Stem Cells from Human Exfoliated Deciduous Teeth (SHED) Differentiate in vivo and Promote Facial Nerve Regeneration.

Post-traumatic lesions with transection of the facial nerve present limited functional outcome even after repair by gold-standard microsurgical techniques. Stem cell engraftment combined with surgical repair has been reported as a beneficial alternative. However, the best association between the source of stem cell and the nature of conduit, as well as the long-term postoperative cell viability are still matters of debate. We aimed to assess the functional and morphological effects of stem cells from human exfoliated deciduous teeth (SHED) in polyglycolic acid tube (PGAt) combined with autografting of rat facial nerve on repair after neurotmesis. The mandibular branch of rat facial nerve submitted to neurotmesis was repaired by autograft and PGAt filled with purified basement membrane matrix with or without SHED. Outcome variables were compound muscle action potential (CMAP) and axon morphometric. Animals from the SHED group had mean CMAP amplitudes and mean axonal diameters significantly higher than the control group ( p < 0.001). Mean axonal densities were significantly higher in the control group ( p = 0.004). The engrafted nerve segment resected 6 weeks after surgery presented cells of human origin that were positive for the Schwann cell marker (S100), indicating viability of transplanted SHED and a Schwann cell-like phenotype. We conclude that regeneration of the mandibular branch of the rat facial nerve was improved by SHED within PGAt. The stem cells integrated and remained viable in the neural tissue for 6 weeks since transplantation, and positive labeling for S100 Schwann-cell marker suggests cells initiated in vivo differentiation.

Evaluation of a recombinant Trypanosoma cruzi mucin-like antigen for serodiagnosis of Chagas' disease.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and is one of the most important endemic problems in Latin America. Lately, it has also become a health concern in the United States and Europe. Currently, a diagnosis of Chagas' disease and the screening of blood supplies for antiparasite antibodies are achieved by conventional serological tests that show substantial variation in the reproducibility and reliability of their results. In addition, the specificity of these assays is curtailed by antigenic cross-reactivity with sera from patients affected by other endemic diseases, such as leishmaniasis. Here we used a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to evaluate a recombinant protein core of a mucin-like molecule (termed trypomastigote small surface antigen [TSSA]) for the detection of specific serum antibodies in a broad panel of human sera. The same samples were evaluated by CL-ELISA using as the antigen either a mixture of native T. cruzi trypomastigote mucins or an epimastigote extract and, for further comparison, by conventional serologic tests, such as an indirect hemagglutination assay and indirect immunofluorescence assay. TSSA showed ∼87% sensitivity among the seropositive Chagasic panel, a value which was increased up to >98% when only parasitologically positive samples were considered. More importantly, TSSA showed a significant increase in specificity (97.4%) compared to those of currently used assays, which averaged 80 to 90%. Overall, our data demonstrate that recombinant TSSA may be a useful antigen for the immunodiagnosis of Chagas' disease.

False-positive results of a rapid K39-based strip test and Chagas disease.

BACKGROUND: The definitive diagnosis of visceral leishmaniasis (VL) requires invasive procedures with demonstration of amastigotes in tissue or promastigotes in culture. Unfortunately, these approaches require laboratory materials not available in poor countries where the disease is endemic. The correct diagnosis of VL is important, and made more difficult by the fact that several common tropical diseases such as malaria, disseminated tuberculosis, and enteric fever share the same clinical presentation. Serological tests have been developed to replace parasitological diagnosis in the field. A commercially available K39-based strip test for VL has been developed for this purpose. The endemic area of leishmaniasis in Brazil overlaps the endemic area of Chagas disease, a disease that can cause false-positive serological test results. The aim of this study was to evaluate the incidence of false-positive exams using a rapid test for VL in patients with Chagas disease. METHODS: A rapid test based on the recombinant K39 antigen of Leishmania was used in: (1) 30 patients with confirmed Chagas disease, (2) 30 patients with a serological diagnosis of Chagas disease by ELISA, indirect immunofluorescence, indirect hemagglutination, and chemiluminescence, (3) 30 healthy patients from a non-endemic area as the control group, (4) 30 patients with confirmed VL, and (5) 20 patients with proved cutaneous leishmaniasis. RESULTS: The sensitivity and specificity of the rapid strip test were 100% when compared with healthy volunteers and those with confirmed Chagas disease. One false-positive result occurred in the group with Chagas disease diagnosed by serological tests (specificity of 96%). CONCLUSION: The rapid test based on recombinant K39 is a useful diagnostic assay, and a false-positive result rarely occurs in patients with a serological diagnosis of Chagas disease.

