Protection of aouts monkeys after immunization with recombinant antigens of Plasmodium falciparum

The genus Aotus spp. (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aouts kenotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containg partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-strech protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challange, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and mixture of two sequences of SERP in the presence of the modified Al(OH)3 adjuvant conferred significant protection against a challange infection with P. falciparum blood stages (2-5 x 10 (elevado a sexta potência) i. RBC). Monkey immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals frm a severe experimental P. falciparum (strain Palo Alto) infection.

Protective immunity induced in mice by F8.1 and F8.2 antigens purified from Schistosoma mansoni eggs

Schistosoma mansoni soluble egg antigens (SEA) were fractionated by isoelectric focusing, resulting in 20 components, characterized by pH, absorbance and protein concentration. The higher absorbance fractions were submitted to electrophoresis, and fraction 8 (F8) presented a specific pattern of bands on its isoelectric point. Protein 3 was observed only on F8, and so, it was utilized to rabbit immunization, in order to evaluate its capacity of inducing protective immunity. IgG antibodies from rabbit anti-F8 serum were coupled to Sepharose, and used to obtain the specific antigen by affinity chromatography. This antigen, submitted to electrophoresis, presented two proteic bands (F8.1 and F8.2), which were transferred to nitrocellulose membrane (PVDF) and sequenciated. The homology of F8.2 to known proteins was determined using the Basic Local Alignment Search Tool program (BLASTp). Significant homologies were obtained for the rabbit cytosolic Ca2+ uptake inhibitor, and for the bird a1-proteinase inhibitor. Immunization of mice with F8.1 and F8.2, in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system.

Induction of protective immunity against Schistosoma mansoni infection by antigens purified from PIII, a fraction of adult worm, associated to the downregulation of granuloma formation

This study was performed in order to define Schistosoma mansoni antigens able to function as modulator agents in BALB/c mice granulomatous hypersensitivity to parasite egg. The antigens P-24, P-35 and P-97 were purified by affinity chromatography from a fraction of S. mansoni adult worm antigenic preparation, denominated PIII, involved in the inhibition of granulomatous response to eggs. Immunization of mice with these antigens, in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system. In vitro blastogenesis assays revealed that purified antigens were able to induce significant proliferation of spleen cells from S. mansoni-infected mice. This protection was correlated to significant decrease in granuloma size induced by PIII. From these results, we concluded that PIII preparation contains antigens capable of mediating protective anti-parasite immunity and down-regulating granulomatous hypersensitivity to S. mansoni eggs.

Preparação e caracterização de pós cerâmicos de aluminatos de lítio

The preparation of gamma-LiAlO2 by coprecipitation and sol-gel synthesis was investigated. Ceramic powders obtained by coprecipitation synthesis were prepared from aqueous solutions of aluminum and lithium nitrates using sodium hydroxide as precipitant agent. By sol-gel synthesis, the ceramic powders were prepared from hydrolysis of aluminum isopropoxide. The materials obtained by two routes of synthesis were dried at 80ºC and calcined at 550, 750, 950 and 1150ºC. The characterization was done by X-ray diffraction, infrared spectroscopy, emission and absorption atomic spectrometry, helium picnometry, specific surface area (BET method) and scanning electronic microscopy. Mixtures of crystalline phases were obtained by coprecipitation synthesis: 80ºC- LiAl2(OH)7.2H2O + Al(OH)3; 550 and 750ºC- alpha-LiAlO2 + eta-Al2O3; 950 and 1150ºC- gamma-LiAlO2 + LiAl5O8. Chemical analysis showed molar ration Al/Li @ 3. Crystalline single-phases were obtained by sol-gel synthesis above 550ºC: 550ºC-alpha-LiAlO2; 750, 950 and 1150ºC-gamma-LiAlO2. These powders presented molar ration Al/Li @ 1. Thus, gamma-LiAlO2 crystalline phase was obtained at 750ºC by sol-gel synthesis while by coprecipitation synthesis, a mixture of crystalline phases was obtained. These results showed the superiority of the sol-gel synthesis for the preparation of pure gamma-LiAlO2.

