The relationship between host defense peptides and adrenal steroids. An account of reciprocal influences.

AIM: ß-defensins 2 and -3 (HBD-2 and HBD-3) and cathelicidin LL-37 are host defense peptides (HDPs) that play a crucial role in the immune response against mycobacteria. Given our former studies in tuberculosis patients wherein their plasma levels of such peptides correlated with steroid hormone concentrations, we now studied the reciprocal influence of cortisol and/or dehydroepiandrosterone (DHEA) on HDPs biosynthesis and LL-37 on adrenal steroidogenesis. MAIN METHODS: Cultures of macrophages derived from the THP-1 line were treated with cortisol (10-6M) and/or DHEA (10-6M and 10-7M) and stimulated with irradiated M. tuberculosis (Mi) or infected M. tuberculosis strain H37Rv to assess cytokine production, HDPs, reactive oxygen species (ROS) and colony forming units. Cultures of NCI-H295-R adrenal line were treated with LL37 (5, 10, and 15 µg/ml) for 24 h to further measure cortisol and DHEA levels together with steroidogenic enzyme transcripts. KEY FINDINGS: In macrophages, M. tuberculosis produced an increase of IL-1ß, TNFα, IL-6, IL-10, LL-37, HBD-2, and HBD-3 levels, irrespective of DHEA treatment. Adding cortisol to M. tuberculosis-stimulated cultures (with or without DHEA) decreased the amounts of these mediators, compared to only stimulated cultures. Although M. tuberculosis reduced ROS levels, DHEA increased these values in addition to diminishing intracellular mycobacterial growth (no matter cortisol treatment). In turn, studies on adrenal cells showed that LL-37 reduced the production of cortisol and DHEA besides modifying transcripts for some steroidogenic enzymes. SIGNIFICANCE: while adrenal steroids seem to influence the production of HDPs, the former compounds are also likely to modulate adrenal biogenesis.

Effect of mycobacterial proteins that target mitochondria on the alveolar macrophages activation during Mycobacterium tuberculosis infection.

Purpose of the study: During the early and progressive (late) stages of murine experimental pulmonary tuberculosis, the differential activation of macrophages contributes to disease development by controlling bacterial growth and immune regulation. Mycobacterial proteins P27 and PE_PGRS33 can target the mitochondria of macrophages. This study aims to evaluate the effect of both proteins on macrophage activation during mycobacterial infection. Materials and methods: We assess both proteins for mitochondrial oxygen consumption, and morphological changes, as well as bactericide activity, production of metabolites, cytokines, and activation markers in infected MQs. The cell line MH-S was used for all the experiments. Results: We show that P27 and PE_PGRS33 proteins modified mitochondrial dynamics, oxygen consumption, bacilli growth, cytokine production, and some genes that contribute to macrophage alternative activation and mycobacterial intracellular survival. Conclusions: Our findings showed that these bacterial proteins partially contribute to promoting M2 differentiation by altering mitochondrial metabolic activity.

1,4-Benzoquinone antimicrobial agents against Staphylococcus aureus and Mycobacterium tuberculosis derived from scorpion venom.

Two 1,4-benzoquinone derivatives, found in the venom of the scorpion Diplocentrus melici following exposure to air, have been isolated, characterized, synthesized, and assessed for antimicrobial activities. Initially a white, viscous liquid, the extracted venom colors within minutes under ambient conditions. From this colored mixture, two compounds, one red, the other blue, were isolated and purified using chromatography. After a variety of NMR and mass spectrometry experiments, the red compound was determined to be 3,5- dimethoxy-2-(methylthio)cyclohexa-2,5-diene-1,4-dione, and the blue compound was determined to be 5-methoxy-2,3- bis(methylthio)cyclohexa-2,5-diene-1,4-dione. Because extremely small amounts of these compounds were isolated from the scorpion venom, we developed laboratory syntheses from commercially available precursors, allowing us to produce sufficient quantities for crystallization and biological assays. The red benzoquinone is effective against Staphylococcus aureus [minimum inhibitory concentration (MIC) = 4 µg/mL], while the blue benzoquinone is active against Mycobacterium tuberculosis (MIC = 4 µg/mL) and even against a multidrug-resistant (MDR) strain with nearly equal effectiveness. The bactericidal effects of both benzoquinones show comparable activity to commercially available antibiotics used against these pathogens and were cytotoxic to neoplastic cell lines, suggesting their potential as lead compounds for the development of novel antimicrobial and anticancer drugs. Importantly, the blue benzoquinone was also effective in vivo with mouse models of MDR tuberculosis infection. After treatment for 2 mo, four mice with late-stage active MDR tuberculosis had a significant decrease in pulmonary bacillary loads and tissue damage. Healthy mice served as negative controls and tolerated treatment well, without adverse side effects.

