Granulomatous Reactivation during the Course of a Leprosy Infection: Reaction or Relapse

Background: Leprosy is a chronic granulomatous infectious disease and is still endemic in many parts of the world. It causes disabilities which are the consequence of nerve damage. This damage is in most cases the result of immunological reactions. Objectives: To investigate the differences between a type 1 leprosy (reversal) reaction and relapse on using histopathology. Methods: The histopathological changes in 167 biopsies from 66 leprosy patients were studied. The patients were selected when their sequential biopsies demonstrated either different patterns or maintained the same pattern of granulomatous reaction over more than two years during or after the treatment of leprosy. Results: In 57 of the patients studied, a reactivation was seen which coincided with a decrease in the bacteriological index (BI), suggesting that this reactivation (reversal reaction or type 1 leprosy reaction) coincides with an effective capacity for bacteriological clearance. In nine patients, an increase of the bacteriologic index (IB) or persistence of solid bacilli occurred during the reactivation, indicating proliferative activity, suggestive of a relapse. The histopathological aspects of the granulomas were similar in both groups. Conclusion: Bacterioscopy provided the only means to differentiate a reversal reaction from a relapse in patients with granulomatous reactivation. The type 1 leprosy reaction may be considered as a part effective immune reconstitution (reversal, upgrading reaction) or as a mere hypersensitivity reaction (downgrading reaction) in a relapse.

Prolonged Survival of Allografts Induced by Mycobacterial Hsp70 Is Dependent on CD4+CD25+Regulatory T Cells

Background: Heat shock proteins (Hsps) are stress induced proteins with immunomodulatory properties. The Hsp70 of Mycobacterium tuberculosis (TBHsp70) has been shown to have an anti-inflammatory role on rodent autoimmune arthritis models, and the protective effects were demonstrated to be dependent on interleukin-10 (IL-10). We have previously observed that TBHsp70 inhibited maturation of dendritic cells (DCs) and induced IL-10 production by these cells, as well as in synovial fluid cells. Methodology/Principal Findings: We investigated if TBHsp70 could inhibit allograft rejection in two murine allograft systems, a transplanted allogeneic melanoma and a regular skin allograft. In both systems, treatment with TBHsp70 significantly inhibited rejection of the graft, and correlated with regulatory T cells (Tregs) recruitment. This effect was not tumor mediated because injection of TBHsp70 in tumor-free mice induced an increase of Tregs in the draining lymph nodes as well as inhibition of proliferation of lymph node T cells and an increase in IL-10 production. Finally, TBHsp70 inhibited skin allograft acute rejection, and depletion of Tregs using a monoclonal antibody completely abolished this effect. Conclusions/Significance: We present the first evidence for an immunosuppressive role for this protein in a graft rejection system, using an innovative approach - immersion of the graft tissue in TBHsp70 solution instead of protein injection. Also, this is the first study that demonstrates dependence on Treg cells for the immunosuppressive role of TBHsp70. This finding is relevant for the elucidation of the immunomodulatory mechanism of TBHsp70. We propose that this protein can be used not only for chronic inflammatory diseases, but is also useful for organ transplantation management.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Cost-effectiveness analysis of PCR for the rapid diagnosis of pulmonary tuberculosis