Coupling of palmitate to ovalbumin inhibits the induction of oral tolerance

Oral tolerance is a phenomenon that may occur in animals exposed to protein antigens for the first time by the oral route. They become unable to produce immune responses at the levels normally observed when they are immunized parenterally with antigen in the presence of adjuvants. Lipids have been used as adjuvants for both parenteral and oral immunization. In the present study we coupled ovalbumin with palmitate residues by incubating the protein with the N-hydroxysuccinimide palmitate ester and tested the preparation for its ability to induce oral tolerance. This was performed by giving 20 mg of antigen to mice by the oral route 7 days prior to parenteral immunization in the presence of Al(OH)3. Mice were bled one week after receiving a booster that was given 2 weeks after primary immunization. Specific antibodies were detected by ELISA. Despite the fact that the conjugates are as immunogenic as the unmodified protein when parenterally injected in mice, they failed to induce oral tolerance. This discrepancy could be explained by differences in the intestinal absorption of the two forms of the antigen. In fact, when compared to the non-conjugated ovalbumin, a fast and high absorption of the lipid-conjugated form of ovalbumin was observed by "sandwich" ELISA.

Immunization by subcutaneous implants of polyester-polyurethane sponges coupled with antigen

A new protocol is described for immunization of outbred Swiss mice. The procedure is based on subcutaneous implantation of antigen-coupled polyester-polyurethane sponges cut into disks of 10 mm in diameter vs 2 mm in thickness. Antigen coupling was performed by overnight incubation of the sponge with a solution of ovalbumin (Ova) (2 mg/ml) diluted in sodium carbonate buffer, pH 9.6. The amount of ovalbumin that was taken up by the sponge was between 71.4 to 82.5 µg. This was estimated by comparing the Ova absorbance at 280 nm in coating buffer solutions before and after incubation. To compare the efficiency of the proposed method, experimental groups immunized with the antigen in the presence of adjuvants (10 µg in Al(OH)3 or 100 µg in complete Freund's adjuvant (CFA)) were run in parallel. The data obtained after the 3rd week of immunization indicate that both cellular and humoral immune responses were achieved. These were assayed by antigen-induced footpad swelling and ELISA (specific antibodies), respectively. The levels of both immune responses elicited were similar to the responses observed in mice immunized with ovalbumin in the presence of Al(OH)3. The method might represent an advantage when immunizing with pathogenic antigens. Preliminary experiments have suggested that the antigen remains immobilized or bound to the sponge for a long period of time, since there is an increment on the cell population inside the sponges after boosting the animals. If so, the undesirable effects of immunization would be reduced.

Efeito do fluoreto de cálcio e do carbonato de cálcio na composição química da solução percolada de um solo ácido

Durante oito meses, foram estudados, em casa de vegetação, os efeitos da aplicação de CaF2 (p.a.) e de CaCO3 (p.a.) sobre a concentração de Al e de outros elementos químicos na solução percolada de amostras de um solo ácido, coletado na região de Piracicaba, SP. Os valores de pH aumentaram na solução percolada de amostras de solos que receberam tanto CaCO3 como CaF2. Os tratamentos com CaF2 foram mais eficientes na retirada de Al do solo do que os tratamentos com CaCO3. Na testemunha e nos tratamentos com CaCO3, a maior parte do Al estava, respectivamente, na forma livre Al3+ ou formando complexos Al-OH, ao passo que, nos tratamentos com CaF2 , mais de 99,5% do Al estava complexado com o fluoreto.

Determinação direta e simultânea de Al, As, Fe, Mn e Ni em cachaça por espectrometria de absorção atômica em forno de grafite

Este trabalho propõe um método simples, rápido e confiável para determinação direta e simultânea de Al, As, Fe, Mn e Ni em cachaça por espectrometria de absorção atômica em forno de grafite (GFAAS). A superfície superior da plataforma do tubo de grafite foi revestida com filme à base de tungstênio (WxCyOz).O programa de aquecimento otimizado (temperatura, tempo de rampa, tempo de patamar) foi o seguinte: secagem 1 (100ºC, 5 s, 5 s); secagem 2 (120ºC, 5 s, 5 s); pirólise (1300ºC, 10 s, 30 s); atomização (2200ºC, 1 s, 6 s) e limpeza (2550ºC, 1 s, 3s). Os desvios padrões relativos (n=3) foram < 4,4%, < 0,7%, < 11%, < 6,0%, < 1,2% para os elementos Al, As, Fe, Mn e Ni, respectivamente. A exatidão foi avaliada por meio de testes de adição e recuperação dos analitos em 8 amostras de cachaças comerciais, e as recuperações situaram-se nos seguintes intervalos: 80 - 105% (Al), 81 - 92% (As), 82 - 108% (Fe), 83 - 106% (Mn), 83 - 108% (Ni). Os limites de detecção calculados foram 9,7 µg L-1 Al, 2,3 µg L-1 As, 12 µg L-1 Fe, 14 µg L-1 Mn e 0,8 µg L-1 Ni.