The ctpF Gene Encoding a Calcium P-Type ATPase of the Plasma Membrane Contributes to Full Virulence of Mycobacterium tuberculosis.

Identification of alternative attenuation targets of Mycobacterium tuberculosis (Mtb) is pivotal for designing new candidates for live attenuated anti-tuberculosis (TB) vaccines. In this context, the CtpF P-type ATPase of Mtb is an interesting target; specifically, this plasma membrane enzyme is involved in calcium transporting and response to oxidative stress. We found that a mutant of MtbH37Rv lacking ctpF expression (MtbΔctpF) displayed impaired proliferation in mouse alveolar macrophages (MH-S) during in vitro infection. Further, the levels of tumor necrosis factor and interferon-gamma in MH-S cells infected with MtbΔctpF were similar to those of cells infected with the parental strain, suggesting preservation of the immunogenic capacity. In addition, BALB/c mice infected with Mtb∆ctpF showed median survival times of 84 days, while mice infected with MtbH37Rv survived 59 days, suggesting reduced virulence of the mutant strain. Interestingly, the expression levels of ctpF in a mouse model of latent TB were significantly higher than in a mouse model of progressive TB, indicating that ctpF is involved in Mtb persistence in the dormancy state. Finally, the possibility of complementary mechanisms that counteract deficiencies in Ca2+ transport mediated by P-type ATPases is suggested. Altogether, our results demonstrate that CtpF could be a potential target for Mtb attenuation.

Close Related Drug-Resistance Beijing Isolates of Mycobacterium tuberculosis Reveal a Different Transcriptomic Signature in a Murine Disease Progression Model.

Mycobacterium tuberculosis (MTB) lineage 2/Beijing is associated with high virulence and drug resistance worldwide. In Colombia, the Beijing genotype has circulated since 1997, predominantly on the pacific coast, with the Beijing-Like SIT-190 being more prevalent. This genotype conforms to a drug-resistant cluster and shows a fatal outcome in patients. To better understand virulence determinants, we performed a transcriptomic analysis with a Beijing-Like SIT-190 isolate (BL-323), and Beijing-Classic SIT-1 isolate (BC-391) in progressive tuberculosis (TB) murine model. Bacterial RNA was extracted from mice lungs on days 3, 14, 28, and 60. On average, 0.6% of the total reads mapped against MTB genomes and of those, 90% against coding genes. The strains were independently associated as determined by hierarchical cluster and multidimensional scaling analysis. Gene ontology showed that in strain BL-323 enriched functions were related to host immune response and hypoxia, while proteolysis and protein folding were enriched in the BC-391 strain. Altogether, our results suggested a differential bacterial transcriptional program when evaluating these two closely related strains. The data presented here could potentially impact the control of this emerging, highly virulent, and drug-resistant genotype.

Effect of Curcumin in Experimental Pulmonary Tuberculosis: Antimycobacterial Activity in the Lungs and Anti-Inflammatory Effect in the Brain.

