DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 mug of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN,viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

Translation termination and protein folding pathway genes are not correlated in gastric cancer

Background: The eukaryotic release factor 3 (eRF3) has been shown to affect both tubulin and actin cytoskeleton, suggesting a role in cytoskeleton assembly, mitotic spindle formation and chromosome segregation. Also, direct interactions between eRF3 and subunits of the cytosolic chaperonin CCT have been described. Moreover, both eRF3a and CCT subunits have been described to be up-regulated in cancer tissues. Our aim was to evaluate the hypothesis that eRF3 expression levels are correlated with the expression of genes encoding proteins involved in the tubulin folding pathways. Methods: Relative expression levels of eRF1, eRF3a/GSPT1, PFDN4, CCT2, CCT4, and TBCA genes in tumour samples relative to their adjacent normal tissues were investigated using real time-polymerase chain reaction in 20 gastric cancer patients. Results: The expression levels of eRF3a/GSPT1 were not correlated with the expression levels of the other genes studied. However, significant correlations were detected between the other genes, both within intestinal and diffuse type tumours. Conclusions: eRF3a/GSPT1 expression at the mRNA level is independent from both cell translation rates and from the expression of the genes involved in tubulin-folding pathways. The differences in the patterns of expression of the genes studied support the hypothesis of genetically independent pathways in the origin of intestinal and diffuse type gastric tumours.

Differential expression of GSPT1 GGCn alleles in cancer

The human eukaryotic release factor 3a (eRF3a), encoded by the G1 to S phase transition 1 gene (GSPT1; alias eRF3a), is upregulated in various human cancers. GSPT1 contains a GGCn polymorphism in exon 1, encoding a polyglycine expansion in the N-terminal of the protein. The longer allele, GGC12, was previously shown to be associated to cancer. The GGC12 allele was present in 2.2% of colorectal cancer patients but was absent in Crohn disease patients and in the control group. Real-time quantitative RT-PCR analysis showed that the GGC12 allele was present at up to 10-fold higher transcription levels than the GGC10 allele (P < 0.001). No GSPT1 amplifications were detected, and there was no correlation between the length of the alleles and methylation levels of the CpG sites inside the GGC expansion. Using flow cytometry, we compared the levels of apoptosis and proliferation rates between cell lines with different genotypes, but detected no significant differences. Finally, we used a cytokinesis-block micronucleus assay to evaluate the frequency of micronuclei in the same cell lines. Cell lines with the longer alleles had higher frequencies of micronuclei in binucleated cells, which is probably a result of defects in mitotic spindle formation. Altogether, these findings indicate that GSPT1 should be considered a potential proto-oncogene.

Characterization of a polyubiquitin gene in T. thermophila and of ubiquitin gene expression during sexual reproduction and under stress conditions

A 5-unit polyubiquitin gene, TTU3, was isolated from a T. thermophila genomic library and sequenced. This gene presents an extra triplet coding for Phe, a AGAGA motif and a putative HSE element in its 5'-non-coding region. The ubiquitin gene expression in this ciliate was investigated by Northern blot hybridization in conjugating cells or cells under stress conditions. Exponentially growing cells express two ubiquitin mRNAs of 0.75 and 1.8 kb and a new species of 1.4 kb is induced under hyperthermic stress. During sexual reproduction of the cells (conjugation) the 1.8-kb mRNA is still transcribed whereas the steady-state population of the 0.75 mRNA transcripts is strongly diminished. Southern blot analysis suggests that ubiquitin in T. thermophila constitutes a large family of about ten members.

Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.

