) and cysteine 46 (C46) of M. tuberculosis MenJ, conserved in known archaeal geranylgeranyl reductases and putative menaquinone saturases, are essential to the hydrogenation of MK-9. The mutation of either C46 to serine (C46S) or W215 to leucine (W215L) in MenJ completely abolishes the catalytic activity in vitro, and menJ knockout strains of M. tuberculosis expressing either the C46S or W215L mutant protein are unable to convert MK-9 to MK-9(II-H2) but survive inside the J774A.1 cells. Thus, surprisingly, the survival of M. tuberculosis in J774A.1 cells is dependent on the expression of MenJ rather than its oxidoreductase activity, the conversion of MK-9 to MK-9(II-H2) as previously hypothesized. Overall, the current data suggest that MenJ is a moonlighting protein.

Background Mycobacterium abscessus causes chronic pulmonary infections. Owing to its resistance to most classes of antibiotics, treatment is complex and cure rates are only 45%. Tigecycline is active against M. abscessus, but severe toxicity and the need for IV administration limit its use. Objectives To assess the potential of inhaled tigecycline as a treatment for M. abscessus pulmonary disease, by measuring its efficacy in a mouse model of chronic M. abscessus pulmonary disease, establishing the intracellular activity of tigecycline against M. abscessus in human macrophages and measuring the activity of tigecycline in the sputum of cystic fibrosis patients. Methods We infected GM-CSF knockout mice with M. abscessus by intrapulmonary aerosol. Infected mice were treated with tigecycline in 0.25, 1.25 and 2.5 mg doses, by inhalation, or untreated, for 28 days. Tigecycline was added to human peripheral blood-derived macrophages infected with M. abscessus to assess its intracellular activity. We performed a time–kill kinetics experiment of tigecycline against M. abscessus with and without sputum of cystic fibrosis patients. Results Inhaled tigecycline proved highly effective against M. abscessus in GM-CSF knockout mice. The effect was dose dependent. Tigecycline showed potent activity against M. abscessus in macrophages and retained most of its activity in the presence of sputum of cystic fibrosis patients. Conclusions Inhaled tigecycline may represent a viable treatment option for M. abscessus pulmonary disease, where treatment outcomes are currently very poor. A stable and safe formulation is required to proceed to further pharmacodynamic studies and ultimately clinical trials.

Nontuberculous mycobacterial (NTM) pulmonary infections are a global health concern and a significant contributor to lung disease. Systemic therapies of a cocktail of antibiotics administered over a long period often lead to adverse reactions and/or treatment failure. NTM pathogens, such as Mycobacterium abscessus (Mabs), are notoriously difficult to treat due to resistance to many traditional antibiotics. However, the antibiotic tigecycline has demonstrated efficacy in vitro and in vivo against Mabs strains varying in drug susceptibility. Tigecycline exhibits instability in aqueous medium, posing delivery challenges, and has caused severe adverse gastrointestinal effects following intravenous administration, requiring treatment discontinuation. To mitigate both of these concerns, inhalation therapies using dry powder aerosols are proposed as an alternative administration route and means of delivery. Tigecycline dry powder formulations were prepared, characterized, and optimized to develop a therapeutic aerosol with low moisture, high dispersibility, and a large fraction of particles in the respirable size range (1–5 μm). The addition of lactose, leucine, and phosphate buffer salts was investigated to achieve additional stability, dispersibility, and tolerability. Preliminary delivery of the dry powders to Mabs-infected mice for 30 min per day over 7 d demonstrated a 0.91-log (87.7%) decrease in lung bacterial burden.

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 non-replicating, 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 following 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. Importantly, 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.

