Characterization of novel immunodominant antigens of Mycobacterium tuberculosis

Seven novel antigens of Mycobacterium tuberculosis, which had previously been identified based on reactivity to sera from patients with tuberculosis, were characterized. Nucleotide sequence analysis of the genes encoding these seven antigens identified one of them as the FtsH and a second as the aminoimidazole ribotide synthase of M. tuberculosis. Antisera raised to the recombinant forms of each of these seven antigens were used to study the distribution of these proteins within mycobacterial species as well as to determine their subcellular localization and hydrophobicity. Four of the seven antigens were conserved only among pathogenic strains of mycobacteria. Of the seven proteins studied, FtsH and a second protein of unknown identity were localized in membranes. Two were cytosolic, while two others, which had a high proline content, were tightly associated with the cell wall. One protein was secreted. This secreted protein could be identified by serum from a majority of tuberculosis patients but not BCG-vaccinated individuals, suggesting its potential use in the immunodiagnosis of tuberculosis.

Scientists in Control: a progressive approach to preparing work ready graduates in QC, EQA and method evaluation

Introduction QC, EQA and method evaluation are integral to delivery of quality patient results. To ensure QUT graduates have a solid grounding in these key areas of practice, a theory-to-practice approach is used to progressively develop and consolidate these skills. Methods Using a BCG assay for serum albumin, each student undertakes an eight week project analysing two levels of QC alongside ‘patient’ samples. Results are assessed using both single rules and Multirules. Concomitantly with the QC analyses, an EQA project is undertaken; students analyse two EQA samples, twice in the semester. Results are submitted using cloud software and data for the full ‘peer group’ returned to students in spreadsheets and incomplete Youden plots. Youden plots are completed with target values and calculated ALP values and analysed for ‘lab’ and method performance. The method has a low-level positive bias, which leads to the need to investigate an alternative method. Building directly on the EQA of the first project and using the scenario of a lab that services renal patients, students undertake a method validation comparing BCP and BCG assays in another eight-week project. Precision and patient comparison studies allow students to assess whether the BCP method addresses the proportional bias of the BCG method and overall is a ‘better’ alternative method for analysing serum albumin, accounting for pragmatic factors, such as cost, as well as performance characteristics. Results Students develop understanding of the purpose and importance of QC and EQA in delivering quality results, the need to optimise testing to deliver quality results and importantly, a working knowledge of the analyses that go into ensuring this quality. In parallel to developing these key workplace competencies, students become confident, competent practitioners, able to pipette accurately and precisely and organise themselves in a busy, time pressured work environment.

Cyclosporine A in the treatment of Interstitial Cystitis

Painful bladder syndrome/interstitial cystitis (PBS/IC) is a chronic urinary bladder disorder of unknown etiology characterized by symptoms of bladder pain and urinary frequency. PBS/IC is a chronic disease in which drug therapy has not led to significant success over the course of time. If the symptoms of PBS/IC are refractory to standard treatments, a possible cure might demand surgical intervention involving cystectomy. The eventual autoimmune etiology in mind, immunosuppressive drug therapy with cyclosporine A (CyA) was started to patients with refractory PBS/IC. CyA is a potent anti-inflammatory drug, a calcineurin inhibitor which inhibits T lymphocyte IL-2 produc-tion. T cells are present in abundance in inflammation of the bladder in PBS/IC. On the basis of a pilot, short-term study with CyA on PBS/IC, use of CyA was continued empirically over the long term. We conducted a prospective, randomized, six-month study in 64 patients comparing the effect of CyA with the FDA approved treatment, pentosan polysulfate sodium (PPS). We measured the drug effect on patient s symptoms, the potassium sensitivity test, and on urinary biomarkers. We further tested the impact of CyA, PPS, DMSO and BCG therapy on a health-related quality of life questionnaire and evaluated the response rate to treatment with these therapies. Long-term use of CyA was safe and effective in PBS/IC patients. The good clinical effect matured individually during the years in which CyA was continued. Cessation of medication led to the reappearance of symptoms, and restarting CyA to renewed alleviation, so that CyA was administered as continuous medication. The response rate to CyA increased during the study period, comprising 75% of CyA patients at six months. 19% of patients responded to PPS therapy. Adverse effects were more common in the CyA group, underlining the importance of monitoring the drug safety and appropriate titration of the dose. The potassium sensitivity test is positive in the majority of PBS/IC patients. Successful therapy of PBS/IC can alter nerve sensitivity to external potassium. This effect was seen more often after CyA therapy. Successful treatment of PBS/IC with CyA resulted to decreasing urinary levels of EGF. IL-6 levels in urine were higher among older patient with a longer history of PBS/IC. In these patients, reduced levels of urinary IL-6 were measured after CyA therapy. Patients who experience the best treatment response have improved quality of life according to the post-treatment health-related quality of life (HRQOL) questionnaire. CyA had more impact on the ma-jority of the aspects of QoL than PPS. Despite DMSO therapy being more successful than BCG in the count of responders, DMSO and BCG had equal effects on the HRQOL questionnaire.

