Behavior of alfa-tomatine and tomatidine against several genera of trypanosomatids from insects and plants and Trypanosoma cruzi / Comportamento de alfa-tomatina e tomatidina contra vários gêneros de tripanossomatídeos de insetos e plantas e Trypanosoma cruzi

Glycoalkaloids are important secondary metabolites accumulated by plants as protection against pathogens. One of them, α-tomatine, is found in high concentrations in green tomato fruits, while in the ripe fruits, its aglycone form, tomatidine, does not present a protective effect, and it is usual to find parasites of tomatoes like Phytomonas serpens in these ripe fruits. To investigate the sensitivity of trypanosomatids to the action of α-tomatine, we used logarithmic growth phase culture of 20 trypanosomatids from insects and plants and Trypanosoma cruzi. The lethal dose 50% (LD50) was determined by mixing 107 cells of the different isolates with α-tomatine at concentrations ranging from 10-3 to 10-8 M for 30 min at room temperature. The same tests performed with the tomatidine as a control showed no detectable toxicity against the same trypanosomatid cultures. The tests involved determination of the percentage (%) survival of the protozoan cultures in a Neubauer chamber using optical microscopy. The LD50 values varied from 10-4 to 10-6 M α-tomatine. Slight differences were detected among the LD50 values of the analyzed samples, and none of them showed evidence of resistance to the action of tomatinase, as shown by some pathogenic fungi.

Os glicoalcaloides são metabólitos secundários importantes produzidos pelas plantas e estão envolvidos em sua proteção contra agentes patogênicos. Um deles, α-tomatina, é encontrado em altas concentrações em frutos de tomate verde, enquanto que, nos frutos maduros, sua forma aglicona, tomatidina, não apresenta um efeito protetor, sendo comum encontrar parasitas de tomates como Phytomonas serpens nesses frutos maduros. Para investigar a sensibilidade dos tripanossomatídeos à ação da α-tomatina, utilizamos formas de cultura em fase logarítmica de 20 tripanossomatídeos de plantas e insetos e Trypanosoma cruzi. A dose letal 50% (DL50) foi determinada, misturando 107 células das formas de cultura com concentrações de 10-3 a 10-8 M de α-tomatina durante trinta minutos a temperatura ambiente. Testes realizados com a tomatidina como controle não mostraram toxicidade detectável contra os mesmos tripanossomatídeos. Os testes foram avaliados pela porcentagem (%) de sobrevivência das formas de cultura dos protozoários observados por microscopia óptica em câmara de Neubauer. Os resultados da determinação de DL50 mostraram que esta variou entre 10-4 a 10-6 M de α-tomatina. Pequenas diferenças foram observadas entre os valores de DL50 das amostras analisadas, e nenhuma delas mostrou evidência de resistência pela ação da tomatinidase, como demonstrado em alguns fungos patogênicos.

Behavior of alfa-tomatine and tomatidine against several genera of trypanosomatids from insects and plants and Trypanosoma cruzi

Glycoalkaloids are important secondary metabolites accumulated by plants as protection against pathogens. One of them, a-tomatine, is found in high concentrations in green tomato fruits, while in the ripe fruits, its aglycone form, tomatidine, does not present a protective effect, and it is usual to find parasites of tomatoes like Phytomonasserpens in these ripe fruits. To investigate the sensitivity of trypanosomatids to the action of a-tomatine, we used logarithmic growth phase culture of 20 trypanosomatids from insects and plants and Trypanosoma cruzi. The lethal dose 50% (LD50) was determined by mixing 107 cells of the different isolates with a-tomatine at concentrations ranging from 10-3 to 10-8 M for 30 min at room temperature. The same tests performed with the tomatidine as a control showed no detectable toxicity against the same trypanosomatid cultures. The tests involved determination of the percentage (%) survival of the protozoan cultures in a Neubauer chamber using optical microscopy. The LD50 values varied from 10-4 to 10-6 M a-tomatine. Slight differences were detected among the LD50 values of the analyzed samples, and none of them showed evidence of resistance to the action of tomatinase, as shown by some pathogenic fungi.

