Myelination key factor krox-20 is downregulated in Schwann cells and murine sciatic nerves infected by Mycobacterium leprae.

Schwann cells (SCs) critically maintain the plasticity of the peripheral nervous system. Peripheral nerve injuries and infections stimulate SCs in order to retrieve homeostasis in neural tissues. Previous studies indicate that Mycobacterium leprae (ML) regulates the expression of key factors related to SC identity, suggesting that alterations in cell phenotype may be involved in the pathogenesis of neural damage in leprosy. To better understand whether ML restricts the plasticity of peripheral nerves, the present study sought to determine the expression of Krox-20, Sox-10, c-Jun and p75NTR in SC culture and mice sciatic nerves, both infected by ML Thai-53 strain. Primary SC cultures were stimulated with two different multiplicities of infection (MOI 100:1; MOI 50:1) and assessed after 7 and 14 days. Sciatic nerves of nude mice (NU-Foxn1nu ) infected with ML were evaluated after 6 and 9 months. In vitro results demonstrate downregulation of Krox-20 and Sox-10 along with the increase in p75NTR-immunolabelled cells. Concurrently, sciatic nerves of infected mice showed a significant decrease in Krox-20 and increase in p75NTR. Our results corroborate previous findings on the interference of ML in the expression of factors involved in cell maturation, favouring the maintenance of a non-myelinating phenotype in SCs, with possible implications for the repair of adult peripheral nerves.

Myelination key factor krox­20 is downregulated in Schwann cells and murine sciatic nerves infected by Mycobacterium leprae

Schwann cells (SCs) critically maintain the plasticity of the peripheral nervous system. Peripheral nerve injuries and infections stimulate SCs in order to retrieve homeostasis in neural tissues. Previous studies indicate that Mycobacterium leprae (ML) regulates the expression of key factors related to SC identity, suggesting that alterations in cell phenotype may be involved in the pathogenesis of neural damage in leprosy. To better understand whether ML restricts the plasticity of peripheral nerves, the present study sought to determine the expression of Krox­20, Sox­10, c­Jun and p75NTR in SC culture and mice sciatic nerves, both infected by ML Thai­53 strain. Primary SC cultures were stimulated with two different multiplicities of infection (MOI 100:1; MOI 50:1) and assessed after 7 and 14 days. Sciatic nerves of nude mice (NU­Foxn1nu) infected with ML were evaluated after 6 and 9 months. In vitro results demonstrate downregulation of Krox­20 and Sox­10 along with the increase in p75NTR­immunolabelled cells. Concurrently, sciatic nerves of infected mice showed a significant decrease in Krox­20 and increase in p75NTR. Our results corroborate previous findings on the interference of ML in the expression of factors involved in cell maturation, favouring the maintenance of a non­myelinating phenotype in SCs, with possible implications for the repair of adult peripheral nerves(AU).

