Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis.

Leishmune, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group (p=0.0253), suggesting that the combination of additional chemotherapy with Leishmune-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p=0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune in the control and cure of CVL.

The mutation G298A®Ala100Thr on th coding sequence of the Duffy antigen/chemokine receptor gene in non-caucasian Brazilians

Ala100Thr has been suggested to be a Caucasian genetic marker on the FY*B allele. As the Brazilian population has arisen from miscegenation among Portuguese, Africans, and Indians, this mutation could possibly be found in Euro- and Afro-Brazilians, or in Brazilian Indians. Fifty-three related individuals and a random sample of 100 subjects from the Brazilian population were investigated using the polymerase chain reaction and four restriction fragment length polymorphisms. Confirming the working hypothesis, among the related individuals three Afro-Brazilians (two of them a mother and daughter) and a woman of Amerindian descent had the Ala100Thr mutation on the FY*B allele. Five non-related Euro-Brazilians also carried the mutation. All nine individuals presented the Fy(a-b+) phenotype. We conclude that the Ala100Thr mutation can occur in populations other than Caucasians and that this mutation does not affect Duffy expression on red blood cells. Gene frequencies for this allele in the non-related individuals were in agreement with those of other populations. The Duffy frequencies of two Amerindian tribes were also investigated.

Occurrence of Leishmania donovani parasitemia in plasma of infected hamsters.

Intracardiac transfusion of plasma, mononuclear cell fraction and blood of infected hamster donors induced visceral leishmaniasis in normal hamster receptors. At the moment of transfusion, the donors already showed all the typical signs of the disease: ascites, cachexia, as well as splenomegaly and a high parasite load in the spleen and liver. All transfused hamsters developed typical visceral leishmaniasis between 90 and 120 days, indicating that all blood products were infectious. Transfusion of the mononuclear cell fraction induced the highest values of parasitic load (spleen, 766 Leishman Donovan Units (LDU); liver, 2650 LDU), splenomegaly and hepatomegaly (spleen-liver/body relative weight: 1.130 and 6.870, respectively). Animals that received the plasma fraction also developed visceral leishmaniasis, showing similar parasitic load (spleen, 107 LDU; liver, 220 LDU) and spleen-liver/body relative weight (1.005 and 6.35, respectively) than those transfused with whole blood. The finding of typical Leishmania donovani infection in animals transfused with plasma demonstrates the possibility of the extracellular location of parasites, free in this blood fraction deprived of red and white blood cells. Fluorescence-assisted cell sorter analysis (FACS) of plasma showed the presence of particles corresponding in size to amastigotes, which fluoresced strongly with the serum of a patient with Kala-azar (73%), but not with normal serum.

Short report: occurrence of Leishmania donovani DNA in donated blood from seroreactive Brazilian blood donors.

Human visceral leishmaniasis (kala-azar) transmitted by blood transfusion has been described in previous reports. Seroprevalence of antibodies to Leishmania donovani was shown to be related to prior blood transfusions in multiply transfused hemodialysis patients in Natal, Rio Grande do Norte, Brazil. In this study, a possible correlation between seroreactivity and the presence of L. donovani DNA was investigated in asymptomatic healthy blood donors. Sera were tested using the fucose mannose ligand (FML) ELISA, which was shown to have a sensitivity of 100%, a specificity of 96-100%, reliability, and diagnostic and prognostic potential for the detection of human and canine kala-azar, respectively. Leishmanial DNA was assessed by the polymerase chain reaction (PCR) and dot-blot hybridization techniques in blood and bone marrow samples. Among 21 FML-seroreactive asymptomatic blood donors, 5 (24%) were positive by the PCR and 9 (43%) were positive in a dot-blot assay of blood samples, showing a significant correlation (chi2 = 14.24, P < 0.01). No Leishmania DNA was detected in 20 FML non-reactive blood donors. Our results point to the need for control of transmission of kala-azar by blood transfusion in areas endemic for this disease.

Prevalence of anti-Leishmania donovani antibody among Brazilian blood donors and multiply transfused hemodialysis patients.