Bacille Calmette-Guerin (BCG) is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease. However, BCG has limited efficacy, necessitating the development of better vaccines. Non-tuberculous mycobacteria (NTMs) are opportunistic pathogens present ubiquitously in the environment. TB endemic countries experience higher exposure to NTMs, but previous studies have not elucidated the relationship between NTM exposure and BCG efficacy against TB. Therefore, we develop a mouse model (BCG + NTM) to simulate human BCG immunization regime and continuous NTM exposure. BCG + NTM mice exhibit superior and prolonged protection against pulmonary TB, with increased B cell influx and anti-Mtb antibodies in serum and airways, compared with BCG alone. Notably, spatial transcriptomics and immunohistochemistry reveal that BCG + NTM mice formed B cell aggregates with features of germinal center development, which correlate with reduced Mtb burden. Our studies suggest a direct relationship between NTM exposure and TB protection, with B cells playing a crucial role.

As exemplified by the COVID-19 pandemic, highly infective respiratory viruses can spread rapidly in the population because of lack of effective approaches to control viral replication and spread. Niclosamide (NCM) is an old anthelminthic drug (World Health Organization essential medicine list) with pleiotropic pharmacological activities. Several recent publications demonstrated that NCM has broad antiviral activities and potently inhibits viral replication, including replication of SARS-CoV-2, SARS-CoV, and dengue viruses. Unfortunately, NCM is almost completely insoluble in water, which limits its clinical use. We developed a highly scalable and cost-effective nanoparticle formulation of NCM (nano NCM) using only FDA-approved excipient and demonstrated potency against SARS-CoV-2 infection in cells (Vero E6 and ACE2-expressing lung epithelium cells). Our ultimate goal is to develop the nano NCM formulation for treatment of COVID-19 patients.

Tuberculosis (TB) patients diagnosed as drug-resistant TB have limited treatment options. Nix-TB trial is testing three-oral-drugs, namely Bedaquiline (B), Pretomanid (Pa) and Linezolid (L) (BPaL regimen) against drug-resistant TB. The BPaL regimen shows excellent favorable outcomes, but toxic effects are observed due to the long-term administration of Linezolid. Spectinamide-1599 (1599) is a potent protein inhibitor of Mycobacterium tuberculosis (Mtb) without known adverse effects and cross-resistance with other TB drugs. We hypothesized that inhaled 1599 could replace L in the BPaL regimen and provide similar or higher efficacy as BPaS regimen. Therefore, we tested BPaL and BPaS regimens in Balb/c (TB resistant) and C3HeB/FeJ (TB susceptible) mice infected with low-dose Mtb, and evaluated bacterial burden using colony-forming units (CFUs). Compared to untreated mice, CFUs in lungs of BPaL and BPaS treated Balb/c mice were decreased by >1 and 5 logs after 2- and 4-weeks treatment, respectively. In C3HeB/FeJ mice, 4-weeks of BPaL and BPaS treatment decreased CFUs by >3 logs compared to untreated control. However, no statistically significant differences were observed between treatment groups in both the mice strains. Cellular profiling using flow cytometry showed increased monocytes (CD11bhi CD14hi CCR2hi) and neutrophils (CD11b+ Ly6G+) in the bone marrow of BPaS mice compared to BPaL. These results along with cytokine profiling of the two treatment groups showed enhanced inflammatory response in BPaS mice than BPaL. We concluded that inhaled 1599 is a potential replacement for Linezolid if combined as BPaS regimen, and further studies are warranted. This project was funded by National Institute of Health under the funding number NIH-NIAID-Allergy and Infect Diseases AI120670.

Plague ecology is characterized by sporadic epizootics, then periods of dormancy. Building evidence suggests environmentally ubiquitous amebae act as feral macrophages and hosts to many intracellular pathogens. We conducted environmental genetic surveys and laboratory co-culture infection experiments to assess whether plague bacteria were resistant to digestion by 5 environmental ameba species. First, we demonstrated that Yersinia pestis is resistant or transiently resistant to various ameba species. Second, we showed that Y. pestis survives and replicates intracellularly within Dictyostelium discoideum amebae for ˃48 hours postinfection, whereas control bacteria were destroyed in