Filariose bancroftiana no Brasil

Foi feita revisão dos estudos sobre a distribuição geográfica e prevalências da filariose linfática bancroftiana no Brasil. Entre 1951 a 1958 foram realizados inquéritos hemoscópicos e entomológicos no País, sendo a transmissão autóctone comprovada em apenas 11 Municípios: Ponta Grossa (SC), 14,5%; Belém (PA), 9,8%; Barra de Laguna (SC), 9,4%; Recife (PE), 6,9%; Castro Alves (BA), 5,9%; Florianópolis (SC), 1,4%; São Luís (MA), 0,6%; Salvador (BA), 0,4%; Maceió (AL), 0,3%; Manaus (AM), 0,2% e Porto Alegre (RS), 0,1%. A distribuição da parasitose era urbana e focal, principalmente em cidades litorâneas. As ações de controle implementadas levaram a uma apreciável queda nas taxas de microfilarêmicos sendo consideradas, a partir de então, áreas endêmicas para filariose linfática no Brasil somente as cidades de Belém e Recife. Estudos recentes mostram transmissão ativa da parasitose, com prevalências superiores às detectadas no passado, em Maceió, cujo foco era considerado extinto, e em Recife. O conhecimento da situação atual da bancroftose no Brasil é de fundamental importância para a implementação de ações de controle da endemia envolvendo o Ministério da Saúde e órgãos de saúde pública.

Identificación y tipificación de biotipos y serotipos de Yersinia enterocolitica

OBJETIVO: Conocer la existencia de Yersinia enterocolitica en suínos visiblemente sanos y sacrificados para el consumo humano. MÉTODOS: Fueron estudiadas 100 muestras de tejido linfático obtenidas en el momento del sacrifício, en un matadero del Estado de México. Fueron realizados muestreos pilotos de 20 casos, de los cuales 20% fueron positivos, permitiendo obtener una muestra estudiada (n=100). Las muestras colectadas de tejido linfático fueron acondicionadas para el aislamiento de Yersinia enterocolitica en caldo de Rappaport y en medio de cultivo de Salmonella-Shigella y MacConkey. Las identificaciones fueron efectuadas por medio de pruebas bioquímicas y serológicas, utilizándose en el caso los antisueros O:3, O:8 y O:9 para la biotipificación correspondiente. RESULTADOS: Fueron obtenidos 22 aislamientos tipificándose 8 serotipos pertenecientes al O:3 y 8 al O:9 correspondientes al biotipo 1; y, en 6 muestras no fue posible la serotipificación. No se encontró en el total de los aislados el serotipo O:8. CONCLUSIONES: En base en la metodología, se registró la presencia de Y. enterocolitica y sus serogrupos en tejido linfático de porcinos por la primera vez en México; esto es importante porque el patógeno y sus serotipos aislados están comprometidos con mayor frecuencia con problemas de salud pública.

Sobre um método de concentração de enterovírus em água de esgoto pelo hidróxido de alumínio

O presente trabalho descreve um estudo experimental em que foi testada a eficiência de um método para concentração de enterovírus em água de esgoto por adsorção ao hidróxido de alumínio. Adicionou-se o gel de A1 (OH)3 (na proporção de 1g para 3,78 litros) à água de esgoto previamente clarificada e com o pH acertado em 5,0. Depois de agitada por 1 hora, a mistura foi filtrada em membrana Millipore tipo AP-20. O hidróxido de alumínio, o qual permaneceu retido na membrana, foi retirado com auxílio de uma espátula e a ele se adicionou meio de Eagle com 10% de soro fetal, sendo o pH acertado em torno de 7,2 - 7,4 para se processar a eluição. A mistura foi centrifugada a 20.000g por 15 minutos, o sedimento desprezado e o sobrenadante inoculado em células da linhagem LLC-MK2 para quantificação viral. Paralelamente, foram também inoculadas em cultura de células as amostras de esgoto brutas, isto é, antes de qualquer procedimento para concentração. O método descrito se mostrou eficiente para concentrar enterovírus em água de esgoto, sendo que tornou possível detectar vírus mesmo de amostras contendo cerca de 1 unidade infecciosa por ml, das quais não se conseguiu isolamento de vírus sem concentração prévia.