Tuberculosis (TB) is one of the ten leading causes of death worldwide. Patients with TB have been observed to suffer from depression and anxiety linked to social variables. Previous experiments found that the substantial pulmonary inflammation associated with TB causes neuroinflammation, neuronal death, and behavioral impairments in the absence of brain infection. Curcumin (CUR) is a natural product with antioxidant, anti-inflammatory and antibacterial activities. In this work, we evaluated the CUR effect on the growth control of mycobacteria in the lungs and the anti-inflammatory effect in the brain using a model of progressive pulmonary TB in BALB/c mice infected with drug-sensitive mycobacteria (strain H37Rv). The results have shown that CUR decreased lung bacilli load and pneumonia of infected animals. Finally, CUR significantly decreased neuroinflammation (expression of TNFα, IFNγ and IL12) and slightly increased the levels of nuclear factor erythroid 2-related to factor 2 (Nrf2) and the brain-derived neurotrophic factor (BDNF) levels, improving behavioral status. These results suggest that CUR has a bactericidal effect and can control pulmonary mycobacterial infection and reduce neuroinflammation. It seems that CUR has a promising potential as adjuvant therapy in TB treatment.

Mycobacterium tuberculosis Infection Induces BCSFB Disruption but No BBB Disruption In Vivo: Implications in the Pathophysiology of Tuberculous Meningitis.

Central nervous system (CNS) tuberculosis is the most lethal and devastating form among the diseases caused by Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis bacilli enter the CNS are still unclear. However, the BBB and the BCSFB have been proposed as possible routes of access into the brain. We previously reported that certain strains of M. tuberculosis possess an enhanced ability to cause secondary CNS infection in a mouse model of progressive pulmonary tuberculosis. Here, we evaluated the morphostructural and molecular integrity of CNS barriers. For this purpose, we analyzed through transmission electron microscopy the ultrastructure of brain parenchymal microvessels and choroid plexus epithelium from animals infected with two mycobacterial strains. Additionally, we determined the expression of junctional proteins and cytokines by immunological techniques. The results showed that the presence of M. tuberculosis induced disruption of the BCSFB but no disruption of the BBB, and that the severity of such damage was related to the strain used, suggesting that variations in the ability to cause CNS disease among distinct strains of bacteria may also be linked to their capacity to cause direct or indirect disruption of these barriers. Understanding the pathophysiological mechanisms involved in CNS tuberculosis may facilitate the establishment of new biomarkers and therapeutic targets.

16α-Bromoepiandrosterone as a new candidate for experimental diabetes-tuberculosis co-morbidity treatment.

Tuberculosis (TB) is the leading cause of death from a single bacterial infectious agent and is one of the most relevant issues of public health. Another pandemic disease is type II diabetes mellitus (T2D) that is estimated to affect half a billion people in the world. T2D is directly associated with obesity and a sedentary lifestyle and is frequently associated with immunosuppression. Immune dysfunction induced by hyperglycemia increases infection frequency and severity. Thus, in developing countries the T2D/TB co-morbidity is frequent and represents one of the most significant challenges for the health-care systems. Several immunoendocrine abnormalities are occurring during the chronic phase of both diseases, such as high extra-adrenal production of active glucocorticoids (GCs) by the activity of 11-ß-hydroxysteroid dehydrogenase type 1 (11-ßHSD1). 11-ßHSD1 catalyzes the conversion of inactive cortisone to active cortisol or corticosterone in lungs and liver, while 11-ß-hydroxysteroid dehydrogenase type 2 (11-ßHSD2) has the opposite effect. Active GCs have been related to insulin resistance and suppression of Th1 responses, which are deleterious factors in both T2D and TB. The anabolic adrenal hormone dehydroepiandrosterone (DHEA) exerts antagonistic effects on GC signaling in immune cells and metabolic tissues; however, its anabolic effects prohibit its use to treat immunoendocrine diseases. 16α-bromoepiandrosterone (BEA) is a water miscible synthetic sterol related to DHEA that lacks an anabolic effect while amplifying the immune and metabolic properties with important potential therapeutic uses. In this work, we compared the expression of 11-ßHSD1 and the therapeutic efficacy of BEA in diabetic mice infected with tuberculosis (TB) (T2D/TB) with respect to non-diabetic TB-infected mice (TB). T2D was induced by feeding mice with a high-fat diet and administering a single low-dose of streptozotocin. After 4 weeks of T2D establishment, mice were infected intratracheally with a high-dose of Mycobacterium tuberculosis strain H37Rv. Then, mice were treated with BEA three times a week by subcutaneous and intratracheal routes. Infection with TB increased the expression of 11-ßHSD1 and corticosterone in the lungs and liver of both T2D/TB and TB mice; however, T2D/TB mice developed a more severe lung disease than TB mice. In comparison with untreated animals, BEA decreased GC and 11-ßHSD1 expression while increasing 11-ßHSD2 expression. These molecular effects of BEA were associated with a reduction in hyperglycemia and liver steatosis, lower lung bacillary loads and pneumonia. These results uphold BEA as a promising effective therapy for the T2D/TB co-morbidity.

