The role of neurotrophin polymorphisms and susceptibility to neural damage in leprosy.

OBJECTIVES: Mycobacterium leprae is able to infect Schwann cells leading to neural damage. Neurotrophins are involved in nervous system plasticity and impact neural integrity during diseases. Investigate the association between single nucleotide polymorphisms in neurotrophin genes and leprosy phenotypes, especially neural damage. DESIGN: We selected single nucleotide polymorphisms in neurotrophins or their receptors genes associated with neural disorders: rs6265 and rs11030099 of brain-derived neurotrophic factor (BDNF), rs6330 of BDNF, rs6332 in NT3 and rs2072446 of P75NTR. The association of genetic frequencies with leprosy phenotypes was investigated in a case-control study. RESULTS: An association of the BDNF single nucleotide polymorphism rs11030099 with the number of affected nerves was demonstrated. The "AA+AC" genotypes were demonstrated to be protective against nerve impairment. However, this variation does not affect BDNF serum levels. BDNF is an important factor for myelination of Schwann cells and polymorphisms in this gene can be associated with leprosy outcome. Moreover, rs11030099 is located in the binding region for micro-RNA (miRNA) 26a that could be involved in control of BDNF expression. We demonstrated different expression levels of this miRNA in polar forms of leprosy. CONCLUSION: Our findings demonstrate for the first time an association between the polymorphism rs11030099 in the BDNF gene and neural commitment in leprosy and may indicate a possible role of miRNA-26a acting synergistically to these genetic variants in neural damage development.

COX/iNOS dependence for angiotensin-II-induced endothelial dysfunction.

Vascular dysfunction induced by angiotensin-II can result from direct effects on vascular and inflammatory cells and indirect hemodynamic effects. Using isolated and functional cultured aortas, we aimed to identify the effects of angiotensin-II on cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) and evaluate their impact on vascular reactivity. Aortic rings from mice were incubated overnight in culture medium containing angiotensin-II (100 nmol/L) or vehicle to induce vascular disfunction. Vascular reactivity of cultured arteries was evaluated in a bath chamber. Immunofluorescence staining for COX-1 and COX-2 was performed. Nitric oxide (NO) formation was approached by the levels of nitrite, a NO end product, and using a fluorescent probe (DAF). Oxidative and nitrosative stress were determined by DHE fluorescence and nitrotyrosine staining, respectively. Arteries cultured with angiotensin-II showed impairment of endothelium-dependent relaxation, which was reversed by the AT1 receptor antagonist. Inhibition of COX and iNOS restored vascular relaxation, suggesting a common pathway in which angiotensin-II triggers COX and iNOS, leading to vasoconstrictor receptors activation. Moreover, using selective antagonists, TP and EP were identified as the receptors involved in this response. Endothelium-dependent contractions of angiotensin-II-cultured aortas were blunted by ibuprofen, and increased COX-2 immunostaining was found in the arteries, indicating endothelium release of vasoconstrictor prostanoids. Angiotensin-II induced increased reactive oxygen species and NO production. An iNOS inhibitor prevented NO enhancement and nitrotyrosine accumulation in arteries stimulated with angiotensin-II. These results confirm that angiotensin-II causes vascular inflammation that culminates in endothelial dysfunction in an iNOS and COX codependent manner.

The Aspergillus fumigatus Mucin MsbA Regulates the Cell Wall Integrity Pathway and Controls Recognition of the Fungus by the Immune System.

Aspergillus fumigatus is a filamentous fungus which causes invasive pulmonary aspergillosis in immunocompromised individuals. In fungi, cell signaling and cell wall plasticity are crucial for maintaining physiologic processes. In this context, Msb2 is an important signaling mucin responsible for activation of a variety of mitogen-activated protein kinase (MAPK)-dependent signaling pathways that regulate cell growth in several organisms, such as the cell wall integrity (CWI) pathway. Here, we aimed to characterize the MSB2 homologue in A. fumigatus Our results showed that MsbA plays a role in the vegetative and reproductive development of the fungus, in stress adaptation, and in resistance to antifungal drugs by modulating the CWI pathway gene expression. Importantly, cell wall composition is also responsible for activation of diverse receptors of the host immune system, thus leading to a proper immune response. In a model of acute Aspergillus pulmonary infection, results demonstrate that the ΔmsbA mutant strain induced less inflammation with diminished cell influx into the lungs and lower cytokine production, culminating in increased lethality rate. These results characterize for the first time the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis and an important regulator of virulence.IMPORTANCEAspergillus fumigatus is an opportunistic fungus with great medical importance. During infection, Aspergillus grows, forming hyphae that colonize the lung tissue and invade and spread over the mammal host, resulting in high mortality rates. The knowledge of the mechanisms responsible for regulation of fungal growth and virulence comprises an important point to better understand fungal physiology and host-pathogen interactions. Msb2 is a mucin that acts as a sensor and an upstream regulator of the MAPK pathway responsible for fungal development in Candida albicans and Aspergillus nidulans Here, we show the role of the signaling mucin MsbA in the pathogen A. fumigatus, as a core sensor for cell wall morphogenesis, fungal growth, and virulence. Moreover, we show that cell wall composition, controlled by MsbA, is detrimental for fungal recognition and clearance by immune cells. Our findings are important for the understanding of how fungal sensors modulate cell physiology.