Behavior of alfa-tomatine and tomatidine against several genera of trypanosomatids from insects and plants and Trypanosoma cruzi

Glycoalkaloids are important secondary metabolites accumulated by plants as protection against pathogens. One of them, a-tomatine, is found in high concentrations in green tomato fruits, while in the ripe fruits, its aglycone form, tomatidine, does not present a protective effect, and it is usual to find parasites of tomatoes like Phytomonasserpens in these ripe fruits. To investigate the sensitivity of trypanosomatids to the action of a-tomatine, we used logarithmic growth phase culture of 20 trypanosomatids from insects and plants and Trypanosoma cruzi. The lethal dose 50% (LD50) was determined by mixing 107 cells of the different isolates with a-tomatine at concentrations ranging from 10-3 to 10-8 M for 30 min at room temperature. The same tests performed with the tomatidine as a control showed no detectable toxicity against the same trypanosomatid cultures. The tests involved determination of the percentage (%) survival of the protozoan cultures in a Neubauer chamber using optical microscopy. The LD50 values varied from 10-4 to 10-6 M a-tomatine. Slight differences were detected among the LD50 values of the analyzed samples, and none of them showed evidence of resistance to the action of tomatinase, as shown by some pathogenic fungi.

Protease and phospholipase inhibition protect Veneza zonata (Hemiptera Coreidae) against septicemia caused by parasite trypanosomatid 563DT.

Veneza zonata (Hemiptera Coreidae) is an insect which causes losses in several crops, and it is also an important vector of lower trypanosomatids. V. zonata specimens were collected on rural properties in Londrina, state of Paraná, Southern Brazil. Inoculation of Leptomonas 563DT into V. zonata hemocoel caused insect death within approximately 24 h, with large bacterial proliferation into their hemocoels. Some bacteria which were found in the digestive tract of those insects, such as Escherichia coli, Providencia rettgeri, and Kluyveria ascorbata, were also found in their hemolymph, which suggests that trypanosomatid crossing into hemocoel caused mechanical lesions in the digestive tract that allowed intestinal bacteria to infect the hemolymph, thereby leading to lethal septicemia. In this study we analysed proteolytic activities from the 563DT Leptomonas strain, which is pathogenic for V. zonata, aiming at evaluating the potential use of this Leptomonas strain for the biocontrol of the insect. The proteolytic action was evaluated on cells and on culture supernatants of trypanosomatids. We also evaluated the gelatinolytic activities, the action over natural and synthetic substrates for aminopeptidases, and the action of protease inhibitors during all trypanosomatid growth stages. A significant reduction in the number of insect deaths was observed when Leptomonas 563DT were incubated with inhibitors of proteases and phospholipases before being inoculated into the insects, which suggests that those enzymes are involved in the pathogenic mechanism.

Phytomonas serpens, a tomato parasite, shares antigens with Trypanosoma cruzi that are recognized by human sera and induce protective immunity in mice.

The immune cross-reactivity between Trypanosoma cruzi, the protozoan that causes Chagas' disease, and Phytomonas serpens, a trypanosomatid that infects tomatoes, was studied. Sera from patients with Chagas' disease presented a strong reactivity with P. serpens antigens by conventional serological assays such as indirect immunofluorescence (IIF) and direct agglutination test (DAT), confirmed after cross-absorption experiments. The results show that this protozoan is highly immunogenic and that rabbit and mouse hyperimmune serum raised against T. cruzi or P. serpens was able to recognize both T. cruzi and P. serpens antigens in immunofluorescence and agglutination assays. The antigenic cross-reactivity between T. cruzi and P. serpens was also demonstrated in vivo. BALB/c mice immunized by the intraperitoneal or oral route with P. serpens and later challenged with a lethal inoculum of T. cruzi blood forms showed a significant decrease in parasitemia and increase in survival compared to controls. A practical implication of these findings is that the ingestion by humans or animals of living plant trypanosomatids present in naturally infected edible fruits could potentially prime the immune response to T. cruzi antigens and interfere with the development of T. cruzi infection.

As diretrizes curriculares nacionais dos cursos de biomedicina / National curriculum directives for biomedical education

Texto elaborado a partir da oficina de trabalho nº11 realizada nos dias 24 e 25/05/2003, coordenada pelos autores, durante o V Congresso Nacional da Rede Unida.

Evaluation of antigens from various Leishmania species in a Western blot for diagnosis of American tegumentary leishmaniasis.