The prevalence of anti-Leishmania donovani antibodies was investigated in 1,500 Brazilian blood donors and multiply transfused hemodialysis patients. Sera were tested using the fucose-mannose ligand (FML) ELISA, which was shown to have 100% sensitivity and 96% specificity for kala-azar. Among 1,194 volunteer blood donors, seroreactivity was 9%, increasing to 25% in a periurban kala-azar focus. However, higher positivity (37%) was found in multiply transfused hemodialysis patients from Natal, where kala-azar is constantly present in low numbers (endemic), with sporadic outbreaks in localized regions (endemic and epidemic). Risk factors included blood transfusion, which was significantly associated with the presence of anti-Leishmania antibodies (chi2 = 8.567, P < 0.005), but did not include potential exposure to sandfly bites (chi2 = 0.033, P > 0.1). The prevalence significantly decreased to 7% in hemodialysis patients from Rio de Janeiro, where kala-azar is only occasionally seen, and was 0% in patients undergoing continuous ambulatorial peritoneal dialysis. The prospective analysis of 27 FML-seroreactive donors from Natal revealed amastigotes of Leishmania in the bone marrow of one subject while four had clinical complaints, including splenomegaly and hepatosplenomegaly. Our results point to the need for control of blood transfusion as a possible route for transmission of kala-azar in endemic areas.

Electrophoretic variation of hair proteins

Hair follicle cells secrete a complex assortment of proteins that form the hair shaft, and can be classified into two major groups. The lowsulfur proteins are keratins that contribute to the backbone of intermediate filaments, and the high-sulfur proteins are associated with these filaments. In the present investigation we describe a comparative electrophoretic study of normal human hair proteins from 182 individuals, including some families. Hair proteins were extracted in urea buffer (pH 9.3), examined by 1O per cent polyacrylamide gel electrophoresis (pH 8.8) in the presence of sodium dodecyl sulfate and stained with Coomassie brilliant blue. Eighteen bands appeared and were reproducible in most individuals, with apparent molecular mass ranging from 10.0 to approximately 100 kDa. Based on the most prominent bands, an electrophoretic profile defined as the "frequent profile" was observed. This profile was observed in 180 individuais and consisted of 6 prominent bands, 4 of them of apparent molecular mass in the 407O-kDa range, which is characteristic of keratins (61.9 ñ 1.02, 58.5 ñ 1.21, 47.9 ñ 1.58, and 45.4 ñ 1.53 kDa), and 2 bands with lower molecular mass (18.9 ñ 0.75 and 13.7 ñ 0.91 kDa). In 2 samples from unrelated women, an additional band of 42.1 ñ 1.72 kDa appeared. The meaning of this variant is still under investigation.

An improved method for the isolation or erythrocyte antigen band-3

An improved method for isolation of human and Rhesus monkey band-3 separated by sodium dodecyl sulfate poliacrylamide gel electrophoresis (SDS-PAGE) is described. Purified band-3 was obtained from human hemoglobin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution + sonication (CE + S). The section of the gel corresponding to the antigen was cut out, mechanically disrupted and incubated in 1 per cent NaHCO3, containing 1 per cent SDS, for 2h, with shaking, at room temperature, followed by overnight incubation at 4ºC. The preparation was subsequently sonicated and clarified by centrifugation. Supernatants were dialyzed against distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibration curve was developed for assay of CE+S material using densitometric evaluation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghost grave 3.15 mg of purified band-3 after CE+S, corresponding to an 8.4 per cent yield. Rabbits were immunized with 50µg CE+S antigen. Serawere collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cells by treatment with protease type VI from Streptomyces griseus (1h at 37ºC), followed by extensive washing and hypotonic lysis. Specific antibodies recognized band-3 and its proteolytic fragments 60 and 63 kDa in human ghosts obtained from different blood donors, confirming the genetic polymorphism. Analogous serum obtained against the Rhesus monkey band-3 proteolytic fragment 63 kDa recognized the human antigen and its respective fragments. These results indicate the existence of similarities between these two species of band-3, suggesting the potential use of this technique in taxonomic and phylogenetic studies. Purification by CE+S is an efficient and rapid method for isolation of band-3 and its fragments with satisfactory yield and maintenance of both their immunogenic and antigenic properties

The 60 and 63 kDa proteolytic peptides of the red cell membrane band-3 protein: their prevalence in human and non-human primates.