In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Current animal models study TB during adulthood but animal models for infant TB are scarce. Here we propose that minipigs can be used as an animal model to study adult, adolescent and infant TB including natural transmission. In these studies, two-month old minipigs (representing infant age in humans) and six-month old minipigs (representing adolescence in humans) were infected via the aerosol route with hyper-virulent clinical strain W-Beijing Mycobacterium tuberculosis (Mtb) HN878 and were monitored for 11 or 36 weeks post-challenge, respectively. In the same studies, infected and unchallenged animals were housed together. Viable bacteria were recovered from pulmonary and thoracic lymph nodes from both - infected and their initially unchallenged natural contacts. Bacillary load, gross lesions and histopathology revealed similarities to the spectrum of disease observed in human TB. The study did not reach terminal end point, thus it was not possible to annotate definitive clinical symptoms of active TB. The results demonstrated that minipigs are experimental hosts of Mtb HN878, and the pathology developed in their lungs resembles pathological findings described in human TB. Importantly, within communities of Mtb infected minipigs natural transmission occurs.

Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis. Despite intensive TB control campaigns, there are sporadic outbreaks of bovine TB in regions declared TB free. It is unclear how M. bovis is able to survive in the environment for long periods of time. We hypothesized that Free-living amoebae (FLA), as ubiquitous inhabitants of soil and water, may act as long-term reservoirs of M. bovis in the environment. In our model, M. bovis would be taken up by amoebal trophozoites, which are the actively feeding, replicating and mobile form of FLA. Upon exposure to hostile environmental conditions, infected FLA will encyst and provide an intracellular niche allowing their M. bovis cargo to persist for extended periods of time. Here, we show that five FLA species (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba lenticulata, Vermamoeba vermiformis and Dictyostellium discoideum) are permissive to M. bovis infection and that the M. bovis bacilli may survive within the cysts of four of these species for over 60 days. We further show that exposure of M. bovis-infected trophozoites and cysts to Balb/c mice leads to pulmonary TB. This work describes for the first time that FLA carrying M. bovis can transmit TB.

PURPOSE: Human tuberculosis (TB) is a global health problem that causes nearly 2 million deaths per year. Anti-TB therapy exists, but it needs to be administered as a cocktail of antibiotics for six months. This lengthy therapy results in low patient compliance and is the main reason attributable to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. METHODS: One alternative approach is to combine anti-TB multidrug therapy with inhalational TB therapy. The aim of this work was to develop and characterize dry powder formulations of spectinamide 1599 and ensure in vitro and in vivo delivered dose reproducibility using custom dosators. RESULTS: Amorphous dry powders of spectinamide 1599 were successfully spray dried with mass median aerodynamic diameter (MMAD) = 2.32 ± 0.05 μm. The addition of L-leucine resulted in minor changes to the MMAD (1.69 ± 0.35 μm) but significantly improved the inhalable portion of spectinamide 1599 while maintaining amorphous qualities. Additionally, we were able to demonstrate reproducibility of dry powder administration in vitro and in vivo in mice. CONCLUSIONS: The corresponding systemic drug exposure data indicates dose-dependent exposure in vivo in mice after dry powder intrapulmonary aerosol delivery in the dose range 15.4 – 32.8 mg/kg.

Pharmacokinetic/pharmacodynamic studies of anti-tuberculosis agents in animal models of tuberculosis are hampered by the frequent necessity to perform sample bioanalysis outside the biosafety level-3 environment. Thus, each specimen has to undergo tedious and time-consuming sample sterilization procedures that may also affect drug stability. Here, we tested treatment of Mycobacterium tuberculosis (Mtb) infected samples with methanol to sterilize samples while preserving drug integrity for further pharmacokinetic/pharmacodynamic evaluations. Tissue samples harvested from Mtb infected mice were homogenized, incubated in methanol, and tested for sterility. Once sterility was confirmed, the samples were used to determine concentrations of the anti-tuberculosis drug spectinamide-1599 in lung homogenates using liquid chromatography coupled with mass spectrometry. The results demonstrate that methanol sterilizes tissue samples harvested from Mtb infected mice without altering the integrity of the drug in the tissue.