Fetoscopic tracheal occlusion for severe congenital diaphragmatic hernia: retrospective study / Oclusão traqueal por fetoscopia em hérnia diafragmática congênita grave: estudo retrospectivo

Abstract Background and objectives: The temporary fetal tracheal occlusion performed by fetoscopy accelerates lung development and reduces neonatal mortality. The aim of this paper is to present an anesthetic experience in pregnant women, whose fetuses have diaphragmatic hernia, undergoing fetoscopic tracheal occlusion (FETO). Method: Retrospective, descriptive study, approved by the Institutional Ethics Committee. Data were obtained from medical and anesthetic records. Results: FETO was performed in 28 pregnant women. Demographic characteristics: age 29.8 ± 6.5; weight 68.64 ± 12.26; ASA I and II. Obstetric: IG 26.1 ± 1.10 weeks (in FETO); 32.86 ± 1.58 (reversal of occlusion); 34.96 ± 2.78 (delivery). Delivery: cesarean section, vaginal delivery. Fetal data: Weight (g) in the occlusion and delivery times, respectively (1045.82 ± 222.2 and 2294 ± 553); RPC in FETO and reversal of occlusion: 0.7 ± 0.15 and 1.32 ± 0.34, respectively. Preoperative maternal anesthesia included ranitidine and metoclopramide, nifedipine (VO) and indomethacin (rectal). Preanesthetic medication with midazolam IV. Anesthetic techniques: combination of 0.5% hyperbaric bupivacaine (5-10 mg) and sufentanil; continuous epidural predominantly with 0.5% bupivacaine associated with sufentanil, fentanyl, or morphine; general. In 8 cases, there was need to complement via catheter, with 5 submitted to PC and 3 to BC. Thirteen patients required intraoperative sedation; ephedrine was used in 15 patients. Fetal anesthesia: fentanyl 10-20 mg.kg-1 and pancuronium 0.1-0.2 mg.kg-1 (IM). Neonatal survival rate was 60.7%. Conclusion: FETO is a minimally invasive technique for severe congenital diaphragmatic hernia repair. Combined blockade associated with sedation and fetal anesthesia proved safe and effective for tracheal occlusion.

Resumo Justificativa e objetivos: A oclusão traqueal fetal temporária feita por meio da fetoscopia acelera o desenvolvimento pulmonar e reduz a mortalidade neonatal. O objetivo deste trabalho é apresentar experiência anestésica em gestantes cujos fetos eram portadores de hérnia diafragmática e foram submetidos à oclusão traqueal por fetoscopia (FETO). Método: Estudo retrospectivo, descritivo, aprovado pelo Comitê de Ética da Instituição. Os dados foram obtidos das fichas anestésicas e dos prontuários. Resultados: A FETO foi feita em 28 gestantes. Características demográficos: idade 29,8 ± 6,5; peso 68,64 ± 12,26; ASA I e II. Obstétricas: IG 26,1 ± 1,10 semana (na FETO); 32,86 ± 1,58 (desoclusão); 34,96 ± 2,78 (parto). Via de parto: cesárea, parto vaginal. Dados fetais: peso (g) nos momentos da oclusão e nascimento, respectivamente (1.045,82 ± 222,2 e 2294 ± 553); RPC na FETO e desoclusão: 0,7 ± 0,15 e 1,32 ± 0,34, respectivamente. Anestesia materna: pré-operatório incluiu ranitidina e metoclopramida; nifedipina (VO) e indometacina (retal). Medicação pré-anestésica com midazolam EV. Técnicas anestésicas: bloqueio combinado com bupivacaína 0,5% hiperbárica 5-10 mg associada ao sufentanil; peridural contínua predominantemente com bupivacaína 0,5% associada a sufentanil, fentanil ou morfina; geral. Em oito casos houve necessidade de complementação pelo cateter, cinco nas submetidas a PC e três a BC. No intraoperatório 13 pacientes necessitaram de sedação; efedrina foi usada em 15 pacientes. Anestesia fetal: fentanil 10 a 20 mg.kg-1 e pancurônio 0,1-0,2 mg.kg-1 (IM). A taxa de sobrevida neonatal foi de 60,7%. Conclusão: A FETO constitui técnica minimamente invasiva para correção de hérnia diafragmática congênita grave. O bloqueio combinado associado à sedação e anestesia fetal se mostrou seguro e eficaz para a oclusão traqueal.

