For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

O tratamento de primeira escolha para leishmaniose tegumentar americana é o antimonial pentavalente. Embora este tratamento seja na maioria das vezes efetivo e indicado, devem ser consideradas as desvantagens tais como efeitos colaterais, longa duração do tratamento e contra-indicação para cardiopatas, nefropatas, idosos, grávidas e outras condições. Com o advento da vacina antileishmaniose tegumentar americana para fins profiláticos e terapêuticos, associando-a ou não a outros fármacos, muitas pesquisas têm sido desenvolvidas, sendo a vacina a principal entre os atuais recursos no tratamento e prevenção da leishmaniose tegumentar americana. Em conclusão, a associação do antimônio com a vacina (imunoquimioterapia) apresentou o mesmo índice de cura em relação ao tratamento padrão (100%), e ainda reduziu o volume do sal em 17,9% e o tempo de cura significativamente, de 87 para 62 dias; conseqüentemente, reduzindo os efeitos colaterais.

The first choice of treatment for American cutaneous leishmaniasis is the pentavalent antimonial drug. Although it has been shown that this treatment is mostly effective and indicated, some disadvantages should be taken into account such as side effects, long term treatment inconveniences and counter-indication for patients suffering from cardiopathy, nephropathy; yet, aging, pregnancy and other conditions. With the advent of the vaccine anti-American cutaneous leishmaniasis as a prophylactic measure, studies on therapy using the vaccine associated or not with other drugs have been performed by many investigators and it is currently among the alternative treatments and prevention measures for American cutaneous leishmaniasis. In conclusion, the association between antimony and vaccine (immunochemotherapy) showed the same cure rate when compared with the standard treatment (100%) and it was also able to reduce the salt volume in 17.9% and treatment length from 87 to 62 days, decreasing side effects.

Estudos anteriores revelaram que uma vacina preparada com promastigotas mortas de cinco cepas de Leishmania pode induzir uma imunidade protetora para a leishmaniose tegumentar americana no homem e em modelos experimentais. Um dos problemas do uso desta vacina é a complexidade de sua composição e a necessidade de se incorporar diferentes cepas de Leishmania. Por esta razão, extratos antigênicos de cada uma das cinco cepas constituintes da vacina foram preparados e usados individualmente em estudos imunológicos com camundongos C57BL/10. A Leishvacin® e a vacina monovalente PH8 induziram os maiores níveis de Interferon-g (IFN-gama) detectado no sobrenadante de células esplênicas dos animais vacinados. Cada grupo imunizado com vacinas monovalentes desenvolveram uma imunidade protetora seis meses após a infecção desafio com promastigotas de Leishmania (Leishmania) amazonensis e nenhuma diferença estatística foi observada entre os grupos vacinados. Pode-se concluir que vacinas compostas por cepas isoladas de Leishmania protegem camundongos C57BL/10 contra, pelo menos, da infecção por L. (L.) amazonensis.

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin® and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection.

