Harmful Effects of Granulocytic Myeloid-Derived Suppressor Cells on Tuberculosis Caused by Hypervirulent Mycobacteria.

BACKGROUND: The role of myeloid-derived suppressor cells (MDSCs) in patients with severe tuberculosis who suffer from uncontrolled pulmonary inflammation caused by hypervirulent mycobacterial infection remains unclear. METHODS: This issue was addressed using C57BL/6 mice infected with highly virulent Mycobacterium bovis strain MP287/03. RESULTS: CD11b+GR1int population increased in the bone marrow, blood and lungs during advanced disease. Pulmonary CD11b+GR1int (Ly6GintLy6Cint) cells showed granularity similar to neutrophils and expressed immature myeloid cell markers. These immature neutrophils harbored intracellular bacilli and were preferentially located in the alveoli. T-cell suppression occurred concomitantly with CD11b+GR1int cell accumulation in the lungs. Furthermore, lung and bone marrow GR1+ cells suppressed both T-cell proliferation and interferon γ production in vitro. Anti-GR1 therapy given when MDSCs infiltrated the lungs prevented expansion and fusion of primary pulmonary lesions and the development of intragranulomatous caseous necrosis, along with increased mouse survival and partial recovery of T-cell function. Lung bacterial load was reduced by anti-GR1 treatment, but mycobacteria released from the depleted cells proliferated extracellularly in the alveoli, forming cords and clumps. CONCLUSIONS: Granulocytic MDSCs massively infiltrate the lungs during infection with hypervirulent mycobacteria, promoting bacterial growth and the development of inflammatory and necrotic lesions, and are promising targets for host-directed therapies.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

PCR in the diagnosis of cutaneous tuberculosis

Seeking to improve the laboratory diagnosis of Cutaneous Tuberculosis, a study was carried out on the application of PCR technique in macerated, decontaminated(with 4% H2SO4 for elimination of normal microbiot), neutralized (with 4% NaOH)biopsies tissues samples stored at -20ºC. Of the 37 samples submitted for study, 16.22% were positive by microscopy for acid-fast bacilli (concentrated method) and in 43.24% the Mycobacterium tuberculosis was isolated in Löwenstein-Jensen medium. Using a M. tuberculosis complex specific primer set (gene sequence for 16S rDNA), the mycobacterial DNA was detected in 24.32% of the biopsies. The sensitivity and specificity of PCR were 43.7% and 90.4%, respectively. Due to low sensitivity and discrepant results between bacteriological techniques and PCR methodology, the samples were repeated in a new PCR with primers for the IS6110 target. The sensitivity and specificity of PCR for the IS6110 target obtained 100% in comparison with the culture method. The results confirm the effectiveness of PCR methodology using primers for the IS6110 gene sequence and permit the PCR method to be applied to frozen cutaneous biopsies sent by services that do not identify the M. tuberculosis by the biology molecular method.

Visando melhorar o diagnóstico laboratorial da Tuberculose Cutânea, foi realizado o estudo da aplicação da técnica de PCR em amostras de tecidos cutâneos macerados, descontaminados (com H2SO4 4% para eliminação da microbiota normal), neutralizados (com NaOH 4%) e armazenadas a -20ºC. Das 37 amostras submetidas ao estudo, 16,22% apresentavam baciloscopias positivas para bacilos álcool-ácidos resistentes (método concentrado) e em 43,24% houve o isolamento do Mycobacterium tuberculosis em meio de cultivo Löwenstein-Jensen. Utilizando-se de primers para a região 16S rDNA do M. tuberculosis, o DNA micobacteriano foi detectado em 24,32% das biópsias. A sensibilidade e especificidade da PCR foram 43,7% e 90,4%, respectivamente. Devido à baixa sensibilidade e resultados divergentes entre as técnicas bacteriológicas e PCR (para a seqüência 16S rDNA), as amostras foram repetidas em um novo PCR com primers para a região IS6110. Tanto a sensibilidade como a especificidade da PCR com primers para IS6110 alcançaram 100% em relação ao cultivo. Os resultados confirmam a eficácia da PCR utilizando primers para a seqüência IS6110 e oferecem a possibilidade da técnica ser aplicada em amostras congeladas enviadas por serviços que não identificam o M. tuberculosis por técnicas de biologia molecular.

Occurrence of low seric gammaglobulin levels in calves which received colostrum from nipple pail or mothers / Ocorrência de baixos níveis de gamaglobulina sérica em bezerros que receberam colostro no balde ou nas mães

Transfer of passive immunity in 24 calves fed with colostrum, was studied at the end of the first day of life. Results show that gammaglobulin concentrations in calver were: 2,90 ± 0,32 and 2,12 ± 0,91 for animals fed from mothers or nipple pail, respectively. Among the 24 calves, five presented problems in immunoglobulin absortion.

A transferência de imunidade passada a bezerros foi estudada através da determinação do nível sérico de gamaglobulina, em 20 bezerros do tipo "Mantiqueira" e 4 da raça "Holandesa", variedade malhada de preto. Após os exames laboratoriais do soro sanguíneo coletado às 24 horas de vida, encontrou-se concentração média de gamaglobulina de 2,90 ± 0,32 de soro para os animais que ficaram com as mães e 2,12 ± 0,91 para os alimentados no balde. Dentre os 24 bezerros, cinco apresentaram problemas de absorção de imunoglobulinas.

Occurrence of low seric gammaglobulin levels in calves which received colostrum from nipple pail or mothers / Ocorrência de baixos níveis de gamaglobulina sérica em bezerros que receberam colostro no balde ou nas mães

Transfer of passive immunity in 24 calves fed with colostrum, was studied at the end of the first day of life. Results show that gammaglobulin concentrations in calver were: 2,90 ± 0,32 and 2,12 ± 0,91 for animals fed from mothers or nipple pail, respectively. Among the 24 calves, five presented problems in immunoglobulin absortion.  

A transferência de imunidade passada a bezerros foi estudada através da determinação do nível sérico de gamaglobulina, em 20 bezerros do tipo "Mantiqueira" e 4 da raça "Holandesa", variedade malhada de preto. Após os exames laboratoriais do soro sanguíneo coletado às 24 horas de vida, encontrou-se concentração média de gamaglobulina de 2,90 ± 0,32 de soro para os animais que ficaram com as mães e 2,12 ± 0,91 para os alimentados no balde. Dentre os 24 bezerros, cinco apresentaram problemas de absorção de imunoglobulinas.