The Therapeutic Effect of Intranasal Administration of Dexamethasone in Neuroinflammation Induced by Experimental Pulmonary Tuberculosis.

Tuberculosis (TB) is an important infectious disease and a public health problem. The organs most frequently affected by TB are the lungs; despite this, it has been reported that TB patients suffer from depression and anxiety, which have been attributed to social factors. In previous experimental work, we observed that the extensive pulmonary inflammation characteristic of TB with high cytokine production induces neuroinflammation, neuronal death and behavioral abnormalities in the absence of brain infection. The objective of the present work was to reduce this neuroinflammation and avoid the psycho-affective disorders showed during pulmonary TB. Glucocorticoids (GCs) are the first-line treatment for neuroinflammation; however, their systemic administration generates various side effects, mostly aggravating pulmonary TB due to immunosuppression of cellular immunity. Intranasal administration is a route that allows drugs to be released directly in the brain through the olfactory nerve, reducing their doses and side effects. In the present work, dexamethasone's (DEX) intranasal administration was evaluated in TB BALB /c mice comparing three different doses (0.05, 0.25 and 2.5 mg/kg BW) on lung disease evolution, neuroinflammation and behavioral alterations. Low doses of dexamethasone significantly decreased neuroinflammation, improving behavioral status without aggravating lung disease.

Hepatocyte growth factor enhances the clearance of a multidrug-resistant Mycobacterium tuberculosis strain by high doses of conventional chemotherapy, preserving liver function.

Tuberculosis (TB) is one of the deadliest infectious diseases in humankind history. Although, drug sensible TB is slowly decreasing, at present the rise of TB cases produced by multidrug-resistant (MDR) and extensively drug-resistant strains is a big challenge. Thus, looking for new therapeutic options against these MDR strains is mandatory. In the present work, we studied, in BALB/c mice infected with MDR strain, the therapeutic effect of supra-pharmacological doses of the conventional primary antibiotics rifampicin and isoniazid (administrated by gavage or intratracheal routes), in combination with recombinant human hepatocyte growth factor (HGF). This high dose of antibiotics administered for 3 months, overcome the resistant threshold of the MDR strain producing a significant reduction of pulmonary bacillary loads but induced liver damage, which was totally prevented by the administration of HGF. To address the long-term efficiency of this combined treatment, groups of animals after 1 month of treatment termination were immunosuppressed by glucocorticoid administration and, after 1 month, mice were euthanized, and the bacillary load was determined in lungs. In comparison with animals treated only with a high dose of antibiotics, animals that received the combined treatment showed significantly lower bacterial burdens. Thus, treatment of MDR-TB with very high doses of primary antibiotics particularly administrated by aerial route can produce a very good therapeutic effect, and its hepatic toxicity can be prevented by the administration of HGF, becoming in a new treatment modality for MDR-TB.

Identification of Mycobacterium tuberculosis CtpF as a target for designing new antituberculous compounds.