Pulmonary Mycobacterium abscesssus (pAMB) infection is particularly challenging to treat because this bacillus displays intrinsic antibiotic resistance to most front-line drugs. Tigecycline, a glycyclcycline tetracycline, shows potent bactericidal effects against pMAB, while successfully evading bacterial resistance mechanisms. Tigecycline is FDA approved as a twice daily IV administration but provokes intolerable side effects in patients. More than its potent antimicrobial effects, tigecycline has demonstrated immunomodulatory properties observed in preclinical animal models of bacterial infection. We hypothesized that inhaled therapy of tigecycline reduces side effects and improve efficacy against pMAB. GM-CSF knockout mice with pMAB were treated daily for 28 days with aerosols of tigecycline at 0.25, 1.25 and 2.50 mg/dose. Thereafter, bacterial burden was assessed, and samples were prepared for immunophenotyping of lymphocyte and myeloid cell populations through H&E staining and IHC techniques. The results demonstrated that treatment with aerosols of tigecycline to mice with pAMB was tolerated and highly effective at reducing bacterial burden. Furthermore, this therapy was associated with the influx of macrophages and lymphocytes in both perivascular zones and granulomatous-like formations. There was also an increase in alveoli lined by hypertrophied cells with vesicular nuclei, suggesting induced Type II cell hyperplasia. To summarize, inhaled tigecycline not only demonstrated bactericidal effects against pMAB, but it invokes unique immunomodulatory effects that appear to aid the significant reduction in pulmonary bacteria.

Many excellent and free training resources exist to improve the computational reproducibility of biomedical research. However, most of these materials target researchers at the stage of data analysis, and they typically provide much less guidance on principles and techniques to improve the reproducibility of the earlier steps of experimental data recording and experimental data pre-processing that are critical for ensuring the reproducibility of immunological research. Many common existing practices---including the use of spreadsheets with embedded formulas that concurrently record and analyze experimental data, problematic management of project files, and reliance on proprietary, vendor-supplied point-and-click software for data pre-processing---interfere with the transparency, reproducibility, and efficiency of laboratory-based immunological research projects. Under an NIH grant, we are creating new training modules as a resource for laboratory-based biomedical researchers to learn how simple computational principles can improve reproducibility at the stages of data recording and pre-processing in immunological research. In this poster, we will highlight several simple approaches that we have identified through this project to improve the reproducibility of immunological research, including strategies for re-designing data collection spreadsheet templates to more easily integrate into a reproducible data analysis workflow and the use of reproducible reporting tools within a laboratory setting.

Here we demonstrate that aerosols of host directed therapies [HDT] administered during a chronic Mycobacterium tuberculosis (Mtb) infection have bactericidal effect. The pulmonary bacterial load of C57BL/6 mice chronically infected with Mtb was reduced by 1.7 and 0.6 log10CFU after two weeks of treatment via aerosol delivery with ST3-H2A2, [a selective peptide inhibitor of the STAT3 N-terminal domain] or IL10R1-7 [selective peptide inhibitor for the IL-10Ra] respectively and when compared to control mice treated with IL10R1-14 [peptide inhibitor used as negative control] or untreated mice infected with Mtb. Accordingly, when compared to control mice, the bactericidal capacity in mice was enhanced upon treatment with peptide inhibitors ST3-H2A2 and IL10R1-7 as evidenced by higher pulmonary activities of nitric oxide synthase, NADPH oxidase and lysozyme enzymes and decreased arginase enzyme activity. This therapy also modulated important checkpoints [Bcl2, Beclin-1, Atg 5, bax] in the apoptosis-autophagy pathways. Thus, even in the absence of antibiotics, targeting of the host pulmonary IL-10-STAT3 pathway can significantly reduce the Mtb bacilli load in the lungs, modulate the host own bactericidal capacity and apoptosis and autophagy pathways. Our approach here also allows targeting checkpoints of the lungs to determine their specific contribution in pulmonary immunity or pathogenesis.