Pathogen-specific TLR2 Protein Activation Programs Macrophages to Induce Wnt-beta-Catenin Signaling

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus non-pathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-beta-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-beta-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-beta-catenin signaling as iNOS(-/-) mice demonstrated compromised ability to trigger activation of Wnt-beta-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-beta-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T(Reg) cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-beta-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress Major histocompatibility complex II expression during Mycobacterium bovis Bacillus Calmette-Guerin infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guerin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-gamma-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Asymmetric cell division in Mycobacterium tuberculosis and its unique features

Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.

Combined inhalation and oral supplementation of Vitamin A and Vitamin D: A possible prevention and therapy for tuberculosis

Tuberculosis is continuing as a problem of mankind. With evolution, MDR and XDR forms of tuberculosis have emerged from drug sensitive strain. MDR and XDR strains are resistant to most of the antibiotics, making the management more difficult. BCG vaccine is not providing complete protection against tuberculosis. Therefore new infections are spreading at a tremendous rate. At the present moment there is experimental evidence to believe that Vitamin A and Vitamin D has anti-mycobacterial property. It is in this context, we have hypothesized a host based approach using the above vitamins that can cause possible prevention and cure of tuberculosis with minimal chance of resistance or toxicity. (C) 2015 Elsevier Ltd. All rights reserved.

Avaliação do consumo alimentar no período reprodutivo e variação de peso no pós-parto

O presente estudo tem como tema central o ganho de peso e o consumo alimentar no período reprodutivo. A tese está dividida em três partes: i) revisão da literatura científica sobre os fatores de risco para o ganho de peso excessivo na gestação e para a sua retenção no pós-parto; ii) análise quantitativa e qualitativa do consumo alimentar (energia, nutrientes e alimentos) do período gestacional para o pós-parto; e iii) avaliação prospectiva da associação entre a dieta hiperproteica (≥ 1.2 g/kg) e a variação de peso pós-gestacional. A revisão da literatura apontou como principais determinantes da variação de peso no pós-parto os seguintes fatores: ganho de peso gestacional, peso pré-gestacional, dieta, atividade física, lactação, idade, escolaridade, renda, paridade e raça. As partes ii e iii correspondem a dois artigos, sendo que a população do estudo empregada nas duas análises foi baseada em uma coorte de mulheres no pós-parto atendidas no Centro Municipal de Saúde (CMS) Marcolino Candau, localizado no Município do Rio de Janeiro. Dentre as 709 mulheres convidadas para participar da pesquisa, 479 ingressaram na coorte. As mulheres foram recrutadas através dos serviços de pré-natal e na rotina pediátrica de imunização do BCG, oferecidos pelo CMS, e imediatamente após o parto, na maternidade central de referência. A coleta de dados ocorreu entre maio de 1999 e abril de 2001, sendo 15 meses de recrutamento e 9 meses de seguimento. As mulheres foram entrevistadas aos 15 dias, 2, 6 e 9 meses do pós-parto. Foram consideradas elegíveis para as duas analises as mulheres com idade entre 18 e 45 anos de idade, entrevistadas até 30 dias do pós-parto, sem doenças crônicas pré-existentes, com gravidez de 35 ou mais semanas gestacionais e sem gestação gemelar. O estudo sobre a composição da dieta do período gestacional para o pós-parto contou com 276 mulheres que responderam aos dois questionários de freqüência de consumo alimentar (QFCA) aplicados aos 15 dias e aos 6 meses do seguimento. Os resultados mostraram que as mulheres consumiram dietas mais energéticas na gravidez e aquelas que mais restringiram o consumo de energia no pós-parto, apresentaram um incremento na densidade de proteínas da dieta. As análises das medidas repetidos do peso foram realizadas para avaliar o efeito da dieta hiperproteica (≥ 1.2 g/kg) sobre a variação de peso no pós-parto, para as quais haviam dados de uma amostra de 430 mulheres. Os resultados mostraram que as mulheres com dieta hiperproteica perderam mais peso do que as mulheres com dieta normo ou hipoproteica (< 1.2 g/kg) (226 g/mês versus 123 g/mês). As duas analises indicam que um pequeno incremento no consumo de proteínas promove maior perda de peso. Estudos longitudinais futuros devem incluir avaliação relativa a segurança das dietas hiperproteicas no pós-parto.