On the adjuvant effect of aluminum hydroxide for mice

Linear relationships were found between the dose of A1(OH)3 adjuvant and the titer of anti-OVA antibodies formed by BDF1 mice. Mice immunized with OVA, DNP-KLH and then boosted with DNP-OVA formed anti-DNP antibodies only when A1(OH)3 was added to the injection of DNP-KLH; addition of A1(OH)3 to the priming injection of OVA decreased, rather than increased antibody formation.

Charaterization of Leishmania major Friedlin Telomeric Terminus

Here we have characterized Leishmania major (Friedlin) telomeric terminus (the very end) using recombinants obtained by a vector-adaptor cloning protocol. As in L. donovani, the last nine nucleotides of L. major terminus are 5'-GGTTAGGGT-OH 3', differing from Trypanosoma cruzi and T. brucei terminus 5'GGGTTAGGG-OH 3', thus indicating that these sequences are genus specific. We have also made a comparative analysis between L. major and L. donovani telomere-associated sequences, and described a novel non-repeated telomeric associated sequence common to L. major low molecular weight chromosomal bands.

Variáveis de acidez em função da mineralogia da fração argila do solo

A acidez do solo e variáveis associadas são muito importantes nos manejos dos solos, especialmente nas regiões tropicais e subtropicais. Com o objetivo de estudar algumas variáveis da acidez do solo e verificar a influência da mineralogia da fração argila sobre a relação entre pH em CaCl2 e grau de saturação por bases (V), amostraram-se os horizontes B e C de diferentes classes de solo no Estado do Paraná: Latossolo Bruno ácrico húmico (LBw-1), Latossolo Bruno distrófico húmico (LBw-2), Latossolo Vermelho distroférrico húmico (LVdf), Cambissolo Háplico alumínico típico (CXa) e Cambissolo Húmico distroférrico típico (CHd). A fração argila foi estudada por difratometria de raios X, análise termodiferencial, análise termogravimétrica e análises químicas. As amostras de solo foram incubadas por 60 dias, após terem recebido doses crescentes de óxido de cálcio, para atingir valores preestabelecidos do grau de saturação por bases (natural, 25, 45, 60, 70, 125 e 150 %). Em seguida, determinaram-se os teores de cátions trocáveis e os valores de pH em H2O e solução de CaCl2 1 mol L-1. Os solos apresentaram o seguinte comportamento quanto à mineralogia da fração argila: mais esmectítico/vermiculítico - CXa; mais oxídico, principalmente óxidos de Fe - LVdf e óxidos de Al - LBw1; mais caulinítico, com menores teores de óxidos de Fe e Al - CHd e LBw2. Essa diversidade mineralógica foi determinante na relação pH em CaCl2 e grau de saturação por bases (V) dos solos. As curvas que mostraram essa relação foram não lineares, o que significa poder tamponante diferenciado ao longo da faixa de pH em CaCl2 estudada; formato convexo dos solos mais oxídicos e côncavo dos solos cauliníticos e com argila 2:1. O formato convexo das curvas foi conseqüência da formação de cargas negativas e dissociação de H+ preferencialmente a valores elevados de pH em CaCl2 (acima de 5), devido à menor acidez (PCZ mais alto) dos radicais Fe-OH e Al-OH. O formato côncavo foi atribuído a cargas permanentes e a maior dissociação de H+ a valores menores de pH em CaCl2 (abaixo de pH 5), uma vez que o radical Si-OH é considerado um ácido forte (PCZ baixo). Para um mesmo pH em CaCl2 verificou-se a seguinte seqüência nos valores de V em função da mineralogia da fração argila: solos com predomínio de óxidos de Fe e Al < solos com predomínio de caulinita < solos cauliníticos com minerais 2:1.

Avaliação das técnicas de precipitação química e encapsulamento no tratamento e destinação conjunta de resíduos líquidos contendo cromo e vidrarias de laboratório

In this paper, we present procedures for the treatment and final disposal of residual solutions containing chromium, in order to evaluate the effectiveness of chemical precipitation of the metal and the potential of the glass encapsulation technique, using broken laboratory glassware. The results demonstrated that pH-values convenient for chemical precipitation are between 10 - 11. With regard to Cr(OH)3 encapsulation, the leaching and solubilization tests allowed to classify the waste as non-dangerous and non-inert. Finally, it is pointed out that the adoption of waste management practices in universities should be encouraged, helping to train professionals skilled in good laboratory practices.