Background: Tuberculosis is one of the most prominent health problems in the world, causing 1.75 million deaths each year. Rapid clinical diagnosis is important in patients who have comorbidities such as Human Immunodeficiency Virus (HIV) infection. Direct microscopy has low sensitivity and culture takes 3 to 6 weeks [1-3]. Therefore, new tools for TB diagnosis are necessary, especially in health settings with a high prevalence of HIV/TB co-infection. Methods: In a public reference TB/HIV hospital in Brazil, we compared the cost-effectiveness of diagnostic strategies for diagnosis of pulmonary TB: Acid fast bacilli smear microscopy by Ziehl-Neelsen staining (AFB smear) plus culture and AFB smear plus colorimetric test (PCR dot-blot). From May 2003 to May 2004, sputum was collected consecutively from PTB suspects attending the Parthenon Reference Hospital. Sputum samples were examined by AFB smear, culture, and PCR dot-blot. The gold standard was a positive culture combined with the definition of clinical PTB. Cost analysis included health services and patient costs. Results: The AFB smear plus PCR dot-blot require the lowest laboratory investment for equipment (US$ 20,000). The total screening costs are 3.8 times for AFB smear plus culture versus for AFB smear plus PCR dot blot costs (US$ 5,635,760 versus US$ 1,498, 660). Costs per correctly diagnosed case were US$ 50,773 and US$ 13,749 for AFB smear plus culture and AFB smear plus PCR dot-blot, respectively. AFB smear plus PCR dot-blot was more cost-effective than AFB smear plus culture, when the cost of treating all correctly diagnosed cases was considered. The cost of returning patients, which are not treated due to a negative result, to the health service, was higher in AFB smear plus culture than for AFB smear plus PCR dot-blot, US$ 374,778,045 and US$ 110,849,055, respectively. Conclusion: AFB smear associated with PCR dot-blot associated has the potential to be a cost-effective tool in the fight against PTB for patients attended in the TB/HIV reference hospital.

Silica-based cationic bilayers as immunoadjuvants

Background: Silica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design. Results: Firstly, the silica/DODAB assembly was characterized at 1 mM NaCl, pH 6.3 or 5 mM Tris. HCl, pH 7.4 and 0.1 mg/ml silica over a range of DODAB concentrations (0.001-1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.05 mM DODAB is enough to produce cationic bilayer-covered particles with good colloid stability. Secondly, conditions for maximal adsorption of bovine serum albumin (BSA) or a recombinant, heat-shock protein from Mycobacterium leprae (18 kDa-hsp) onto DODAB-covered or onto bare silica were determined. At maximal antigen adsorption, cellular immune responses in vivo from delayed-type hypersensitivity reactions determined by foot-pad swelling tests (DTH) and cytokines analysis evidenced the superior performance of the silica/DODAB adjuvant as compared to alum or antigens alone whereas humoral response from IgG in serum was equal to the one elicited by alum as adjuvant. Conclusion: Cationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum, better performance regarding cellular immune responses and employs very low, micromolar doses of cationic and toxic synthetic lipid.

Bacterial community in the rhizosphere and rhizoplane of wild type and transgenic eucalyptus

The rhizosphere is a niche exploited by a wide variety of bacteria. The expression of heterologous genes by plants might become a factor affecting the structure of bacterial communities in the rhizosphere. In a greenhouse experiment, the bacterial community associated to transgenic eucalyptus, carrying the Lhcb1-2 genes from pea (responsible for a higher photosynthetic capacity), was evaluated. The culturable bacterial community associated to transgenic and wild type plants were not different in density, and the Amplified Ribosomal DNA Restriction Analysis (ARDRA) typing of 124 strains revealed dominant ribotypes representing the bacterial orders Burkholderiales, Rhizobiales, and Actinomycetales, the families Xanthomonadaceae, and Bacillaceae, and the genus Mycobacterium. Principal Component Analysis based on the fingerprints obtained by culture-independent Denaturing Gradient Gel Electrophoresis analysis revealed that Alphaproteobacteria, Betaproteobacteria and Actinobacteria communities responded differently to plant genotypes. Similar effects for the cultivation of transgenic eucalyptus to those observed when two genotype-distinct wild type plants are compared.