Avaliação da resposta imune humoral frente a antígenos de Strongyloides venezuelensis / Evaluation of the humoral immune response to Strongyloides venezuelensis antigens

A estrongiloidíase afeta 30 milhões de pessoas em 70 países. Usualmente, o diagnóstico dessa enteroparasitose é realizado por testes parasitológicos baseados no hidro termotropismo das larvas eliminadas nas fezes, porém esses têm se mostrado pouco sensíveis. Neste trabalho, extratos antigênicos foram testados pelas técnicas de ELISA, Immunoblotting e IFI, utilizando larvas filarióides de Strongyloides venezuelensis, parasita de roedores, que mostram reação cruzada com epítopos de Strongyloides stercoralis. Sensibilidade de 89, 85, 57 por cento para a reação de ELISA e de 100, 100 e 96 por cento, para o Immunoblotting com os antígenos SAL, ZWIP e ZW, e especificidade de 90, 60 e 81 por cento para o ELISA e 96, 92 e 91 por cento para o Immunoblotting para os mesmos antígenos, foram encontradas nestes ensaios.

Strongyloidiasis affects 30 million people in 70 countries. This enteral parasitosis is usually diagnosed using parasitological tests based on hydrotropism or thermotropism of larvae eliminated in feces, but these tests have been shown to have low sensitivity. In this study, antigenic extracts were tested by means of ELISA, immunoblotting and IFI, using filariform larvae of Strongyloides venezuelensis, a parasite of rodents that shows cross-reactions with Strongyloides stercoralis epitopes. Sensitivity of 89, 85 and 57 percent for the ELISA reaction and 100, 100 and 96 percent for immunoblotting with the SAL, ZWIP and ZW antigens, and specificity of 90, 60 and 81 percent for ELISA and 96, 92 and 91 percent for immunoblotting with the same antigens, were found in these assays.

[Evaluation of the humoral immune response to Strongyloides venezuelensis antigens]. / Avaliação da resposta imune humoral frente a antígenos de Strongyloides venezuelensis.

Strongyloidiasis affects 30 million people in 70 countries. This enteral parasitosis is usually diagnosed using parasitological tests based on hydrotropism or thermotropism of larvae eliminated in feces, but these tests have been shown to have low sensitivity. In this study, antigenic extracts were tested by means of ELISA, immunoblotting and IFI, using filariform larvae of Strongyloides venezuelensis, a parasite of rodents that shows cross-reactions with Strongyloides stercoralis epitopes. Sensitivity of 89, 85 and 57% for the ELISA reaction and 100, 100 and 96% for immunoblotting with the SAL, ZWIP and ZW antigens, and specificity of 90, 60 and 81% for ELISA and 96, 92 and 91% for immunoblotting with the same antigens, were found in these assays.

[Behavior of the chemiluminescent ELISA method in relation to results considered discordant via three conventional techniques for diagnosing Chagas disease]. / Comportamento do método quimioluminescente-ELISA em relação a resultados considerados discordantes por meio de três técnicas convencionais para diagnóstico da doença de Chagas.