Characterization of MicroRNA Expression Profiles and Identification of Potential Biomarkers in Leprosy.

Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy.

[Fetoscopic tracheal occlusion for severe congenital diaphragmatic hernia: retrospective study]. / Oclusão traqueal por fetoscopia em hérnia diafragmática congênita grave: estudo retrospectivo.

BACKGROUND AND OBJECTIVES: The temporary fetal tracheal occlusion performed by fetoscopy accelerates lung development and reduces neonatal mortality. The aim of this paper is to present an anesthetic experience in pregnant women, whose fetuses have diaphragmatic hernia, undergoing fetoscopic tracheal occlusion (FETO). METHOD: Retrospective, descriptive study, approved by the Institutional Ethics Committee. Data were obtained from medical and anesthetic records. RESULTS: FETO was performed in 28 pregnant women. Demographic characteristics: age 29.8±6.5; weight 68.64±12.26; ASA I and II. Obstetric: IG 26.1±1.10 weeks (in FETO); 32.86±1.58 (reversal of occlusion); 34.96±2.78 (delivery). Delivery: cesarean section, vaginal delivery. Fetal data: Weight (g) in the occlusion and delivery times, respectively (1045.82±222.2 and 2294±553); RPC in FETO and reversal of occlusion: 0.7±0.15 and 1.32±0.34, respectively. Preoperative maternal anesthesia included ranitidine and metoclopramide, nifedipine (VO) and indomethacin (rectal). Preanesthetic medication with midazolam IV. Anesthetic techniques: combination of 0.5% hyperbaric bupivacaine (5-10mg) and sufentanil; continuous epidural predominantly with 0.5% bupivacaine associated with sufentanil, fentanyl, or morphine; general. In 8 cases, there was need to complement via catheter, with 5 submitted to PC and 3 to BC. Thirteen patients required intraoperative sedation; ephedrine was used in 15 patients. Fetal Anesthesia: fentanyl 10 to 20mg·kg-1 and pancuronium 0,1-0,2mg·kg-1 (IM). Neonatal survival rate was 60.7%. CONCLUSION: FETO is a minimally invasive technique for severe congenital diaphragmatic hernia repair. Combined blockade associated with sedation and fetal anesthesia proved safe and effective for tracheal occlusion.

Characterization of MicroRNA expression profiles and identification of potential biomarkers in leprosy

Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy.

Putative virulence factors of Corynebacterium pseudotuberculosis FRC41: vaccine potential and protein expression.

BACKGROUND: Corynebacterium pseudotuberculosis, a facultative intracellular bacterial pathogen, is the etiological agent of caseous lymphadenitis (CLA), an infectious disease that affects sheep and goats and it is responsible for significant economic losses. The disease is characterized mainly by bacteria-induced caseous necrosis in lymphatic glands. New vaccines are needed for reliable control and management of CLA. Thus, the putative virulence factors SpaC, SodC, NanH, and PknG from C. pseudotuberculosis FRC41 may represent new target proteins for vaccine development and pathogenicity studies. RESULTS: SpaC, PknG and NanH presented better vaccine potential than SodC after in silico analyses. A total of 136 B and T cell epitopes were predicted from the four putative virulence factors. A cluster analysis was performed to evaluate the redundancy degree among the sequences of the predicted epitopes; 57 clusters were formed, most of them (34) were single clusters. Two clusters from PknG and one from SpaC grouped epitopes for B and T-cell (MHC I and II). These epitopes can thus potentially stimulate a complete immune response (humoral and cellular) against C. pseudotuberculosis. Several other clusters, including two from NanH, grouped B-cell epitopes with either MHC I or II epitopes. The four target proteins were expressed in Escherichia coli. A purification protocol was developed for PknG expression. CONCLUSIONS: In silico analyses show that the putative virulence factors SpaC, PknG and NanH present good potential for CLA vaccine development. Target proteins were successfully expressed in E. coli. A protocol for PknG purification is described.