Two murine models of sepsis: immunopathological differences between the sexes-possible role of TGFß1 in female resistance to endotoxemia.

Endotoxic shock (ExSh) and cecal ligature and puncture (CLP) are models that induce sepsis. In this work, we investigated early immunologic and histopathologic changes induced by ExSh or CLP models in female and male mice. Remarkable results showed that females supported twice the LD100 of LPS for males, CLP survival and CFU counts were similar between genders, high circulating LPS levels in ExSh mice and low levels of IgM anti-LPS in males. In the serum of ExSh males, TNF and IL-6 increased in the first 6 h, in CLP males at 12 h. In the liver of ExSh mice, TNF increased at 1.5 and 12 h, IL-1 at 6 h. TGFß1 increased in females throughout the study and at 12 h in males. In CLP mice, IL-6 decreased at 12 h, TGFß1 increased at 6-12 h in males and at 12 h in females. In the lungs of ExSh males, IL-1ß increased at 1.5-6 h and TGFß1 at 12 h; in females, TNF decrease at 6 h and TGFß1 increased from 6 h; in CLP females, TNF and IL-1ß decreased at 12 h and 1.5 h, respectively, and TGFß1 increased from 6 h; in males, TGFß1 increased at 12 h. In the livers of ExSh mice, signs of inflammation were more common in males; in the CLP groups, inflammation was similar but less pronounced. ExSh females had leucocytes with TGFß1. The lungs of ExSh males showed patches of hyaline membranes and some areas of inflammatory cells, similar but fewer and smaller lesions were seen in male mice with CLP. In ExSh females, injuries were less extent than in males, similar pulmonary lesions were seen in female mice with CLP. ExSh males had lower levels of TGFß1 than females, and even lower levels were seen in CLP males. We conclude that the ExSh was the most lethal model in males, associated with high levels of free LPS, low IgM anti-LPS, exacerbated inflammation and target organ injury, while females showed early TGFß1 production in the lungs and less tissue damage. We didn't see any differences between CLP mice.

Repeated Episodes of Ischemia/Reperfusion Induce Heme-Oxygenase-1 (HO-1) and Anti-Inflammatory Responses and Protects against Chronic Kidney Disease.

Preconditioning episodes of ischemia/reperfusion (IR) induce protection against acute kidney injury (AKI), however their long-term effect still unknown. We evaluated AKI to chronic kidney disease (CKD) transition, after three-mild or three-severe episodes of IR. AKI was induced by single bilateral IR (1IR), or three episodes of IR separated by 10-day intervals (3IR) of mild (20 min) or severe (45 min) ischemia. Sham-operated rats served as controls. During 9-months, the 1IR group (20 or 45 min) developed CKD evidenced by progressive proteinuria and renal fibrosis. In contrast, the long-term adverse effects of AKI were markedly ameliorated in the 3IR group. The acute response in 3IR, contrasted with the 1IR group, that was characterized by an increment in heme oxygenase-1 (HO-1) and an anti-inflammatory response mediated by a NFkB-p65 phosphorylation and IL-6 decrease, together with an increase in TGF-ß, and IL-10 expression, as well as in M2-macrophages. In addition, three episodes of IR downregulated endoplasmic reticulum (ER) stress markers expression, CHOP and BiP. Thus, repeated episodes of IR with 10-day intervals induced long-term renal protection accompanied with HO-1 overexpression and M2-macrophages increase.

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.

Nrf2 protects the lung against inflammation induced by titanium dioxide nanoparticles: A positive regulator role of Nrf2 on cytokine release.

Titanium dioxide nanoparticles (TiO2 NPs) have been classified as possibly carcinogenic to humans and they are an important nanomaterial widely used in pharmaceutical and paint industries. Inhalation is one of the most important routes of exposure in occupational settings. Several experimental models have shown that oxidative stress and inflammation are key mediators of cell damage. In this regard, Nrf2 modulates cytoprotection against oxidative stress and inflammation, however, its role in inflammation induced by TiO2 NPs exposure has been less investigated. The aim of this work was to investigate the role of Nrf2 in the cytokines produced after 4 weeks of TiO2 NPs exposure (5 mg/kg/2 days/week) using wild-type and Nrf2 knockout C57bl6 mice. Results showed that Nrf2 protects against inflammation and oxidative damage induced by TiO2 NPs exposure, however, Nrf2 is a positive mediator in the expression of IFN-γ, TNF-α, and TGF-ß in bronchial epithelium and alveolar space after 4 weeks of exposure. These results suggest that Nrf2 has a central role in up-regulation of cytokines released during inflammation induced by TiO2 NPs and those cytokines are needed to cope with histological alterations in lung tissue.