An enhanced regimen as post-exposure chemoprophylaxis for leprosy: PEP++

The ongoing transmission of Mycobacterium (M.) leprae reflected in a very slow decline in leprosy incidence, forces us to be innovative and conduct cutting-edge research. Single dose rifampicin (SDR) as post-exposure prophylaxis (PEP) for contacts of leprosy patients, reduces their risk to develop leprosy by 60%. This is a promising new preventive measure that can be integrated into routine leprosy control programmes, as is being demonstrated in the Leprosy Post-Exposure Programme that is currently ongoing in eight countries.The limited (60%) effectiveness of SDR is likely due to the fact that some contacts have a preclinical infection beyond the early stages for which SDR is not sufficient to prevent the development of clinical signs and symptoms of leprosy. An enhanced regimen, more potent against a higher load of leprosy bacteria, would increase the effectiveness of this preventive measure significantly.The Netherlands Leprosy Relief (NLR) is developing a multi-country study aiming to show that breaking the chain of transmission of M. leprae is possible, evidenced by a dramatic reduction in incidence. In this study the assessment of the effectiveness of an enhanced prophylactic regimen for leprosy is an important component. To define the so called PEP++ regimen for this intervention study, NLR convened an Expert Meeting that was attended by clinical leprologists, public health experts, pharmacologists, dermatologists and microbiologists.The Expert Meeting advised on combinations of available drugs, with known efficacy against leprosy, as well as on the duration of the intake, aiming at a risk reduction of 80-90%. To come to a conclusion the Expert Meeting considered the bactericidal, sterilising and bacteriostatic activity of the potential drugs. The criteria used to determine an optimal enhanced regimen were: effectiveness, safety, acceptability, availability, affordability, feasibility and not inducing drug resistance.The Expert Meeting concluded that the enhanced regimen for the PEP++ study should comprise three standard doses of rifampicin 600 mg (weight adjusted when given to children) plus moxifloxacin 400 mg given at four-weekly intervals. For children and for adults with contraindications for moxifloxacin, moxifloxacin should be replaced by clarithromycin 300 mg (weight adjusted).

Estudo clínico, epidemiológico e imunológico para leishmaniose tegumentar americana em centro de referência em dermatologia / Clinical, epidemiological and immunological study to american tegumentary leishmaniasis in a dermatology reference center

Leishmaniose Tegumentar Americana (LTA) é uma doença infecciosa, causada por protozoários do gênero Leishmania. É uma das doenças infectoparasitárias mais incidentes no mundo. No presente trabalho realizou-se um estudo transversal retrospectivo das características clínicas, epidemiológicas e imunológicas de portadores de Leishmaniose Tegumentar Americana.Foram utilizados prontuários de 34 pacientes com diagnóstico de LTA. A análise estatística foi realizada pelo Teste de Spearman. O sexo masculino foi acometido em 68% e o feminino 32%. A idade variou de 1 a 92 anos. A forma cutânea localizada ocorreu em 79,5%,sendo as úlceras a forma clínica mais comum (56%).Principal área acometida foi face (44%). O tempo para o diagnóstico foi menor que 10 meses em 68% dos indivíduos.Intradermorreação de Montenegro (IDRM) foi realizada em 29 pacientes, com positividade em 89,6% e a imunofluorescência indireta (IFI) em apenas 16 pacientes, sendo positiva em 13. A idade e o tempode evolução da doença apresentaram associação significativa com IDRM. Entretanto não foi observada associação da IFI com a idade do paciente e o tempo de doença, pelo teste de Spearman. O tratamento foi realizado na maioria dos casos com glucantime (71%),seguido de pentamidina (17%). Os resultados evidenciam que os exames sorológicos constituem uma ferramenta auxiliar e a correlação com achados clínicos e histopatológicos são imprescindíveis.

Introduction: American cutaneous leishmaniasis (ACL) is an infectious disease caused by protozoa of the genus Leishmania. World  leishmaniasis  is an important endemic disease and public health problem in developing countries. Methods: We conducted a retrospective, descriptive and analytical cross-sectional study of 34 patients diagnosed with ACL. Statistical analysis was performed using the nonparametric Spearman’s test. Results: The gender involved was male (68%) and female(32%); the age range of 1 to 92 years old. The most common clinical manifestations were localized cutaneous form (79.5%) and the ulcers (56%).The face was main affected area (44%) and the minor time from onset of symptoms to consultation was 10 months (68%) of patients. Montenegro skin test (MST) was performed in 29 patients, being positive in (89.6%) and the indirect immunofluorescence (IIF) in only 16 patients, being positive in 13. The age and the duration of the disease were significantly associated with MST. Conclusions: It was not observed the IFI association with the patient’s age and disease duration. The treatment was in most cases, meglumine antimoniate (71%), followed Pentamidine (17%). The results demonstrated thatthe serological tests constitute an auxiliary tool andthe correlations with clinical and histopathological findings are essential.