Spectinamides are a novel series of spectinomycin analogs being developed for the treatment of tuberculosis. Intrapulmonary aerosol (IPA) administration of lead spectinamide 1599 has previously been shown to be more efficacious than subcutaneous (SC) administration at comparable doses. The objective of the current study was to characterize the disposition of 1599 in plasma and lungs in mice in order to provide a potential rationale for the observed efficacy differences. 200 mg/kg of 1599 was administered to healthy BALB/c mice by SC injection or by IPA delivery. Plasma and major organs were collected at specified time points until 8 h after dosing. Drug concentrations were measured by LC-MS/MS and analyzed by noncompartmental pharmacokinetic analysis. 1599 demonstrated rapid absorption into plasma after IPA and SC administration, resulting in very similar plasma exposure for both routes. In contrast, drug exposure in the lungs was 48 times higher following IPA as compared to SC administration, which is highly desirable as the lungs are the main site of infection in pulmonary TB. The higher local exposure in the lungs is likely the basis for the increased efficacy after IPA compared to SC administration. Overall, this study supports the pulmonary route as a potential pathway for the treatment of tuberculosis with 1599.

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.

Background Free-living amoebae (FLA) are voracious feeders, consuming bacteria and other microbes during colonization of the phytobiome. FLA are also known to secrete bacteriocidal or bacteriostatic compounds into their growth environment. Methodology and principal findings Here, we explore the impacts of co-cultivation of five FLA species, including Acanthamoeba castellanii, A. lenticulata, A. polyphaga, Dictyostelium discoideum and Vermamoeba vermiformis, on survival of two devastating bacterial pathogens of rice, Xanthomonas oryzae pathovars (pv.) oryzae and oryzicola. In co-cultivation assays, the five FLA species were either bacteriostatic or bactericidal to X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Despite these effects, bacteria were rarely detected inside amoebal cells. Furthermore, amoebae did not disrupt X. oryzae biofilms. The bactericidal effects persisted when bacteria were added to a cell-free supernatant from amoebal cultures, suggesting some amoebae produce an extracellular bactericidal compound. Conclusions/Significance This work establishes novel, basal dynamics between important plant pathogenic bacteria and diverse amoebae, and lays the framework for future mechanistic studies.

Cost-effective animal models that accurately reflect the pathological progression of pulmonary tuberculosis are needed to screen and evaluate novel tuberculosis drugs and drug regimens. Pulmonary disease in humans is characterized by a number of heterogeneous lesion types that reflect differences in cellular composition and organization, extent of encapsulation, and degree of caseous necrosis. C3HeB/FeJ mice have been increasingly used to model tuberculosis infection because they produce hypoxic, well-defined granulomas exhibiting caseous necrosis following aerosol infection with Mycobacterium tuberculosis. A comprehensive histopathological analysis revealed that C3HeB/FeJ mice develop three morphologically distinct lesion types in the lung that differ with respect to cellular composition, degree of immunopathology and control of bacterial replication. Mice displaying predominantly the fulminant necrotizing alveolitis lesion type had significantly higher pulmonary bacterial loads and displayed rapid and severe immunopathology characterized by increased mortality, highlighting the pathological role of an uncontrolled granulocytic response in the lung. Using a highly sensitive novel fluorescent acid-fast stain, we were able to visualize the spatial distribution and location of bacteria within each lesion type. Animal models that better reflect the heterogeneity of lesion types found in humans will permit more realistic modeling of drug penetration into solid caseous necrotic lesions and drug efficacy testing against metabolically distinct bacterial subpopulations. A more thorough understanding of the pathological progression of disease in C3HeB/FeJ mice could facilitate modulation of the immune response to produce the desired pathology, increasing the utility of this animal model., Summary: C3HeB/FeJ mice develop three morphologically distinct lesion types, which differ with respect to bacterial load, cellular composition and degree of immunopathology following low-dose aerosol infection with Mycobacterium tuberculosis.