Virulence and immune response induced by Mycobacterium avium complex strains in a model of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice.

The genus Mycobacterium comprises more than 150 species, including important pathogens for humans which cause major public health problems. The vast majority of efforts to understand the genus have been addressed in studies with Mycobacterium tuberculosis. The biological differentiation between M. tuberculosis and nontuberculous mycobacteria (NTM) is important because there are distinctions in the sources of infection, treatments, and the course of disease. Likewise, the importance of studying NTM is not only due to its clinical significance but also due to the mechanisms by which some species are pathogenic while others are not. Mycobacterium avium complex (MAC) is the most important group of NTM opportunistic pathogens, since it is the second largest medical complex in the genus after the M. tuberculosis complex. Here, we evaluated the virulence and immune response of M. avium subsp. avium and Mycobacterium colombiense, using experimental models of progressive pulmonary tuberculosis and subcutaneous infection in BALB/c mice. Mice infected intratracheally with a high dose of MAC strains showed high expression of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase with rapid bacillus elimination and numerous granulomas, but without lung consolidation during late infection in coexistence with high expression of anti-inflammatory cytokines. In contrast, subcutaneous infection showed high production of the proinflammatory cytokines TNF-α and gamma interferon with relatively low production of anti-inflammatory cytokines such as interleukin-10 (IL-10) or IL-4, which efficiently eliminate the bacilli but maintain extensive inflammation and fibrosis. Thus, MAC infection evokes different immune and inflammatory responses depending on the MAC species and affected tissue.

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.

[Research on conventional and molecular epidemiology of tuberculosis in Orizaba, Veracruz, 1995-2008]. / Investigación sobre epidemiología convencional y molecular de tuberculosis en Orizaba, Veracruz, 1995-2008.

This study describes the achievements of the Mexican Consortium against Tuberculosis, in the Sanitary District of Orizaba, Veracruz, Mexico between 1995 and 2008. In brief, the main results can be classified as follows: 1) Conventional and molecular epidemiology (measurement of burden of disease, trends, risk factors and vulnerable groups, consequences of drug resistance, identification of factors that favor nosocomial and community transmission); 2) Development of diagnostic techniques to detect drug resistance, description of circulating clones and adaptation of simple techniques to be used in the field; 3) Evaluation of usefulness of tuberculin skin test, immunologic responses to BCG, impact of directly observed therapy for tuberculosis (DOTS), and study of immunological biomarkers and 4) Comments on ethical aspects of tuberculosis research. Additionally, we describe the impact on public policies, transference of technology, capacity building and future perspectives.

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.

Induction of bone formation in abdominal implants constituted by collagen sponges embedded with plant-based human transforming growth factor family proteins in ectopic dog model.

BACKGROUND: Trauma, osteomyelitis, bone tumour resections and congenital deformities are the main causes of bone deficiency in which autologous graft is the preferred treatment, but usually the bone supplies are limited. METHODS: An experimental model of heterotopic bone formation in the subcutaneous abdominal area of dogs was developed. This model consists in omentum wrapped implants constituted by collagen type 1 sponges embedded with demineralized bone powder, calcium cloride, thrombin and platelet rich plasma; the implant is totally converted in trabecular bone after four months of implantation. This model was improved by accelerating bone production, after the isolation of the most conspicuous histological constituents (inflammatory, bone and adipose tissues) by laser microdisection and purified from them RNA that was used to determine by RT-PCR the gene expression kinetics of the most important growth bone factors. Then, the most abundant and rapidly synthesized factors were produced by genetic engineering in tobacco plants. RESULTS: Bone morphogenetic proteins 2 and 7 and transforming growth factor-ß1were the most rapidly and highly synthesized factors, and they were efficiently produced in a genetic engineering plant based system in tobacco leaves. Their incorporation as recombinant proteins in the scaffold collagen sponge induced in just one month mature heterotopic bone. CONCLUSION: This study demonstrates for the first time that this plant system is able to produce recombinant bone growth factors in high amount and at low cost, and they were highly efficient to rapidly induce bone formation in abdominal implants potentially useful for autotransplantation.

The protective effect of immunoglobulin in murine tuberculosis is dependent on IgG glycosylation.

