Dulce Mata-Espinosa

Granulocyte–macrophage colony-stimulating factor: not just another haematopoietic growth factor

Abstract Granulocyte–macrophage colony-stimulating factor (GM-CSF) is often used to treat leucopenia. Other haematopoietins may increase the number of circulating leucocytes with higher efficiency, but GM-CSF has additional effects that may be far more relevant than its haematopoietic activity. GM-CSF induces differentiation, proliferation and activation of macrophages and dendritic cells which are necessary for the subsequent T helper cell type 1 and cytotoxic T lymphocyte activation. GM-CSF haematopoietic and non-haematopoietic functions have pro-inflammatory and immune regulatory potential to treat a variety of autoimmune diseases and tumours. On the other hand, GM-CSF deficiency leads to various immune dysfunctions and the current utilization of GM-CSF as haematopoietic factor might be an accurate but very incomplete indication for a cytokine with vast clinical potential.

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

BackgroundNew 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).MethodsThe 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.ResultsThe 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.ConclusionUA and OA showed antimicrobial activity plus an immune-stimulatory effect that permitted the control of experimental pulmonary TB.

Therapeutic Effect of Recombinant Adenovirus Encoding Interferon-γ in a Murine Model of Progressive Pulmonary Tuberculosis

We constructed recombinant adenoviruses encoding murine interferon-γ (AdIFNγ) and tested its therapeutic efficiency in a well characterized model of progressive pulmonary tuberculosis (TB) in Balb/c mice, infected through the trachea with the laboratory drug-susceptible H37Rv strain or multidrug-resistant (MDR) clinical isolate. When the disease was in a late phase, 2 months after infection, we administered by intratracheal cannulation a single dose [1.7 × 109 plaque forming units (pfu)] of AdIFNγ or the control adenovirus. Groups of mice were killed at different time-points and the lungs were examined to determine bacilli colony forming units (CFU), cytokine/chemokine gene expression, and CD4/CD8 subpopulations, and also subjected to automated histomorphometry. In comparison with the control group, after 2 weeks of treatment and during the next 6 months, AdIFNγ-treated animals infected with either the H37Rv strain or the MDR strain showed significantly lower bacilli loads and tissue damage (pneumonia), higher expressions of IFN-γ, tumor necrosis factor (TNF), and inducible nitric oxide synthase (iNOS), and bigger granulomas. When compared with the results from conventional chemotherapy or AdIFNγ treatment alone, the combined treatment with AdIFNγ plus conventional chemotherapy shortened the time taken for reduction of bacillary load. This shows that gene therapy with AdIFNγ efficiently reconstituted the protective immune response and controlled the progress of pulmonary TB produced by MDR or non-MDR strains.We constructed recombinant adenoviruses encoding murine interferon-gamma (AdIFNgamma) and tested its therapeutic efficiency in a well characterized model of progressive pulmonary tuberculosis (TB) in Balb/c mice, infected through the trachea with the laboratory drug-susceptible H37Rv strain or multidrug-resistant (MDR) clinical isolate. When the disease was in a late phase, 2 months after infection, we administered by intratracheal cannulation a single dose [1.7 x 10(9) plaque forming units (pfu)] of AdIFNgamma or the control adenovirus. Groups of mice were killed at different time-points and the lungs were examined to determine bacilli colony forming units (CFU), cytokine/chemokine gene expression, and CD4/CD8 subpopulations, and also subjected to automated histomorphometry. In comparison with the control group, after 2 weeks of treatment and during the next 6 months, AdIFNgamma-treated animals infected with either the H37Rv strain or the MDR strain showed significantly lower bacilli loads and tissue damage (pneumonia), higher expressions of IFN-gamma, tumor necrosis factor (TNF), and inducible nitric oxide synthase (iNOS), and bigger granulomas. When compared with the results from conventional chemotherapy or AdIFNgamma treatment alone, the combined treatment with AdIFNgamma plus conventional chemotherapy shortened the time taken for reduction of bacillary load. This shows that gene therapy with AdIFNgamma efficiently reconstituted the protective immune response and controlled the progress of pulmonary TB produced by MDR or non-MDR strains.