Statins increase rifampin mycobactericidal effect

Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation.

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Increased hepcidin expression in multibacillary leprosy

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

Estudo comparativo entre escores de dor após uso de duas preparações de lidocaína tópica / Comparing the pain ratings of two topical lidocaine preparations

Introdução: Os procedimentos cosmiátricos têm aumentado, e com eles, a busca de analgesia eficiente e segura. Os anestésicos tópicos são opção às anestesias infiltrativas, devendo promover analgesia adequada e atuar na pele íntegra, sem induzir efeitos adversos.Objetivo: Comparar os escores de dor entre duas formulações tópicas de lidocaína, em pacientes submetidos à terapia com laser fracionado de CO2.Métodos: Oito pacientes foram submetidos a uma sessão de laser de CO2 fracionado, após a aplicação de formulação industrializada de lidocaína 4% na hemiface direita e formulação magistral de lidocaína 30% associada à tetracaina 7% na hemiface esquerda. A intensidade da dor foi avaliada através da escala visual analógica de dor (EVA) no final do procedimento.Resultados: Os anestésicos tópicos, nas formulações magistral e industrializada, não apresentaram diferença estatisticamente significativa na avaliação dos escores de dor. Conclusões: Os dados sugerem que fórmulas com grande concentração de anestésicos não são mais eficientes em produzir analgesia do que as formulações industrializadas.

Differential effects of neuromuscular blockers on twitches and tetani in the isolated rat muscle: a multiple comparison study using simultaneous confidence intervals

In the present work a comparative quantitative evaluation of the differential effects of neuromuscular blockers on twitches and tetani was performed, encompassing atracurium, cisatracurium, mivacurium, pancuronium, rocuronium and vecuronium. The sciatic nerve-extensor digitorum longus muscle of the rat was used, in vitro. Twitches were evoked at 0.1 Hz and tetani at 50 Hz. The differential effects of the studied compounds on twitches and tetani were statistically compared using simultaneous confidence intervals for the ratios between mean IC(50) for the block of twitches and mean IC(50) for the block of tetani. The results of ratios of mean IC(50) together with their corresponding 95% simultaneous confidence intervals were vecuronium 2.5 (1.8-3.5); mivacurium 3.8 (3.0-4.9); pancuronium 3.9 (2.0-7.6); rocuronium 6.1 (3.8-9.9); atracurium 9.0 (6.4-12.6); cisatracurium 13.1 (6.0-28.4). Using the criteria that neuromuscular blockers displaying disjunct confidence intervals for the ratios of mean IC(50) differ statistically with regard to differential effects on twitches and tetani, significant differences in ratios of IC(50) were detected in the following cases vecuronium vs. rocuronium, vs. atracurium and vs. cisatracurium and mivacurium vs cisatracurium and vs. atracurium. The results show that the magnitude of the differential effects of neuromuscular blockers on twitches and tetani, as evaluated in the present work in the form of ratios of mean IC(50), does not depend on the chemical structure (comparing steroidal and isoquinolinic compounds), but seems to depend on differential pre- and post-synaptic effects of the compounds. It is also suggested that the greater the ability of a compound to block twitches and tetani in a differential manner, the safer is the compound from the clinical anesthesiology viewpoint.

Cellular mechanisms of atracurium-induced tetanic fade in the isolated rat muscle