A indução de imunidade no homem contra a leishmaniose cutânea tem sido estudada por vários pesquisadores usando uma grande variedade de preparações antigênicas, como: promastigotas vivas ou atenuadas, extratos de promastigotas, antígenos parcialmente purificados e proteínas puras. Neste trabalho foram isoladas 11 proteínas de L. (L.) amazonensis com pesos moleculares variando de 13.5 a 97 kDa por eletroforese em gel de poliacrilamida e por eletroeluição. Estas proteínas foram combinadas em diferentes preparações vacinais com gp63 e BCG. As vacinas foram avaliadas in vitro quanto à capacidade de estimular linfócitos de pessoas vacinadas com Leishvacin<FONT FACE="Symbol">â</font> a produzirem IFN-<FONT FACE="Symbol">g</font> e a estimularem a proliferação de linfócitos de camundongos vacinados. Assim, camundongos C57BL/10 foram vacinados em intervalos de 15 dias com três doses de cada vacina contendo 30 <FONT FACE="Symbol">m</font>g de cada proteína. 100 <FONT FACE="Symbol">m</font>g de BCG foram usados somente na primeira dose. Sete dias após a última dose os animais receberam a primeira infecção desafiado com 105 promastigotas infectantes e um segundo desafio foi administrado 143 dias após, com o mesmo número de parasitas. Sessenta dias após o segundo desafio, proteções de 42,86% foram obtidas com as vacinas constituídas de gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa e gp63+97kDa; 57,14% de proteção foi obtido com a vacina gp63+46+97kDa, gp63+46+97+13.5kDa, gp63+46+33kDa, e 71,43% com a vacina constituída de gp63 mais todas as proteínas. Em contraste, a vacina gp63+46+33kDa não induziu proteção nos camundongos vacinados, indicando que possivelmente a proteína de 40kDa induziu a uma atividade imunossupressora da resposta imunoprotetora. Estes resultados sugerem que uma futura vacina contra a leishmaniose cutânea deverá conter, excluindo-se a proteína de 40kDa, um coquetel de proteínas imunogênicas indutoras de proteção de camundongos contra a leishmaniose cutânea.

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-<FONT FACE="Symbol">g</font> by lymphocyte from subjects vaccinated with Leishvacin<FONT FACE="Symbol">â</font> . The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 <FONT FACE="Symbol">m</font>g of each protein at 15 days interval. One hundred <FONT FACE="Symbol">m</font>g of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-<FONT FACE="Symbol">g</font> production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis.

O diagnóstico da giardíase através da pesquisa de trofozoítos ou cistos do parasita nas fezes apresenta uma elevada percentagem de resultados falso-negativos. No presente trabalho foi feita, pela primeira vez no Brasil, uma avaliação do ProSpecT Giardia Ensaio em Microplaca (Alexon, Inc., BIOBRÁS), utilizando amostras fecais de pacientes de Belo Horizonte, Brasil. Um total de 90 amostras fecais foram testadas, primeiramente como fezes frescas e, então, preservadas em formalina 10%, para um novo teste 2 meses após. Com as amostras frescas, o ensaio imunoenzimático, através de leitura visual ou no espectrofotômetro, foi capaz de identificar todos os 30 pacientes positivos no exame parasitológico de fezes (EPF) e foi negativo em 57 dos 60 pacientes sem G. lamblia (sensibilidade = 100,0%, especificidade = 95,0%). Com as amostras preservadas em formalina 10% durante 2 meses, o teste identificou 27 das 30 amostras fecais positivas (sensibilidade = 90,0%) e foi negativo em 59 das 60 amostras negativas no EPF (especificidade = 98,3%). Comparando-se as leituras das densidades óticas nas amostras fecais positivas para G. lamblia com as amostras com EPF negativo ou contendo outros parasitas intestinais que não a G. lamblia, foi observada uma nítida diferença, tanto nas fezes frescas como nas fezes conservadas em formalina 10% durante 2 meses. O ProSpecT Giardia Ensaio em Microplaca mostrou-se uma boa alternativa para o diagnóstico da G. lamblia, especialmente para aqueles casos em que o EPF foi repetidamente negativo e o paciente apresenta sintomas compatíveis com a giardíase.

It is known that fecal examination to detect Giardia lamblia cysts or trophozoites produces a high percentage of false-negative results. A commercially available immunoenzymatic assay (ProSpecT Giardia Microplate Assay, Alexon, Inc., BIOBRÁS) to detect G. lamblia specific coproantigen was evaluated for the first time in Brazil. A total of 90 specimens were tested. Each specimen was first tested as unpreserved stool, and then it was preserved in 10% Formalin to be tested 2 months later. The assay was able to identify all the 30 positive patients (sensitivity = 100.0%) by visual or spectrophotometric examination in the unpreserved specimens and was negative in 57 of the 60 patients without G. lamblia (specificity = 95.0%). The assay identified 27 of the 30 positive patients (sensitivity = 90.0%) and was negative in 59 of the 60 negatives (specificity = 98.3%) in the preserved stools according to both readings. A marked difference was observed in the optical densities in both groups, preserved and unpreserved stools, when the G. lamblia-positive specimens were compared to the negative or positive for other intestinal parasites than G. lamblia. The assay seems a good alternative for giardiasis diagnosis, especially when the fecal examination was repeatedly negative and the patient presents giardiasislike symptoms.