A Western blot method that uses antigens from culture promastigote forms of Leishmania (Viannia) braziliensis, L. (Leishmania) amazonensis, L. (Leishmania) tropica, and a trypanosomatid (strain 268T) isolated from naturally infected tomatoes was evaluated for laboratory diagnosis of American tegumentary leishmaniasis (ATL). Serum samples were obtained from 108 patients with ATL (group I), 23 chagasic patients (group II), 32 patients with other diseases (group III), and 78 healthy individuals (group IV). The overall analysis showed a sensitivity of 76.90%, 90.40%, 78.50%, and 87.90%, a specificity of 100%, 93.80%, 87.80%, and 77.10%, a positive predictive value of 100%, 94.00%, 89.50%, and 72.50%, a negative predictive value of 75.70%, 90.00%, 75.40%, and 90.20%, and a concordance coefficient kappa of 0.7358, 0.8400, 0.6491, and 0.6287 for L. (V.) braziliensis, L. (L.) amazonensis, L. (L.) tropica, and strain 268T antigens, respectively. The antigenic profile recognized by serum samples from patients with ATL and with Chagas' disease permits serologic distinction between these infections.

Comportamento imunológico e antigênico de cinco amostras de Toxoplasma gondii inoculadas em gatos / Immunogenic and antigenic aspects from five Toxoplasma gondii strains inoculated in cats

A biologia do Toxoplasma gondii demonstra que o gato é o hospedeiro completo, responsável pela disseminaçäo do parasito. Assim, dois gatos domésticos foram imunizados com cada uma das amostras, VPS (humano), LIV-IV e LIV-V (suíno), CPL (caprino) e CN (felino) de T. gondii. Foram utilizados taquizoítas vivos em inóculos endovenosos de 2x10 elevado a 6 (1§ inóculo) e 4x10 elevado a 7 (2§ inóculo - 35 dias após), exceto a amostra VPS, onde um gato morreu no 10§ dia com sinais clínicos agudos da doença, nas outras amostras, nenhum sinal clínico foi constatado durante os 6 meses de observaçäo. O nível de anticorpos na imunizaçäo foi acompanhado através da reaçäo de imunofluorescência indireta (IFI) com conjugado anti-IgG de felino. Os títulos de anticorpos obtidos no 20§ dia variaram de 1:1.024 a 1:4.096 e de 1:1.024 a 1:8.000 no 40§ dia. Somente a amostra VPS expressou títulos de 1:16.000 no 30§ dia da imunizaçäo. Títulos homólogos e heterólogos foram equivalentes sem nenhuma diferença entre as amostras. Quando soros imunes foram adsorvidos com taquisoítas vivos de cada amostra, a reduçäo nos títulos de anticorpos foi demonstrada em ambos homólogos e heterólogos. Esses resultados sugerem que, embora diferente em virulência para gatos, a superfície antigênica é comum entre as amostras do T. gondii, com base no nível de anticorpo demonstrado pela IFI. Esses resultados também demonstram que, aparentemente, näo há correlaçäo entre virulência e as características sorológicas das amostras estudadas no T. gondii. Entretanto, a importância do teste IFI em diagnóstico laboratorial é reforçada.

Doença de Chagas congênita: epidemiologia, diagnóstico laboratorial, prognóstico e tratamento / Congenital Chagas's Disease: edipemiology, laboratorial diagnosis, prognosis and treatment

Os autores revisam os aspectos epidemiológicos, clínicos e os métodos laboratoriais utilizados para o diagnóstico da Doença de Chagas congênita, salientando as limitaçöes quanto à sensibilidade e especificidade dos mesmos, e sugerem métodos alternativos que contribuiriam para um diagnóstico rápido e preciso, essencial para um tratamento eficaz.

Dosagem microbiológica de Tiamina (Vit.B1) em cultivares de soja (Glycine max) L. Merril

Utilizando-se métodos microbiológico, foi quantificada a vitamina tiamina (vit. B1) em cultivares de soja (Glycine Max) safras 1982(47) e 1984(96). Os valores encontrados demonstram que esta vitamina näo apresenta variaçöes quantitativas significativas entre as variedades genéticas analisadas, porém foi observada perda da atividade biológica desta vitamina durante a estocagem dos gräos