Development of a flow cytometry method for detection of bovine multi-cytokines / 生物工程学报

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial­Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy. (AU)

Description of Pediatric Tuberculosis Evaluated in a Referral Center in Istanbul Turkey

PURPOSE: Diagnosis of tuberculosis (TB) in children is more challenging than in adults. This study aimed to describe demographical, clinical and laboratory findings of children diagnosed with tuberculosis in Turkey, including the issues of contact tracing, culture positivity and forms of the disease. MATERIALS AND METHODS: Clinical and laboratory data of 51 children with a mean age of 8.0+/-4.6 years who were diagnosed with TB were retrospectively reviewed. Main diagnostic tools included tuberculin skin test, chest X-ray, sputum/gastric aspirate culture with sensitivity testing, and direct microscopy for acid-fast bacilli on available samples. Clinical characteristics and outcomes of the patients were examined. RESULTS: Thirty-six (70.6%) children were diagnosed with intra-thoracic and 15 (29.4%) with extra-thoracic tuberculosis. Twenty-eight of the patients had a positive Bacillus Calmette-Guerin vaccine scar (28/51, 54.9%) and 23/51 (45.1%) had a positive tuberculin skin test. An adult TB contact was identified in 27 (52.9%) of the cases. On direct microscopy, acid-fast bacilli were found in nine (17.6%) patients and positive culture for Mycobacterium tuberculosis was found in 19 (37.3%). Drug resistance to isoniazid was detected in four (7.8%). One patient with nephrotic syndrome and miliary tuberculosis died during follow-up. All other patients responded well to the treatment. CONCLUSION: Focusing on active contact tracing among all household contacts of tuberculous cases may be helpful in early identification and controlling childhood disease, even in regions with low disease prevalence. Adopting a suspicious and proactive approach in this particular age group is warranted.