Although atracurium is a widely used neuromuscular blocker, we still lack knowledge regarding some of its cellular mechanisms of action. Thus, similar to other clinically used blockers atracurium induces, both in vivo and in vitro, fade of the tetanic contraction. However, the cellular mechanisms underlying this tetanic fade have never been systematically studied. In the present work these mechanisms were investigated in vitro. A sciatic nerve extensor digitorum longus muscle preparation of the rat was used. A combination of myographical and electrophysiological techniques was employed. Indirect twitches were evoked at 0.1 Hz and tetanic contractions at 50 Hz. Trains of end-plate potentials were evoked at a frequency of 50 Hz. The electrophysiological variables used in the analysis of the trains of end-plate potentials were peak amplitude of the first end-plate potential in the train, peak amplitude of plateau end-plate potentials in the train, tetanic run-down of the end-plate potentials' train, quantal content of first and plateau end-plate potentials in the train, quantal size. In the myographical study atracurium, at a concentration of 2.4 microM, induced a complete fade of the tetanic contraction while only slightly affected the twitch. In the electrophysiological study atracurium, at the same 2.4 microM concentration, significantly decreased the amplitude of both first end-plate potentials in the train (control 14.4 mV; atracurium 3.2 mV) and plateau end-plate potentials (control 10.8 mV; atracurium 2.4 mV) and reinforced the tetanic run-down of the train of end-plate potentials, evaluated as the percent loss in amplitude of plateau end-plate potentials compared to first end-plate potentials in the trains (control 25.2%; atracurium 33.2%). Atracurium also significantly decreased the quantal content of first end-plate potentials in the train (control 231; atracurium 68), the quantal content of plateau end-plate potentials (control 159; atracurium 42) and the quantal size (control 0.119 mV; atracurium 0.075 mV). In relative terms the decrease in quantal content was about twice as large as the decrease in quantal size. This indicates that the fade of the tetanic contraction induced by atracurium (2.4 microM) is due to both pre- and postsynaptic blocking effects, the presynaptic one being stronger.

Ação do etanol na fertilidade em fêmeas da espécie Rattus norvegicus albinus e seus possíveis efeitos embriofetotóxicos / Ethanol action on fertility in female Rattus norvegicus albinus species and its possible embryofoetotoxic effects

O etanol ou álcool etílico é um depressor do sistema nervoso central e, em doses altas, é também um agente anestésico. Seu consumo aumentou significantemente nas últimas décadas, particularmente nos países do terceiro mundo. No Brasil, assim como na maioria dos países ocidentais, o etanol, em suas diversas preparações, é uma das substâncias psicoativas mais amplamente utilizadas. O consumo de álcool durante a gestação pode provocar no feto humano uma síndrome de deficiência de crescimento, conhecida como Síndrome Alcoólica Fetal, caracterizada por disfunção neurológica e anomalias faciais, além do aumento de complicações obstétricas. Neste estudo, procuramos avaliar a ação do etanol na fertilidade materna, nos pesos das mães e dos fetos e na incidência de pontos de reabsorção, utilizando como modelo experimental, fêmeas prenhes da espécie Rattus norvegicus albinus, que foram submetidas à ingestão de etanol nas doses de 3,2 e 6,4 mg/peso. Nossos resultados indicam que a administração de etanol em ratas prenhes levou a uma redução do peso médio das placentas, dos fetos e do número de fetos vivos por ninhada, além de aumentar o número de ponbtos de reabsorção. Esses efeitos foram observados nas doses estudadas, que não foram suficientes para causar uma toxicidade materna, visto que não houve diminuição do ganho de peso nas ratas tratadas com etanol durante a gestação.

Antagonism produced by hypertonic nutrient solutions on the depressive effect of carbofuran on isolated guinea pig atria

The action of hyperosmotic nutrient solutions on the depressive effects of carbofuran was studied on spontaneously contracting guinea pig atria. The force and frequency of contraction were recorded using an isotonic lever. Carbofuran 33.0 micrograms ml-1 produced a gradual depressive effect. The time for stabilization of the depressive effect was 6.0 +/- 2.3 min. After the depressive effect of carbofuran was established, NaCl, mannitol, or urea were introduced into the organ bath to increase the osmolarity of the nutrient solution to about 100 mosmol kg-1 of water. This resulted in a reversal of the contraction force and frequency to the initial levels. The addition of the osmotic agents at the same concentrations before the addition of 33.0 micrograms ml-1 carbofuran avoided its depressive effect on guinea pig atria.