When indirect hemagglutination, indirect immunofluorescence and enzyme-linked immunosorbent assay are used together for serologically diagnosing Chagas disease, results that are considered discordant sometimes occur because there is disagreement between what these tests indicate. The availability of the chemiluminescent ELISA method enabled tests on 200 serum samples that had previously produced discordant results from the three above-mentioned methods. CL-ELISA revealed that 193 of these samples were negative and seven were positive. The use of this new procedure provides further support for understanding this subject, but more concrete advances will depend on documentation with blood analyses from people previously demonstrated to be unquestionably infected or uninfected with Trypanosoma cruzi.

Comportamento do método quimioluminescente-ELISA em relação a resultados considerados discordantes por meio de três técnicas convencionais para diagnóstico da doença de Chagas / Behavior of the chemiluminescent ELISA method in relation to results considered discordant via three conventional techniques for diagnosing Chagas disease

Quando utilizadas, em conjunto, a hemaglutinação indireta, a imunofluorescência indireta e ELISA para diagnóstico sorológico da doença de Chagas por vezes ocorrem resultados considerados discordantes, por não haver concordância entre o que indicam essas técnicas. A disponibilidade do método quimioluminescente-ELISA permitiu executá-lo com 200 soros que examinados pelos três testes citados que motivaram a obtenção de resultados discordantes. Com o método quimioluminescente-ELISA sucederam 193 negativos e sete positivos. O emprego desse novo procedimento trouxe mais um subsídio para compreensão do assunto, mas avanço mais concreto dependerá de documentação com soros de pessoas infectadas ou não pelo Trypanosoma cruzi conforme comprovação parasitológica.

When indirect hemagglutination, indirect immunofluorescence and enzyme-linked immunosorbent assay are used together for serologically diagnosing Chagas disease, results that are considered discordant sometimes occur because there is disagreement between what these tests indicate. The availability of the chemiluminescent ELISA method enabled tests on 200 serum samples that had previously produced discordant results from the three abovementioned methods. CL-ELISA revealed that 193 of these samples were negative and seven were positive. The use of this new procedure provides further support for understanding this subject, but more concrete advances will depend on documentation with blood analyses from people previously demonstrated to be unquestionably infected or uninfected with Trypanosoma cruzi.

Observacões sobre o TESA blot no diagnóstico sorológico da doenca de Chagas / Observations on the use of TESA blot for the serological diagnosis of Chagas' disease

Comparamos o TESA blot com a hemaglutinacão indireta, imunofluorescência indireta e ELISA. Nos 30 soros de pessoas infectadas pelo Trypanosoma cruzi, as quatro técnicas foram positivas em todos, e nos 30 não-infectados, totalmente negativas. Nos soros indeterminados e nos de leishmaniose visceral, comprovamos muitas positividades falsas, em quantidade bastante menor com o TESA blot.

[Observations on the use of TESA blot for the serological diagnosis of Chagas' disease]. / Observações sobre o TESA blot no diagnóstico sorológico da doença de Chagas.

TESA blot was compared with indirect hemagglutination, indirect immunofluorescence and ELISA tests. In sera from 30 participants infected with Trypanosoma cruzi, and in 30 non infected the four techniques produced entirely equivalent results, all positive and all negative, respectively. In cases admitted to be inconclusive or in visceral leishmaniasis, frequent false positives were detected. However, TESA blot contributed with the least proportion of them.

Avaliação rotineira de prova de execução rápida (ELISA - ImmunoComb II Chagas Ab) para diagnóstico sorológico da doença de Chagas / An evaluation of a quick test for the serological diagnosis of Chagas disease (ELISA - ImmunoComb II Chagas Ab) in routine work