Three phenotypes based on the polymorphism of band-3 protein from human red cells are described. Limited proteolysis of intact red cells from most individuals (homozygotes) yields a peptide of 60 kDa, but in some cases (heterozygotes), there is also a 63-kDa peptide, and rarely only the single peptide of 63 kDa is found. This is the first description of the 63-kDa homozygote. The interpretation that the three phenotypes are controlled by two alleles of a single autosomal locus, with no dominance, is supported by population and family studies. The frequencies of the allele, which we designate as p63, is 0.041 +/- 0.0068 in Caucasoids and 0.125 +/- 0.0121 in Negroids. The electrophoretic profiles and molecular weights of the peptides obtained with several commercial proteases from Streptomyces griseus are similar to those obtained with chymotrypsin. Whereas band-3 protein in two New-World monkeys (Saimiri and Cebus) resisted pronase attack, an Old-World monkey (Macaca mulatta) was monomorphic for a 63-kDa fragment, and in an ape (Pan troglodytes), a doublet of 62 kDa and 64 kDa was found. Band-3 protein polymorphism appears to be a good marker for genetic differentiation in human populations.

A and AB ABO blood group variants in Brazil.

Relatamos tres casos, dois dos quais pertencentes a mesma irmandade, com a forma completa da sindrome EEC (ectrodactilia-displasia ectodermica-fenda labial/palatina),com discussao sobre a variabilidade clinica e provavel heterogeneidade genetica desta entidade

Dermatoglifos dos indios Erigbactsa (Mato Grosso). / Dermatoglyphs of the Erigbatasa Indians, Mato Grosso

Um total de 59 homens e 40 mulheres foi estudado quanto aos seus dermatoglifos digitais e palmares. O padrao digital mais comumente observado foi o verticilo (53% nos homens, 51% nas mulheres) e o menos frequente a presilha radial (3% a 1% respectivamente). O indice de intensidade de padrao calculado para o total da amostra foi de 14,5 +/- 3,7, e a contagem total de cristas de 131,2 +/- 43,0 (homens e 116,5 +/- 48,8 (mulheres). As frequencias mais baixas, em ambos os sexos, quanto a presenca de padroes, ocorreram na 2a. interdigital.As linhas palmares principais mostraram tendencia a uma disposicao longitudinal, e as mais altas na 4a. interdigital, com o indice respectivo dando um valor, no total da amostra, de 7,1 +/- 1,8.As medidas obtidas, em homens e mulheres, quanto ao angulo atd, foram de 82,6 +/- 10,7 e 85,4 +/- 11,7; enquanto a contagem a-b forneceu os numeros de 81,2 +/- 8,3 e 81,2 +/- 11,0 respectivamente. Esses resultados nao diferem muito dos observados anteriormente em outras populacoes de indigenas sul-americanos. Os dermatoglifos parecem ser especialmente uteis, nas analises interpopulacionais quando sao feitas comparacoes entre grandes grupos raciais

Genetic and taxonomical aspects of Biomphalaria glabrata ABO agglutinins

Foram realizadas titulaçöes de posturas dos dois genótipos pigmentários, albinos totais e pigmentados, de caramujos Biomphalaria glabrata. Ambas as cepas reagiram negativamente com hemácias O normais e nenhuma hemolisina foi detectada. Ambos os genótipos de Santa Luzia (Minas Gerais) tinham similares títulos médios frente a eritrócitos A1, A2 e B normais e nenhuma diferença foi observada entre os genótipos em relaçäo com cada um dos grupos sanguíneos ABO. O padräo sorológico da linhagem albino de Porecatú (Paraná) é similar `aquela de Santa Luzia, mais mostra títulos mais baixos. Por outro lado, os dois genótipos de Rio Capibaribe (Pernambuco) mostraram diferenças significantes e um gradiente marcante nos seus títulos médios frente a hemácias dos diversos grupos ABO. Os caramujos albinos desta localidade näo apresentam atividade anti-B. A ausência de anti-B foi significantemente associada com o genótipo albino da amostra em conjunto (Método exato de Fisher, p = 0,0058). A aglutinina anti-A de esta cepa deu reaçöes semelhantes com a lectina anti-A hel contra um antígeno A fraco de um doador de grupo AB Negroide Brasileiro. Absorçöes seletivas de extratos de caramujos pigmentados permitiram separar aglutininas salinas anti-A e anti-B e aglutininas incompletas anti-A, anti-A,B e anti-O. Títulos mais elevados, em geral, foram obtidos através do uso de células tratadas com enzimas. A açäo da neuraminidase näo segue a tendências de outras enzimas desde que ela näo altera os títulos com as células A1, B e O. As posturas das três espécies do género Biomphalaria, hospedeiro intermediário da Schistossomose mansoni, poderiam ser distinguidas pela presença ou ausência dos seguintes critérios: 1) Aglutinaçäo...