Antibodies have demonstrated having a protective effect in animal models of tuberculosis (TB). These experiments have considered the specificity of antigen recognition and the different isotypes and subclasses as significant contributors of this effect. However, the carbohydrate chain heterogeneity on the Fc region of IgG (Fc-IgG) can play an important role in modulating the immune response. Patients with TB usually have high titers of specific IgG; however, the carbohydrate associated with Fc-IgG usually lacks galactose. To assess the effect of this abnormal IgG in murine pulmonary TB, we evaluated the specificity of recognition to Mycobacterium tuberculosis antigens in vitro and protective effects in vivo comparing human intravenous immunoglobulin (IVIg) and IVIg treated with an endoglycosidase to remove the glycan residues (EndoS-treated IVIg). Our results showed similar antigen recognition. The study of distribution and kinetics of IVIg in serum and bronchial lavage after intraperitoneal (i.p.) administration in mice showed that IVIg circulates for 21 days. Finally, the protective effect of intact and EndoS-treated IVIg administered by i.p was studied in a murine model of progressive TB. IVIg treatment caused reduction in pulmonary bacilli loads, larger granulomas, and less pneumonia, while animals treated with EndoS-treated IVIg were not protected compared with control animals. Thus, IVIg has a protective activity in experimental pulmonary TB, and this effect requires intact Fc oligosaccharides.

Specific bacterial genotypes of Mycobacterium tuberculosis cause extensive dissemination and brain infection in an experimental model.

Meningeal tuberculosis is a severe type of extrapulmonary disease, which is thought to begin with respiratory infection, followed by hematogenous dissemination and brain infection. Host genetic susceptibility factors and specific mycobacterial substrains could be involved in its development. From an epidemiological study in Colombia, we selected three Mycobacterium tuberculosis clinical strains isolated from the cerebrospinal fluid (CSF) of patients with meningeal tuberculosis, and used them to infect BALB/c mice through the intratracheal route. These strains showed a distinctive spoligotype pattern. The course of infection in terms of strain virulence (mice survival, bacillary loads in lungs), bacilli dissemination and extrapulmonary infection (bacilli loads in blood, brain, liver, kidney and spleen), and immune responses (cytokine expression determined by real time PCR in brain and lung) was studied and compared with that induced by the laboratory strain H37Rv and other five clinical strains isolated from patients with pulmonary TB. All the clinical isolates from meningeal TB patients disseminated extensively through the hematogenous route infecting the brain, producing inflammation in the cerebral parenchyma and meninges, whereas H37Rv and clinical isolates from pulmonary TB patients showed very limited efficiency to infect the brain. Thus, it seems that mycobacterial strains with a distinctive genotype are able to disseminate extensively after the respiratory infection and infect the brain.

Investigación sobre epidemiología convencional y molecular de tuberculosis en Orizaba, Veracruz, 1995-2008 / Research on conventional and molecular epidemiology of tuberculosis in Orizaba, Veracruz, 1995-2008

Se describen los resultados de investigación del Consorcio Mexicano contra la Tuberculosis, en la Jurisdicción Sanitaria de Orizaba, Veracruz, entre 1995 y 2008. Las aportaciones principales de los trabajos se refieren a los siguientes rubros: 1. Epidemiología convencional y molecular (medición de la carga de la enfermedad, tendencias, factores de riesgo y grupos vulnerables; descripción de las consecuencias de la farmacorresistencia e identificación de factores que favorecen la transmisión en la comunidad y en los hospitales). 2. Desarrollo de técnicas rápidas para conservación de muestras respiratorias que permitan el aislamiento y diagnóstico de farmacorresistencia de M. tuberculosis en campo. 3. Evaluación de la prueba tuberculínica, respuesta inmunológica al Bacilo de Calmette-Guerin (BCG), biomarcadores de la respuesta inmunitaria y medidas de control. 4. Comentarios en torno a aspectos éticos de la investigación en tuberculosis. Además se describe el impacto en políticas públicas, la transferencia de tecnología, la formación de recursos humanos y las perspectivas a futuro.

This study describes the achievements of the Mexican Consortium against Tuberculosis, in the Sanitary District of Orizaba, Veracruz, Mexico between 1995 and 2008. In brief, the main results can be classified as follows: 1) Conventional and molecular epidemiology (measurement of burden of disease, trends, risk factors and vulnerable groups, consequences of drug resistance, identification of factors that favor nosocomial and community transmission); 2) Development of diagnostic techniques to detect drug resistance, description of circulating clones and adaptation of simple techniques to be used in the field; 3) Evaluation of usefulness of tuberculin skin test, immunologic responses to BCG, impact of directly observed therapy for tuberculosis (DOTS), and study of immunological biomarkers and 4) Comments on ethical aspects of tuberculosis research. Additionally, we describe the impact on public policies, transference of technology, capacity building and future perspectives.