A resposta imune induzida por uma proteína recombinante de Leishmania (Leishmania) amazonensis de 33 kD (Larp33) foi avaliada em linfócitos de indivíduos vacinados com a Leishvacin® e em camundongos através de vacinação. Larp33 foi expressa em Escherichia coli após clonagem de um fragmento genômico de L. (L.) amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de indivíduos vacinados com Leishvacin® e induziu proliferação em linfócitos desses indivíduos em níveis comparáveis ao antígeno total de Leishmania. A análise por imunoblot indicou que Larp33 corresponde a uma proteína de aproximadamente 40 kD expressa em promastigotas de L. (L.) amazonensis e L. (Viannia) braziliensis. Hibridização com sonda de DNA correspondente a parte do fragmento clonado, também demonstrou que o gene codificador desta proteína é expresso em promastigotas destas duas espécies. Larp33 em associação com BCG (Bacille Calmette-Guerin) foi capaz de induzir 75% de proteção em camundongos C75BL/10 e BALB/c, suceptíveis à infecção por L. (L.) amazonensis. Linfócitos dos camundongos protegidos produziram IL-2 e IFN-<FONT FACE="Symbol">g</font> em resposta a Larp33. Nossos resultados indicam que Larp33 é imunogênica para linfócitos de indivíduos vacinados com Leishvacin ® e protetora para camundongos contra a infecção por L. (L.) amazonensis

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-<FONT FACE="Symbol">g</font>, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of ³-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50% of the immunized animals were protected for six months.

Foi realizado o estudo histológico do material obtido nas biópsias do local de inoculação do antígeno para teste de Montenegro (T.M.) nos seguintes grupos de indivíduos: I) Seis pacientes com leishmaniose cutânea comprovados parasitologicamente, com Montenegro positivo; II) Cinco indivíduos normais, não residentes em zona endêmica, com Montenegro negativo; III) Nove soldados participantes de ensaios clínicos com vacina anti-LTA - MAYRINK e cols. 1979 e que tiveram o TM positivo 35 dias após vacinação. IV) Um último grupo constituído de quatro soldados, também participantes de ensaio clínico com a mesma vacina acima, dois vacinados que não mostraram TM positivo 35 dias após vacinação e dois que receberam placebo. As biópsias foram realizadas 48 hs após a inoculação do antígeno. O material foi fixado em formol à 10% (pH 7.2). Histologicamente, excetivando o grupo II (controle negativo), os grupos I-I1I-IV mostraram diferenças quantitativas no infiltrado mononuclear. Os quadros histológicos de cada grupo são descritos e discutidos.

The Montenegro skin test is widely used as a diagnostic method for American cutaneous leishmaniasis (ACL) but little is known about the histological changes that occur in the skin after administration of the antigen. This report is based on histological studies of biopsied material obtained, from inoculation sites, 48 hours after individuals had been given intradermal injections with a standardized Montenegro antigen. The material examined was obtained from four distinctly different test groups: naturally infected patients with parasitologically proved ACL and with positive Montenegro's reaction; individuals without previous history of ACL and not previously tested with Montenegro antigen; participants in anti-ACL vaccine trials who developed positive reactions to Montenegro antigen after vaccination; other participants in vaccine trials who had negative Montenegro responses after vaccination or had served as controls in the trials. The histological pictures of each group are described and discussed. Histologically, the reactions of vaccinated individuals were indistinguishable from those with naturally acquired infections.