The emergence of tuberculosis (TB) produced by multi-drug resistance (MDR) and extensively-drug resistance (XDR) Mycobacterium tuberculosis (Mtb), encourages the development of new antituberculous compounds, as well as the identification of novel drug targets. In this regard, plasma membrane P-type ATPases are interesting targets because they play a crucial role in ion homeostasis and mycobacterial survival. We focused on Mtb CtpF, a calcium P-type ATPase that responds to a broad number of intraphagosomal conditions, as a novel target. In this study, we evaluated the capacity of cyclopiazonic acid (CPA), a well-known inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), to inhibit the ATPase activity of CtpF and the Mtb growth demonstrating that CtpF is a druggable target. A homology modeling of CtpF was generated for molecular docking studies of CtpF with CPA and key pharmacophoric features were identified, which were used to perform a pharmacophore-based virtual screening of the ZINC database, and to identify CtpF inhibitor candidates. Molecular docking-based virtual screening and MM-BGSA calculations of candidates allowed identifying six compounds with the best binding energies. The compounds displayed in vitro minimum inhibitory concentrations (MIC) ranging from 50 to 100 µg/mL, growth inhibitions from 29.5 to 64.0% on Mtb, and inhibitions of Ca2+-dependent ATPase activity in Mtb membrane vesicles (IC50) ranging from 4.1 to 35.8 µM. The compound ZINC63908257 was the best candidate by displaying a MIC of 50 µg/mL and a Ca2+ P-type ATPase inhibition of 45% with IC50 = 4.4 µM. Overall, the results indicate that CtpF is a druggable target for designing new antituberculous compounds.

Experimental Pulmonary Tuberculosis in the Absence of Detectable Brain Infection Induces Neuroinflammation and Behavioural Abnormalities in Male BALB/c Mice.

Tuberculosis (TB) is a chronic infectious disease in which prolonged, non-resolutive inflammation of the lung may lead to metabolic and neuroendocrine dysfunction. Previous studies have reported that individuals coursing pulmonary TB experience cognitive or behavioural changes; however, the pathogenic substrate of such manifestations have remained unknown. Here, using a mouse model of progressive pulmonary TB, we report that, even in the absence of brain infection, TB is associated with marked increased synthesis of both inflammatory and anti-inflammatory cytokines in discrete brain areas such as the hypothalamus, the hippocampal formation and cerebellum accompanied by substantial changes in the synthesis of neurotransmitters. Moreover, histopathological findings of neurodegeneration and neuronal death were found as infection progressed with activation of p38, JNK and reduction in the BDNF levels. Finally, we perform behavioural analysis in infected mice throughout the infection, and our data show that the cytokine and neurochemical changes were associated with a marked onset of cognitive impairment as well as depressive- and anxiety-like behaviour. Altogether, our results suggest that besides pulmonary damage, TB is accompanied by an extensive neuroinflammatory and neurodegenerative state which explains some of the behavioural abnormalities found in TB patients.

Immunotherapeutic effects of recombinant adenovirus encoding interleukin 12 in experimental pulmonary tuberculosis.

High dose of Mycobacterium tuberculosis (Mtb) strain H37Rv administered by intratracheal injection in BALB/c mice induce progressive tuberculosis (TB). In this model, during the first month there is a temporal control of bacillary growth, in coexistence with macrophage activation, granuloma formation and Th-1 response. Then, bacterial proliferation recommences, accompanied by progressive pneumonia and decreasing expression of protective cytokines (IFN-γ and TNF-α). In this model, we studied the IL-12 gene expression kinetics and cellular source. There is a rapid and progressive IL-12 expression peaking at day 14, when granulomas start their formation and numerous macrophages show strong IL-12 immunostaining, while during progressive TB there is a significant decrease of IL-12 expression and occasional macrophages showed IL-12 immunolabeling. In the second part of this study, we determined the immunotherapeutic effect of recombinant adenoviruses that codify IL-12 (AdIL-12). Intratracheal administration of only one dose of AdIL-12 one day before Mtb infection produced significant decrease of bacterial loads, lesser pneumonia and higher expression of TNF-α, IFN-γ and iNOS. When only one dose of AdIL-12 was given in healthy mice cohoused with infected mice with highly virulent and transmissible Mtb, total prevention of infection was conferred. Moreover, when AdIL-12 was administered by intranasal route in animals suffering late active TB after 2 months of infection, a very low pulmonary bacilli burdens was detected. These experimental data confirm that IL-12 is a significant cytokine in the immune protection against Mtb, and gene therapy based in adenoviruses coding this cytokine increased protective immunity and prevent Mtb transmission.