Estudo de aspectos epidemiológicos, clínicos e imunológicos da hanseníase em município hiperendêmico

A epidemiologia da hanseníase tem demonstrado a utilização de ferramentas clínicas, moleculares e imunológicas, para o mapeamento dos principais focos de ocorrência da doenças e de áreas de alto risco de adoecimento. Programas de controle da hanseníase, doença de longo período de incubação, em que os contatos são os principais grupos de risco, devem estabelecer todos os procedimentos disponíveis para o controle desse agravo. Objetiva-se estudar aspectos epidemiológicos clínicos e imunológicos da hanseníase em município hiperendêmico do Maranhão. Estudo de epidemiológico descritivo, realizado com 599 contatos, entre outubro de 2009 a outubro de 2011, no Serviço de Dermatologia no ambulatório do Hospital Universitário da Universidade Federal do Maranhão unidade Presidente Dutra São Luís - MA. Foi observado que a maioria dos contatos foi do gênero feminino com 57,17%, a faixa etária predominante acima de 15 anos, ou seja, (64,59%) com maior frequência na faixa de 20 a 59 anos, idade adulta e produtiva e o tipo de convívio predominante é o intradomiciliar com 74,95%, a forma clínica foi HT (30,00%), a classificação operacional mais frequente foi a MB com 61,90% do gênero feminino; a maioria apresentou somente uma cicatriz vacinal pela BCG, destacou-se o gênero masculino com 63,00%. O teste Elisa apresentou percentual de 63,50% e o ML-Flow 64,00%. Considerando o resultado dos testes Elisa e ML-FLOW sugere-se a utilização das técnicas imunológicas como ferramentas necessárias para o diagnóstico precoce, controle e vigilância dos contatos como forma de interromper a cadeia de transmissão da hanseníase na cidade pesquisada.

Esquemas terapêuticos no carcinoma da bexiga

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Baseline health-related quality-of-life data as prognostic factors in a phase III multicentre study of women with metastatic breast cancer.

The potential value of baseline health-related quality-of-life (HRQOL) and clinical factors in predicting prognosis was examined using data from an international randomised phase III trial which compared doxorubicin and paclitaxel with doxorubicin and cylophosphamide as first line chemotherapy in 275 women with metastatic breast cancer. The European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and the related breast module (QLQ-BR23) were used to assess baseline HRQOL data. The Cox proportional-hazards regression model was used for both univariate and multivariate analyses of survival. In the univariate analyses, performance status (P<0.001) and number of sites involved (P=0.001) were the most important clinical prognostic factors. The HRQOL variables at baseline most strongly associated with longer survival were better appetite, physical and role functioning, as well as less fatigue (P<0.001). The final multivariate model retained performance status (P<0.001) and appetite loss (P=0.005) as the variables best predicting survival. Substantial loss of appetite was the only independent HRQOL factor predicting poor survival and was strongly correlated (/r/>0.5) with fatigue, role and physical functioning. In addition to known clinical factors, appetite loss appears to be a significant prognostic factor for survival in women with metastatic breast cancer. However, the mechanism underlying this association remains to be precisely defined in future studies.

In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Heparin-binding-hemagglutinin-induced IFN-gamma release as a diagnostic tool for latent tuberculosis.

BACKGROUND: The detection of latent tuberculosis infection (LTBI) is a major component of tuberculosis (TB) control strategies. In addition to the tuberculosis skin test (TST), novel blood tests, based on in vitro release of IFN-gamma in response to Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 (IGRAs), are used for TB diagnosis. However, neither IGRAs nor the TST can separate acute TB from LTBI, and there is concern that responses in IGRAs may decline with time after infection. We have therefore evaluated the potential of the novel antigen heparin-binding hemagglutinin (HBHA) for in vitro detection of LTBI. METHODOLOGY AND PRINCIPAL FINDINGS: HBHA was compared to purified protein derivative (PPD) and ESAT-6 in IGRAs on lymphocytes drawn from 205 individuals living in Belgium, a country with low TB prevalence, where BCG vaccination is not routinely used. Among these subjects, 89 had active TB, 65 had LTBI, based on well-standardized TST reactions and 51 were negative controls. HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI. PPD-based tests yielded 90.00% sensitivity and 70.00% specificity for the detection of LTBI, whereas the sensitivity and specificity for the ESAT-6-based tests were 40.74% and 90.91%, and those for the HBHA-based tests were 92.06% and 93.88%, respectively. The QuantiFERON-TB Gold In-Tube (QFT-IT) test applied on 20 LTBI subjects yielded 50% sensitivity. The HBHA IGRA was not influenced by prior BCG vaccination, and, in contrast to the QFT-IT test, remote (>2 years) infections were detected as well as recent (<2 years) infections by the HBHA-specific test. CONCLUSIONS: The use of ESAT-6- and CFP-10-based IGRAs may underestimate the incidence of LTBI, whereas the use of HBHA may combine the operational advantages of IGRAs with high sensitivity and specificity for latent infection.