Inflammatory responses to biomaterials heavily influence the environment surrounding implanted devices, often producing foreign-body reactions. The macrophage is a key immunomodulatory cell type consistently associated with implanted biomaterials and routinely used in short-term in vitro cell studies of biomaterials aiming to reproduce host responses. Inconsistencies within these studies, including differently sourced cells, different durations of culture, and assessment of different activation markers, lead to many conflicting results in vitro that confound consistency and conclusions. We hypothesize that different experimentally popular monocyte–macrophage cell types have intrinsic in vitro culture-specific differences that yield conflicting results. Recent studies demonstrate changes in cultured macrophage cytokine expression over time, leading to the hypothesis that changes in macrophage phenotype also occur in response to extended culture. Here, macrophage cells of different transformed and primary-derived origins were cultured for 21 days on model polymer biomaterials. Cell type-based differences in morphology and cytokine/chemokine expression as well as changes in cell surface biomarkers associated with differentiation stage, activation state, and adhesion were compared. Results reflect consistent macrophage development toward an M2 phenotype via up-regulation of the macrophage mannose receptor for all cell types following 21-day extended culture. Significantly, implanted biomaterials experiencing the foreign-body response and encapsulation in vivo often elicit a shift toward an analogous M2 macrophage phenotype. In vitro “default” of macrophage cultures, regardless of lineage, to this M2 state in the presence of biomaterials at long culture periods is not recognized, but has important implications to in vitro modeling of in vivo host response. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2864–2874, 2015.

Brucella melitensis is the etiologic agent of brucellosis in small ruminants and a common cause of disease in humans. While the protective immune response against this pathogen has been well studied in the mouse model, little is known of the immune response triggered by B. melitensis infection in natural hosts. The objective of the present study was to evaluate the caprine immune response over the course of infection with virulent B. melitensis strain 16 M and reduced virulence vaccine strain Rev. 1. Pregnant goats were infected at 11–14 weeks of gestation with 8 × 106 or 8 × 107 CFU of B. melitensis. Changes in granulocyte, monocyte, and mononuclear cell numbers were monitored by flow cytometry. Proliferative and functional responses of CD4+ T cells and WC1+ γδ T cells were also studied. B. melitensis 16 M infection triggered a pro-inflammatory response characterized by increased numbers of granulocytes, monocytes, and lymphocytes. The relative lymphocytosis was comprised of increases in CD4+ but not WC1+ T cell types in most animals. Little proliferative response was observed in Rev. 1-infected goats. Analysis of lymphocyte function suggested a degree of potential CD4+ T cell anergy, with low levels of CD25 expression and unresponsiveness to mitogen stimulation noted post-infection. The components of the protective response elicited by the Rev. 1 vaccine strain remain undefined. The study suggests a potential WC1+ γδ T cell mediated response, with high percentages of γδ T cells found to produce IFN-γ at various time points over the course of Brucella infection.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.

Many vaccines against childhood diseases are administered early after birth, but vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. In countries with endemic tuberculosis (TB), Bacillus Calmette-Guerin (BCG) is administered as part of the neonatal vaccine regimen because it prevents against the disseminated form of TB in children, although it has variable efficacy against pulmonary TB. Several promising new vaccines against TB are currently being tested in adult animal models. Here we evaluated neonatal piglets as an animal model to test vaccine efficacy. For this purpose, minipigs were vaccinated or not with BCG 48 h after birth and their immune response followed longitudinally until adolescence. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Immunological responses in vaccinated and non-vaccinated animals were further monitored upon infection with a low dose exposure to Mycobacterium tuberculosis strain HN878 via the aerosol route. Comparing the immunological response elicited by BCG vaccination in minipigs vs similar studies in infants, suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.

Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.

Of the non-tuberculous mycobacteria, Mycobacterium abscessus is particularly refractory to antimicrobial therapy and new agents with activity against these pathogens are urgently needed. The screening of candidate antimicrobial agents against M. abscessus requires a relevant and reproducible animal model of chronic infection. Granulocyte-macrophage colony-stimulating factor knockout (GM-CSF KO) mice were used to develop a new animal model of chronic pulmonary M. abscessus infection that can be used for preclinical efficacy testing of antimicrobial drugs.GM-CSF KO mice were infected with a clinical isolate of M. abscessus via intrapulmonary aerosol delivery using a microsprayer device. The clinical condition, histology and cfu of M. abscessus-infected GM-CSF KO mice were evaluated over a period of 4 months. Mice were treated with azithromycin (100 mg/kg) by oral gavage and the clinical condition, histology and bacterial burden was determined after 2 weeks of treatment.We show that pulmonary infection of GM-CSF KO mice with M. abscessus results in a chronic pulmonary infection that lends itself to preclinical testing of new antimicrobial drugs against this bacterium. Azithromycin treatment of M. abscessus-infected GM-CSF KO mice resulted in a lower bacterial burden in the lungs and spleen, weight gain and significant improvement in lung pathology.Intrapulmonary aerosol infection of GM-CSF KO mice with M. abscessus is a useful animal model for studying pathogenesis as well as pre-clinical testing of new compounds against M. abscessus in acute or chronic phases of infection.

Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis. Despite intensive TB control campaigns, there are sporadic outbreaks of bovine TB in regions declared TB free. It is unclear how M. bovis is able to survive in the environment for long periods of time. We hypothesized that Free-living amoebae (FLA), as ubiquitous inhabitants of soil and water, may act as long-term reservoirs of M. bovis in the environment. In our model, M. bovis would be taken up by amoebal trophozoites, which are the actively feeding, replicating and mobile form of FLA. Upon exposure to hostile environmental conditions, infected FLA will encyst and provide an intracellular niche allowing their M. bovis cargo to persist for extended periods of time. Here, we show that five FLA species (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba lenticulata, Vermamoeba vermiformis and Dictyostellium discoideum) are permissive to M. bovis infection and that the M. bovis bacilli may survive within the cysts of four of these species for over 60 days. We further show that exposure of M. bovis-infected trophozoites and cysts to Balb/c mice leads to pulmonary TB. This work describes for the first time that FLA carrying M. bovis can transmit TB.

Brucella melitensis is the causative agent of brucellosis in small ruminants and is of considerable economic and public health importance in many countries worldwide. The control of disease in humans depends on the control of disease in livestock; however, few counties with endemic B. melitensis infection have been able to successfully eradicate this pathogen. This underscores the need for further research on the pathogenesis of both virulent and vaccine strains of B. melitensis in the small ruminant host. The aim of the present study was to characterize clinical effects, tissue colonization, shedding, and humoral immune response following B. melitensis infection in goats. Both virulent (16M) and reduced virulence (Rev. 1) strains of B. melitensis were studied. Pregnant goats were infected at 11–14 weeks of gestation with 8 x 106 or 8 x 107 CFU of B. melitensis. Infection of goats with B. melitensis 16M resulted in an 86% abortion rate. This strain disseminated widely in pregnant does post-infection with none of the 15 sampled tissues spared from colonization. Importantly, we report the first isolation of B. melitensis from muscle tissue in ruminants. Pathogenesis of Rev. 1 infection was variable with two does showing minimal colonization and one doe exhibiting disease similar to that of animals infected with fully virulent 16M. Shedding of B. melitensis in milk occurred in all 16M- and Rev. 1- infected goats. In pregnant animals challenged with virulent B. melitensis, median time to seroconversion was 21 days; however, 2 animals did not seroconvert until after abortion.

Material surfaces that provide biomimetic cues, such as nanoscale architectures, have been shown to alter cell/biomaterial interactions. Recent studies have identified titania nanotube arrays as strong candidates for use in interfaces on implantable devices due to their ability to elicit improved cellular functionality. However, limited information exists regarding the immune response of nanotube arrays. Thus, in this study, we have investigated the short- and long-term immune cell reaction of titania nanotube arrays. Whole blood lysate (containing leukocytes, thrombocytes and trace amounts of erythrocytes), isolated from human blood, were cultured on titania nanotube arrays and biomedical grade titanium (as a control) for 2 hours and 2 and 7 days. In order to determine the in vitro immune response on titania nanotube arrays, immune cell functionality was evaluated by cellular viability, adhesion, proliferation, morphology, cytokine/chemokine expression, with and without lipopolysaccharide (LPS), and nitric oxide release. The results presented in this study indicate a decrease in short- and long-term monocyte, macrophage and neutrophil functionality on titania nanotube arrays as compared to the control substrate. This work shows a reduced stimulation of the immune response on titania nanotube arrays, identifying this specific nanoarchitecture as a potentially optimal interface for implantable biomedical devices.