QSAR Modeling of a Set of Pyrazinoate Esters as Antituberculosis Prodrugs

Tuberculosis is an infection caused mainly by Mycobacterium tuberculosis. A first-line antimycobacterial drug is pyrazinamide (PZA), which acts partially as a prodrug activated by a pyrazinamidase releasing the active agent, pyrazinoic acid (POA). As pyrazinoic acid presents some difficulty to cross the mycobacterial cell wall, and also the pyrazinamide-resistant strains do not express the pyrazinamidase, a set of pyrazinoic acid esters have been evaluated as antimycobacterial agents. In this work, a QSAR approach was applied to a set of forty-three pyrazinoates against M. tuberculosis ATCC 27294, using genetic algorithm function and partial least squares regression (WOLF 5.5 program). The independent variables selected were the Balaban index (I), calculated n-octanol/water partition coefficient (ClogP), van-der-Waals surface area, dipole moment, and stretching-energy contribution. The final QSAR model (N = 32, r(2) = 0.68, q(2) = 0.59, LOF = 0.25, and LSE = 0.19) was fully validated employing leave-N-out cross-validation and y-scrambling techniques. The test set (N = 11) presented an external prediction power of 73%. In conclusion, the QSAR model generated can be used as a valuable tool to optimize the activity of future pyrazinoic acid esters in the designing of new antituberculosis agents.

3D-Pharmacophore mapping of thymidine-based inhibitors of TMPK as potential antituberculosis agents

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis monophosphate kinase (TMPKmt) is essential to DNA replication. Thus, this enzyme represents a promising target for developing new drugs against TB. In the present study, the receptor-independent, RI, 4D-QSAR method has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 81 thymidine analogues, and two corresponding subsets, reported as inhibitors of TMPKmt. The resulting optimized models are not only statistically significant with r (2) ranging from 0.83 to 0.92 and q (2) from 0.78 to 0.88, but also are robustly predictive based on test set predictions. The most and the least potent inhibitors in their respective postulated active conformations, derived from each of the models, were docked in the active site of the TMPKmt crystal structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. Moreover, the QSAR models provide insights regarding a probable mechanism of action of the analogues.

Rational Design and 3D-Pharmacophore Mapping of 5 `-Thiourea-Substituted alpha-Thymidine Analogues as Mycobacterial TMPK Inhibitors

Thymidine monophosphate kinase (TMPK) has emerged as an attractive target for developing inhibitors of Mycobacterium tuberculosis growth. In this study the receptor-independent (RI) 4D-QSAR formalism has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 5`-thiourea-substituted alpha-thymidine inhibitors. Models were developed for the entire training set and for a subset of the training set consisting of the most potent inhibitors. The optimized (RI) 4D-QSAR models are statistically significant (r(2) = 0.90, q(2) = 0.83 entire set, r(2) = 0.86, q(2) = 0.80 high potency subset) and also possess good predictivity based on test set predictions. The most and least potent inhibitors, in their respective postulated active conformations derived from the models, were docked in the active site of the TMPK crystallographic structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. This model identifies new regions of the inhibitors that contain pharmacophore sites, such as the sugar-pyrimidine ring structure and the region of the 5`-arylthiourea moiety. These new regions of the ligands can be further explored and possibly exploited to identify new, novel, and, perhaps, better antituberculosis inhibitors of TMPKmt. Furthermore, the 3D-pharmacophores defined by these models can be used as a starting point for future receptor-dependent antituberculosis drug design as well as to elucidate candidate sites for substituent addition to optimize ADMET properties of analog inhibitors.

Transcriptional responses of host peripheral blood cells to tuberculosis infection

Host responses following exposure to Mycobacterium tuberculosis (TB) are complex and can significantly affect clinical outcome. These responses, which are largely mediated by complex immune mechanisms involving peripheral blood cells (PBCs) such as T-lymphocytes, NK cells and monocyte-derived macrophages, have not been fully characterized. We hypothesize that different clinical outcome following TB exposure will be uniquely reflected in host gene expression profiles, and expression profiling of PBCs can be used to discriminate between different TB infectious outcomes. In this study, microarray analysis was performed on PBCs from three TB groups (BCG-vaccinated, latent TB infection, and active TB infection) and a control healthy group. Supervised learning algorithms were used to identify signature genomic responses that differentiate among group samples. Gene Set Enrichment Analysis was used to determine sets of genes that were co-regulated. Multivariate permutation analysis (p < 0.01) gave 645 genes differentially expressed among the four groups, with both distinct and common patterns of gene expression observed for each group. A 127-probeset, representing 77 known genes, capable of accurately classifying samples into their respective groups was identified. In addition, 13 insulin-sensitive genes were found to be differentially regulated in all three TB infected groups, underscoring the functional association between insulin signaling pathway and TB infection. Published by Elsevier Ltd.