Objetivo. Avaliar, em atividade rotineira, o desempenho de prova rápida (ELISA - ImmunoComb II Chagas Ab - EIC) para o diagnóstico sorológico da doença de Chagas. Métodos. Foi realizada comparação com outros testes habitualmente utilizados: hemaglutinação indireta - HI; imunofluorescência indireta - IFI; ELISA. Quatro grupos de soros compuseram a casuística: de pessoas com com doença de Chagas confirmada sorológica e parasitologicamente; de pessoas sem doença de Chagas, conforme verificação sorológica; correspondentes a resultados tidos como indeterminados quando efetuadas HI, IFI e ELISA; de pessoas com leishmaniose visceral, para apreciação de especificidade. Resultados. Os indicados por EIC mostraram-se totalmente iguais, em termos de positividade ou negatividade, aos obtidos por HI, IFI e ELISA quanto aos soros dos dois grupos primeiro citados. Em relação a indeterminados e a pacientes com leishmaniose visceral, com o EIC sucederam menos duvidosos ou falsos-positivos, mas algumas inadequações ainda persistiram, mantendo obstáculos à obtenção de diagnóstico plenamente aceitável. Conclusões: A prova EIC propiciou resultados absolutamente idênticos aos derivados de HI, IFI e ELISA quando analisados soros de pessoas com doença de Chagas ou não, de acordo com apreciações sorológicas ou confirmatórias da presença de infecção pelo Trypanosoma cruzi. Contudo, considerando os indeterminados e os de pacientes com leishmaniose visceral, o EIC não eliminou inadequações, apesar de tê-las comprovado com menores prevalências.

The surface coat of the mammal-dwelling infective trypomastigote stage of Trypanosoma cruzi is formed by highly diverse immunogenic mucins.

A thick coat of mucin-like glycoproteins covers the surface of Trypanosoma cruzi and plays a crucial role in parasite protection and infectivity and host immunomodulation. The appealing candidate genes coding for the mucins of the mammal-dwelling stages define a heterogeneous family termed TcMUC, which comprises up to 700 members, thus precluding a genetic approach to address the protein core identity. Here, we demonstrate by multiple approaches that the TcMUC II genes code for the majority of trypomastigote mucins. These molecules display a variable, non-repetitive, highly O-glycosylated central domain, followed by a short conserved C terminus and a glycosylphosphatidylinositol anchor. A simultaneous expression of multiple TcMUC II gene products was observed. Moreover, the C terminus of TcMUC II mucins, but not their central domain, elicited strong antibody responses in patients with Chagas' disease and T. crusi infected animals. This highly diverse coat of mucins may represent a refined parasite strategy to elude the mammalian host immune system.

[Evaluation of the sensitivity of the indirect immunofluorescence test at the 1:20 dilution in the diagnosis of Chagas disease]. / Avaliação da sensibilidade da diluição 1 / 20 pela reação de imunofluorescência indireta, no diagnóstico sorológico da doença de Chagas.

Sera from people in the chronic stage of Chagas disease, whose infection had been parasitologically validated, were assayed by using the indirect immunofluorescence test to evaluated its performance at the 1:20 dilution. All tests were consistently positive at 1:20 and higher dilutions, even in the presence of concomitant infection with the human immunodeficiency virus (HIV). It is thus valid, into the light of this experiment, to take into account the remarkable sensitivity of such serological test at the above mentioned dilution.

Avaliaçäo da sensibilidade da diluiçäo 1/20 pela reaçäo de imunofluorescência indireta, no diagnóstico sorológico da doença de Chagas / Evaluation of the sensitivity of the indirect immunofluorescence test at the 1 / 20 dilution in the diagnosis of Chagas disease

Soros de pessoas com doença de Chagas em fase crônica, comprovada parasitologicamente, foram utilizados para realizaçäo de reaçäo de imunofluorescência indireta, a fim de avaliar o comportamento da prova na diluiçäo 1/20. Sistematicamente houve positividade nessa diluiçäo e em maiores, mesmo quando presente co-infecçäo pelo vírus da imunodeficiência humana (HIV). Portanto, é válido valorizar resultados positivos na diluiçäo citada, que a propósito da casuística analisada demonstrou expressiva sensibilidade

A Trypanosoma cruzi small surface molecule provides the first immunological evidence that Chagas' disease is due to a single parasite lineage.