Therapeutic efficacy of liposomes containing 4-(5-pentadecyl-1,3,4-oxadiazol-2-yl)pyridine in a murine model of progressive pulmonary tuberculosis.

BACKGROUND AND OBJECTIVES: Tuberculosis (TB) is one of the deadliest infectious diseases and comprises a global public health concern because co-infection with Human immunodeficiency virus (HIV) and, in particular, the continuous isolation of new Multidrug-resistant strains (MDR), rendering the discovery of novel anti-TB agents a strategic priority. One of the most effective first-line mycobactericidal drugs is Isoniazid (INH). Previously, we reported in vitro anti-mycobacterial activity against sensitive and MDR Mycobacterium tuberculosis strains of a new oxadiazole obtained from the hybridization of INH and palmitic acid. The present study evaluated the therapeutic potential of liposomes including Phosphatidylcholine (PC) and L-α Phosphatidic acid (PA) or PC and Cholesterol (Chol) containing 4-(5-pentadecyl-1,3,4-oxadiazol-2-yl)pyridine in BALB/c male mice infected by intratracheal (i.t.) route with drug-sensitive or MDR M. tuberculosis. METHODS: The lipophilic 4-(5-pentadecyl-1,3,4-oxadiazol-2-yl)pyridine was obtained to mix INH and palmitoyl chloride. The in vivo anti-TB effect of this oxadiazole derivative contained in two different liposomes was tested in BALB/c mice infected with a sensitive strain of M. tuberculosis, initiating treatment 2 months post-infection, by i.t. route, of 50 µg of oxadiazole derivative for 1 month. In a second stage, mice were infected with an MDR (resistant to first-line drugs) and treated with 150 µg of an oxadiazole derivative carried by PC + Chol liposomes for 2 months. The effect of the oxadiazole derivative in vivo was determined by the quantification of lung bacilli loads and histopathology. RESULTS: In comparison with control animals, drug-sensitive, strain-infected mice treated for 1 month with 50 µg of this oxadiazole derivative contained in the liposomes of PC + Chol showed a significant, 80% decrease of live bacilli in lungs, which correlated with the morphometric observation, and the group of MDR clinical isolate-infected mice treated with 150 µg of the oxadiazole derivative contained in the same type of liposome showed significantly lower lung bacillary loads than control mice, producing 90% of bacilli burden reduction after 2 months of treatment. CONCLUSION: These results confirm and extend the reported highly efficient anti-mycobacterial activity of this lipophilic oxidazole derivative when it is carried by liposomes in mice suffering from late progressive pulmonary TB induced by drug-sensitive, and most prominently by, MDR strains.

Mycobacterium bovis BCG as immunostimulating agent prevents the severe form of chronic experimental Chagas disease.

Introduction: There is currently no vaccine against Chagas disease (ChD), and the medications available confer multiple side effects. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) produces balanced Th1, Th2, and Th17 modulatory immune responses and has improved efficacy in controlling chronic infections through nonspecific immunity. We aimed to improve the response to infection by inducing a stronger immune response and greater protection against the parasite by trained immunity. Methods: BALB/c mice were immunized with BCG subcutaneously, and 60 days later, they were infected with Trypanosoma cruzi intraperitoneally. An evaluation of the progression of the disease from the acute to the chronic stage, analyzing various aspects such as parasitemia, survival, clinical status, and humoral and cellular immune response, as well as the appearance of visceral megas and the histopathological description of target organs, was performed. Results: Vaccination reduced parasitemia by 70%, and 100% survival was achieved in the acute stage; although the presentation of clinical signs was reduced, there was no increase in the antibody titer or in the differential production of the isotypes. Conclusion: Serum cytokine production indicated a proinflammatory response in infected animals, while in those who received BCG, the response was balanced by inducing Th1/Th2-type cytokines, with a better prognosis of the disease in the chronic stage.

Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages.

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

Ursolic and oleanolic acids as antimicrobial and immunomodulatory compounds for tuberculosis treatment.

BACKGROUND: New alternatives for the treatment of Tuberculosis (TB) are urgently needed and medicinal plants represent a potential option. Chamaedora tepejilote and Lantana hispida are medicinal plants from Mexico and their hexanic extracts have shown antimycobacterial activity. Bioguided investigation of these extracts showed that the active compounds were ursolic acid (UA) and oleanolic acid (OA). METHODS: The activity of UA and OA against Mycobacterium tuberculosis H37Rv, four monoresistant strains, and two drug-resistant clinical isolates were determined by MABA test. The intracellular activity of UA and OA against M. tuberculosis H37Rv and a MDR clinical isolate were evaluated in a macrophage cell line. Finally, the antitubercular activity of UA and OA was tested in BALB/c mice infected with M. tuberculosis H37Rv or a MDR strain, by determining pulmonary bacilli loads, tissue damage by automated histomorphometry, and expression of IFN-γ, TNF-α, and iNOS by quantitative RT-PCR. RESULTS: The in vitro assay showed that the UA/OA mixture has synergistic activity. The intracellular activity of these compounds against M. tuberculosis H37Rv and a MDR clinical isolate in a macrophage cell line showed that both compounds, alone and in combination, were active against intracellular mycobacteria even at low doses. Moreover, when both compounds were used to treat BALB/c mice with TB induced by H37Rv or MDR bacilli, a significant reduction of bacterial loads and pneumonia were observed compared to the control. Interestingly, animals treated with UA and OA showed a higher expression of IFN-γ and TNF-α in their lungs, than control animals. CONCLUSION: UA and OA showed antimicrobial activity plus an immune-stimulatory effect that permitted the control of experimental pulmonary TB.

The Use of Viral Vectors for Gene Therapy and Vaccination in Tuberculosis.

Tuberculosis (TB), an infection caused by Mycobacterium tuberculosis (Mtb), is one of the primary causes of death globally. The treatment of TB is long and based on several drugs, producing problems in compliance and toxicity, increasing Mtb resistance to first-line antibiotics that result in multidrug-resistant TB and extensively drug-resistant TB. Thus, the need for new anti-TB treatments has increased. Here, we review some model strategies to study gene therapy based on the administration of a recombinant adenovirus that encodes diverse cytokines, such as IFNγ, IL12, GM/CSF, OPN, TNFα, and antimicrobial peptides to enhance the protective immune response against Mtb. These models include a model of progressive pulmonary TB, a model of chronic infection similar to latent TB, and a murine model of pulmonary Mtb transmission to close contacts. We also review new vaccines that deliver Mtb antigens via particle- or virus-based vectors and trigger protective immune responses. The results obtained in this type of research suggest that this is an alternative therapy that has the potential to treat active TB as an adjuvant to conventional antibiotics and a promising preventive treatment for latent TB reactivation and Mtb transmission. Moreover, Ad vector vaccines are adequate for preventing infectious diseases, including TB.

Beneficial or detrimental activity of regulatory T cells, indoleamine 2,3-dioxygenase, and heme oxygenase-1 in the lungs is influenced by the level of virulence of Mycobacterium tuberculosis strain infection.