Topliss method in the optimization of salicylic acid derivatives as potential antimycobacterial agents

The Topliss method was used to guide a synthetic path in support of drug discovery efforts toward the identification of potent antimycobacterial agents. Salicylic acid and its derivatives, p-chloro, p-methoxy, and m-chlorosalicylic acid, exemplify a series of synthetic compounds whose minimum inhibitory concentrations for a strain of Mycobacterium were determined and compared to those of the reference drug, p-aminosalicylic acid. Several physicochemical descriptors (including Hammett`s sigma constant, ionization constant, dipole moment, Hansch constant, calculated partition coefficient, Sterimol-L and -B-4 and molecular volume) were considered to elucidate structure-activity relationships. Molecular electrostatic potential and molecular dipole moment maps were also calculated using the AM1 semi-empirical method. Among the new derivatives, m-chlorosalicylic acid showed the lowest minimum inhibitory concentration. The overall results suggest that both physicochemical properties and electronic features may influence the biological activity of this series of antimycobacterial agents and thus should be considered in designing new p-aminosalicylic acid analogs.

Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.

DNAhsp65 vaccination induces protection in mice against Paracoccidioides brasiliensis infection

Heat-shock proteins are Molecules with extensive data showing their potential as immunomodulators of different types of diseases, The gene of HSP65 from Mycobacterium leprae has shown prophylactic and immunotherapeutic effects against a broad arrays of experimental models including tuberculosis, leishmaniasis, arthritis and diabetes. With this in mind, we tested the DNAhsp65 vaccine using an experimental model of Paraccocidiodomycosis, an important endemic mycosis in Latin America. The intramuscular immunization with DNAhsp65 induced, in BALB/c mice, an increase of Th1-levels cytokines and a reduction of fungal burdens resulted in a marked reduction of collagen and lung remodeling. DNAhsp65 may be an attractive candidate for prevention, therapy and as an adjuvant for mycosis treatment. (C) 2008 Elsevier Ltd. All rights reserved.

Leukotrienes Are Potent Adjuvant during Fungal Infection: Effects on Memory T Cells

Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB(4) induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB(4) is also important in early effector T cell recruitment that is mediated by LTB(4) receptor 1, the high-affinity receptor for LTB(4). The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB(4) and IFN-gamma production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase(-/-), a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-beta as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase(-/-)-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis. The Journal of Immunology, 2008,181: 8544-8551.

Evaluation of Environmental Mycobacteria Contamination in a Specific Pathogen Free Animal Facility from a Tropical Country

P>With the evidence showing the protection variability of bacille Calmette-Guerin, new potential vaccines for tuberculosis have been tested around the world. One of the general concerns in tuberculosis vaccine development is the possibility of priming the host immune system with prior exposure to environmental mycobacteria antigens, which can change the efficacy of subsequent vaccination. As there is a great homology between the species from Mycobacterium genera, the previous contact of experimental animals with environmental mycobacteria could sensitize the mice and, in this way, could influence subsequent vaccine research. The aim of our study was to investigate critical points in an animal facility to search for environmental mycobacteria that eventually could be in direct or indirect contact with the experimental animals. Samples were collected from surfaces of walls, floor, animal cages and shelves and analysed using the Ogawa-Kudoh decontamination method. Samples of drinking water, food and sawdust were collected for analysis by the NALC/NaOH decontamination method. Also, the samples were cultivated directly in broth medium, without any method for decontamination. After decontamination methods, we observed bacterial colony growth in 4.31% of the total of samples analysed. These samples were stained with Ziehl-Neelsen and we did not detect any acid-fast bacilli, suggesting that the animal facility analysed is free from contamination by environmental mycobacteria and is not a source of mycobacterial antigens. Furthermore, our study showed a new paradigm in tuberculosis vaccine development: concern about the animal facility environment in terms of immune system priming of experimental animals by nascent bacterial contaminants.