Chagas' disease is a major health and economic problem caused by the protozoan Trypanosoma cruzi. Multiple independently evolving clones define a complex parasite population that can be arranged into two broad genetic lineages termed T. cruzi I and II. These lineages have different evolutionary origin and display distinct ecological and biological traits. Here we describe a novel molecule termed TSSA for trypomastigote small surface antigen that provides the first immunological marker allowing discrimination between lineages. TSSA is a surface, glycosylphosphatidyl inositol (GPI)-anchored mucin-like protein, highly antigenic during the infection. TSSA sequences from different parasite isolates reveal a population dimorphism that perfectly matches with the two T. cruzi lineages. Interestingly, this dimorphism is restricted to the central region of the molecule, which comprises the immunodominant B cell epitopes. This sequence variability has a major impact on TSSA antigenicity, leading to no immunological cross-reactivity between both isoforms for antibodies present either in immunization or infection sera. Furthermore, the absolute seroprevalence for TSSA in confirmed Chagasic patients is restricted to T. cruzi II isoform, strongly suggesting that human infections are due to this particular subgroup. Even though association of T. cruzi II with Chagas' disease has been proposed based on molecular markers, this is the first immunological evidence supporting this hypothesis. The implications of these results for the future research on Chagas' disease could be envisaged.

Trypanosoma cruzi parasitemia observed in immunocompromised patients: the importance of the artificial xenodiagnosis

Trypanosoma cruzi parasitemia observed in immunocompromised patients (transplant or positive HIV) occurred more frequently by the artificial xenodiagnosis method (10/38) compared with hemoculture (2/38), given the same quantity of blood. Other ways of diagnosis, like mice inoculation (5/38), QBC and buffy coat (2/38), were evaluated also. This result showed the importance of the artificial xenodiagnosis. The other techniques increased only one more patient positive

Avaliaçäo, em modelo experimental, da possibilidade do alopurinol impedir reativaçäo da infecçäo pelo Trypanosoma cruzi na vigência de imunodepressäo / Experimental model for the evaluation of the allopurinol as an agent to avoid the reactivation of Trypanosoma cruzi infection during immunodepression

Pacientes com a doença de Chagas na fase crônica têm sido tratados por meio de transplante de coraçäo, em determinadas circunstâncias, e em virtude de imunodepressäo pode ocorrer reativaçäo da infecçäo devida ao Trypanosoma cruzi. Foi verificado que o alopurinol mostrou-se apto a arrefecer esse acontecimento. Por isso, utilizando modelo experimental, procuramos verificar se tal fármaco é capaz de evitar a reativaçäo parasitária quando usado preventivamente. Verificamos, contudo, que propriedade dessa natureza näo ficou comprovada.

Avaliaçäo de eventual influência da intradermorreaçäo de Montenegro sobre prova sorologica para o diagnóstico da leishmaniose tegumentar americana / Assesment of the possible influence of Montenegro's intradermal reaction (leishmanin skin test) on one serological assay for the diagnosis of American cutaneous and mucosal leishmaniasis

Foi avaliada a eventual influência da intradermorreacao de Montenegro no resultado de prova sorologica imunenzimatica para diagnóstico da leishmaniose tegumentar americana. Houve o intuito de verificar possível interferência, capaz de conturbar atividades assistenciais, científicas e epidemiológicas, entre outras. Näo se verificou qualquer participaçäo, pelo menos com a metodologia usada

Seleçäo de doadores de sangue: divergências de resultados de provas sorológicas para diagnóstico da doença de chagas / Blood donors selection: results divergences of sorologics tests for Chagas desease diagnosis

Resultados de provas sorológicas para diagnóstico da doença de Chagas, interpretados como duvidosos ou inconclusivos e decorrentes de execuçoes em Serviço de Hemoterapia, mereceram avaliaçao por meio de repetiçoes, associadas a análises parasitológicas. Claras discordâncias foram verificadas, indicando que a triagem de doadores de sangue nem sempre é confiável, afigurando-se necessário que se torne mais segura, a fim de evitar impropriedades vigentes.