Tuberculosis (TB) caused by the complex Mycobacterium tuberculosis (Mtb) is the main cause of death by a single bacterial agent. Last year, TB was the second leading infectious killer after SARS-CoV-2. Nevertheless, many biological and immunological aspects of TB are not completely elucidated, such as the complex process of immunoregulation mediated by regulatory T cells (Treg cells) and the enzymes indoleamine 2,3-dioxygenase (IDO) and heme oxygenase 1 (HO-1). In this study, the contribution of these immunoregulatory factors was compared in mice infected with Mtb strains with different levels of virulence. First Balb/c mice were infected by intratracheal route, with a high dose of mild virulence reference strain H37Rv or with a highly virulent clinical isolate (strain 5186). In the lungs of infected mice, the kinetics of Treg cells during the infection were determined by cytofluorometry and the expression of IDO and HO-1 by RT-PCR and immunohistochemistry. Then, the contribution of immune-regulation mediated by Treg cells, IDO and HO-1, was evaluated by treating infected animals with specific cytotoxic monoclonal antibodies for Treg cells depletion anti-CD25 (PC61 clone) or by blocking IDO and HO-1 activity using specific inhibitors (1-methyl-D,L-tryptophan or zinc protoporphyrin-IX, respectively). Mice infected with the mild virulent strain showed a progressive increment of Treg cells, showing this highest number at the beginning of the late phase of the infection (28 days), the same trend was observed in the expression of both enzymes being macrophages the cells that showed the highest immunostaining. Animals infected with the highly virulent strain showed lower survival (34 days) and higher amounts of Treg cells, as well as higher expression of IDO and HO-1 one week before. In comparison with non-treated animals, mice infected with strain H37Rv with depletion of Treg cells or treated with the enzymes blockers during late infection showed a significant decrease of bacilli loads, higher expression of IFN-g and lower IL-4 but with a similar extension of inflammatory lung consolidation determined by automated morphometry. In contrast, the depletion of Treg cells in infected mice with the highly virulent strain 5186 produced diffuse alveolar damage that was similar to severe acute viral pneumonia, lesser survival and increase of bacillary loads, while blocking of both IDO and HO-1 produced high bacillary loads and extensive pneumonia with necrosis. Thus, it seems that Treg cells, IDO and HO-1 activities are detrimental during late pulmonary TB induced by mild virulence Mtb, probably because these factors decrease immune protection mediated by the Th1 response. In contrast, Treg cells, IDO and HO-1 are beneficial when the infection is produced by a highly virulent strain, by regulation of excessive inflammation that produced alveolar damage, pulmonary necrosis, acute respiratory insufficiency, and rapid death.

Adenoviral Vector Codifying for TNF as a Co-Adjuvant Therapy against Multi-Drug-Resistant Tuberculosis.

Mycobacterium tuberculosis is the main causal agent of pulmonary tuberculosis (TB); the treatment of this disease is long and involves a mix of at least four different antibiotics that frequently lead to abandonment, favoring the surge of drug-resistant mycobacteria (MDR-TB), whose treatment becomes more aggressive, being longer and more toxic. Thus, the search for novel strategies for treatment that improves time or efficiency is of relevance. In this work, we used a murine model of pulmonary TB produced by the MDR-TB strain to test the efficiency of gene therapy with adenoviral vectors codifying TNF (AdTNF), a pro-inflammatory cytokine that has protective functions in TB by inducing apoptosis, granuloma formation and expression of other Th1-like cytokines. When compared to the control group that received an adenoviral vector that codifies for the green fluorescent protein (AdGFP), a single dose of AdTNF at the chronic active stage of the disease produced total survival, decreasing bacterial load and tissue damage (pneumonia), which correlated with an increase in cells expressing IFN-γ, iNOS and TNF in pneumonic areas and larger granulomas that efficiently contain and eliminate mycobacteria. Second-line antibiotic treatment against MDR-TB plus AdTNF gene therapy reduced bacterial load faster within a week of treatment compared to empty vector plus antibiotics or antibiotics alone, suggesting that AdTNF is a new potential type of treatment against MDR-TB that can shorten second-line chemotherapy but which requires further experimentation in other animal models (non-human primates) that develop a more similar disease to human pulmonary TB.