Immunotherapeutic effects of recombinant adenovirus encoding granulocyte–macrophage colony-stimulating factor in experimental pulmonary tuberculosis

BALB/c mice with pulmonary tuberculosis (TB) develop a T helper cell type 1 that temporarily controls bacterial growth. Bacterial proliferation increases, accompanied by decreasing expression of interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and inducible nitric oxide synthase (iNOS). Activation of dendritic cells (DCs) is delayed. Intratracheal administration of only one dose of recombinant adenoviruses encoding granulocyte–macrophage colony‐stimulating factor (AdGM‐CSF) 1 day before Mycobacterium tuberculosis (Mtb) infection produced a significant decrease of pulmonary bacterial loads, higher activated DCs and increased expression of TNF‐α, IFN‐γ and iNOS. When AdGM‐CSF was given in female mice B6D2F1 (C57BL/6J X DBA/2J) infected with a low Mtb dose to induce chronic infection similar to latent infection and corticosterone was used to induce reactivation, a very low bacilli burden in lungs was detected, and the same effect was observed in healthy mice co‐housed with mice infected with mild and highly virulent bacteria in a model of transmissibility. Thus, GM‐CSF is a significant cytokine in the immune protection against Mtb and gene therapy with AdGM‐CSF increased protective immunity when administered in a single dose 1 day before Mtb infection in a model of progressive disease, and when used to prevent reactivation of latent infection or transmission.

Effect of cortisol and/or DHEA on THP1-derived macrophages infected with Mycobacterium tuberculosis.

Tuberculosis (TB) is a major health problem requiring an appropriate cell immune response to be controlled. Macrophages play a central role in the response against Mycobacterium tuberculosis (Mtb). Given our prior studies in which adrenal steroids were found to modify the cellular immune responses from TB patients, it was sensible to analyze the immunomodulatory capability of cortisol and DHEA on macrophages infected with Mtb. The human macrophage-like THP-1 cells were infected with the H37Rv strain of Mtb and treated with Cortisol and DHEA at different doses. We monitored phagocytosis, intracellular-bacterial growth, autophagosoma formation, as well as cytokine gene expression and production. Cultures exposed to cortisol showed a decreased production of IL-1β, TNF-α, with DHEA being unable to modify the pattern of cytokine production or to reverse the cortisol inhibitory effects. Interestingly the intra-macrophagic bacterial burden was found reduced by DHEA treatment. While this effect was not related to a different cytokine pattern, in terms their production or mRNA expression, DHEA treatment did promote autophagy in Mtb-infected macrophages, irrespective of Cortisol presence. In essence, the better control of Mtb load by DHEA-treated macrophages seems to be dependent on an autophagic mechanism. The present results are relevant for two reasons as autophagy is not only important for clearance of mycobacteria but also for the prevention of tissue damage.

Gene therapy based in antimicrobial peptides and proinflammatory cytokine prevents reactivation of experimental latent tuberculosis.

Mycobacterium tuberculosis (Mtb) latent infection can lead to reactivation. The design of new strategies to prevent it is an important subject. B6D2F1 mice were infected intratracheally with a low dose of Mtb H37Rv to induce chronic infection. After 7 months, mice were treated with one dose of recombinant adenoviruses encoding TNFα, β defensin-3 and LL37. Immunosupression was induced 1 month later with corticosterone. In comparison with the control group, mice treated with adenoviruses showed significantly less bacterial load and pneumonia, the adenoviruses encoding TNFα and LL37 being the most efficient. Gene therapy based in a proinflammatory cytokine or antimicrobial peptides is a potentially useful system to prevent reactivation of latent tuberculosis.

The BCGΔBCG1419c strain, which produces more pellicle in vitro, improves control of chronic tuberculosis in vivo.

Mycobacterium tuberculosis (Mtb) has been a threat to humans since ancient times, and it is the main causative agent of tuberculosis (TB). Until today, the only licensed vaccine against Mtb is the live attenuated M. bovis Bacillus Calmette-Guérin (BCG), which has variable levels of protection against the pulmonary form of infection. The quest for a new vaccine is a priority given the rise of multidrug-resistant Mtb around the world, as well as the tremendous burden imposed by latent TB. The objective of this study was to evaluate the immunogenicity and capacity of protection of a modified BCG strain (BCGΔBCG1419c) lacking the c-di-GMP phosphodiesterase gene BCG1419c, in diverse mice models. In a previous report, we have shown that BCGΔBCG1419c was capable of increasing biofilm production and after intravenous infection of immunocompetent mice; this strain persisted longer in lungs than parental BCG Pasteur. This led us to hypothesize that BCGΔBCG1419c might therefore possess some advantage as vaccine candidate. Our results in this report indicate that compared to conventional BCG, vaccination with BCGΔBCG1419c induced a better activation of specific T-lymphocytes population, was equally effective in preventing weight loss despite being used at lower dose, reduced tissue damage (pneumonic scores), increased local IFNγ(+) T cells, and diminished bacterial burden in lungs of BALB/c mice infected intratracheally with high dose Mtb H37Rv to induce progressive TB. Moreover, vaccination with BCGΔBCG1419c improved resistance to reactivation after immunosuppression induced by corticosterone in a murine model of chronic infection similar to latent TB. Furthermore, despite showing increased persistence in immunocompetent mice, BCGΔBCG1419c was as attenuated as parental BCG in nude mice. To our knowledge, this is the first demonstration that a modified BCG vaccine candidate with increased pellicle/biofilm production has the capacity to protect against Mtb challenge in chronic and reactivation models of infection.

Efficacy of gene-therapy based on adenovirus encoding granulocyte- macrophage colony-stimulating factor in drug-sensitive and drug- resistant experimental pulmonary tuberculosis

Tuberculosis (TB), although a curable disease, remains a major cause of morbidity and mortality worldwide. It is necessary to develop a short-term therapy with reduced drug toxicity in order to improve adherence rate and control disease burden. Granulocyte-macrophage colony-stimulating factor (GM-CSF) may be a key cytokine in the treatment of pulmonary TB since it primes the activation and differentiation of myeloid and non-myeloid precursor cells, inducing the release of protective Th1 cytokines. In this work, we administrated by intratracheal route recombinant adenoviruses encoding GM-CSF (AdGM-CSF). This treatment produced significant bacterial elimination when administered in a single dose at 60 days of infection with drug sensitive or drug resistant Mtb strains in a murine model of progressive disease. Moreover, AdGM-CSF combined with primary antibiotics produced more rapid elimination of pulmonary bacterial burdens than conventional chemotherapy suggesting that this form of treatment could shorten the conventional treatment.

A significant therapeutic effect of immunoglobulins administered alone, or in combination with conventional chemotherapy, in experimental pulmonary tuberculosis caused by drug-sensitive or drug-resistant strains

The recommended chemotherapy for drug-sensitive tuberculosis (TB) consists of four different antibiotics administrated for 6 months. This long treatment leads to significant compliance problems and consequently to recrudescence of the disease and to the development of multidrug-resistant (MDR) strains. Thus, new alternatives are needed to shorten or simplify the treatment of TB. Antibodies have therapeutic effects in animal models of TB, so their use as adjuvants in drug-sensitive and MDR TB is an interesting alternative. To assess the effect of antibodies, BALB/c mice with active late disease 60 days after infection with drug-sensitive TB strain H37Rv were treated with intravenous immunoglobulin (IVIg), alone or in combination with conventional chemotherapy. When compared with control non-treated animals, IVIg alone produced a significantly decreased burden of pulmonary bacilli. This decrease was even greater when IVIg was used in combination with conventional chemotherapy. The combined therapy also significantly reduced tissue damage (pneumonia) when compared to infected animals treated only with antibiotics. IVIg treatment also caused decreased bacillary burdens in mice infected with an MDR strain. In vitro experiments suggested that improving phagocytosis by efficient opsonization is perhaps the principal mechanism of this beneficial therapeutic effect.

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.