The role of complex chromosome translocations in leukemia

Resumo A tumorigénese é um processo de transformação celular que se desenrola tipicamente em várias etapas. Os diferentes níveis de evolução tumoral resultam da acumulação sucessiva de mutações genéticas numa célula normal que lhe conferem uma vantagem selectiva no respectivo meio tecidular. As mutações podem manifestar-se sob a forma de alterações nucleotídicas pontuais ao nível da sequência de DNA, levando a uma desregulação da função proteíca ou à formação de proteínas não-funcionais, ou através de alterações cromossómicas numéricas ou estruturais. Na leucemia, por exemplo, os genes híbridos que resultam de translocações cromossómicas desempenham um importante papel no processo tumorigénico. Estes genes são transcritos sob a forma de um RNA mensageiro de fusão, o qual é traduzido numa proteína híbrida com função oncogénica. Frequentemente, os subtipos de doença leucémica estão associados com translocações cromossómicas que envolvem 2 pontos de quebra recorrentes e específicos. É disto exemplo a leucemia mielóide crónica, em que uma translocação recíproca entre os cromossomas 9 e 22 conduz à formação de um gene de fusão BCR-ABL1. Em diferentes subtipos de doença, existe também uma pequena proporção de casos que apresenta translocações cromossómicas complexas, que envolvem um ou mais pontos de quebra adicionais em outras localizações genómicas além das que estão implicadas na formação dos genes de fusão. Por vezes, os pontos de quebra estão também associados a delecções extensas de material genético que se pensa terem uma função importante na tumorigénese. No entanto, o papel destas regiões genómicas no desenvolvimento tumoral não tem sido um motivo recorrente de estudo. Neste contexto, o objectivo desta dissertação foi o de determinar o potencial papel tumorigénico de alterações génicas adicionais ocorridas nos pontos de quebra de translocações cromossómicas complexas. Para a prossecução do objectivo proposto, foram estudados 5 rearranjos cromossómicos distintos associados com diferentes tipos de doença hematológica maligna, nomeadamente a leucemia linfoblástica aguda de células B (2 casos), leucemia mielóide aguda, neoplasma mieloproliferativo e síndrome mielodisplásico/neoplasma ieloproliferativo, não classificável. O mapeamento dos pontos de quebra foi efectuado utilizando a hibridação fluorescente in situ e diferentes metodologias de biologia molecular, tendo como base a informação inicial da análise citogenética. Em casos seleccionados, o papel dos novos genes candidatos foi avaliado in vitro utilizando modelos de linhas celulares, nomeadamente no que respeita às funções de controlo da proliferação celular e de regulação transcricional. De entre os 5 casos estudados, quatro deles evidenciaram translocações complexas envolvendo 3 cromossomas, nomeadamente t(12;21;5)(p13;q22;q13), t(12;6;15)(p13;p24~25;q22), t(9;11;19)(p22;q23;p13) e t(X;20;16)(p11;q13;q23). No caso remanescente, foi observada uma translocação dicêntrica dic(9;12)(p11;p11) acompanhada de delecções extensas em ambos os pontos de quebra. Nos casos com t(12;21;5) e t(9;11;19) as translocações estavam associadas com a presença de genes de fusão recorrentes, nomeadamente TV6(12p13)-RUNX1(21q22) e TLL(11q23)-MLLT3(9p22), indicando que se tratavam de rearranjos complexos das translocações t(12;21) e t(9;11) associadas com a leucemia linfoblástica aguda de células B e a leucemia mielóide aguda, respectivamente. O papel dos pontos de quebra adicionais foi estudado em detalhe no caso com t(9;11;19). Através da metodologia de long distance inverse-polymerase chain reaction, foram identificados os pontos de quebra na sequência de DNA dos 3 cromossomas envolvidos na translocação. Além dos pontos de quebra nos genes MLL e MLLT3, foi observado que o local de quebra no cromossoma 19 interrompeu a sequência de um novo gene, designado CCDC94,conduzindo à sua haplo-insuficiência nas células com t(9;11;19). Através de ensaios de reverse transcription-polymerase chain reaction verificámos que o gene CCDC94 é expresso ubiquitariamente em tecidos humanos normais. A análise informática da sequência prevista da proteína CCDC94 indicou uma elevada identidade de aminoácidos com a proteína cwf16, envolvida na regulação do ciclo celular da levedura Schizosaccharomyces pombe. Através da clonagem do DNA complementar de CCDC94 em vectores de expressão, e após a transfecção destes em culturas de linhas celulares in vitro, observámos que este gene codifica uma proteína de localização exclusivamente nuclear. A expressão ectópica da proteína CCDC94 diminuiu a progressão do ciclo celular e a proliferação das células em cultura. Inversamente, a supressão do transcrito do gene CCDC94 através de interferência de RNA conduziu a um aumento significativo da proliferação celular, confirmando que CCDC94 regula negativamente a proliferação e a progressão do ciclo celular. Estes resultados mostram que os pontos de quebra adicionais, presentes em translocações cromossómicas complexas em leucemia, podem resultar na haplo-insuficiência de genes controladores dos mecanismos proliferativos, cooperando desta forma com a acção das proteínas de fusão para proporcionar ao clone leucémico uma proliferação celular descontrolada. Nos restantes 3 casos estudados não foram identificados genes de fusão. Ao invés, todos aqueles apresentaram delecções de extensão variável associadas com os pontos de quebra cromossómicos. No caso com t(12;6;15), identificámos uma delecção de 1.2 megabases de DNA na banda 12p13 que resultou na eliminação de 9 genes incluindo ETV6 e CDKN1B. O gene ETV6 codifica um factor de transcrição que é essencial para a formação das diferentes linhagens hematopoiéticas na medula óssea, enquanto CDKN1B é traduzido numa proteína responsável por bloquear a entrada das células na fase G1 do ciclo celular e,consequentemente, por travar a proliferação celular. Neste contexto, os resultados obtidos indicam que a perda simultânea de ETV6 e de CDKN1B, através de uma translocação cromossómica complexa, constituiu uma acção cooperativa na leucemogénese. A mesma noção pode aplicar-se ao caso com dic(9;12), no qual pelo menos 2 genes que codificam para factores de transcrição importantes na linhagem hematopoiética, PAX5 no cromossoma 9 e ETV6 no cromossoma 12, estavam deleccionados como resultado do rearranjo cromossómico. Dado que o factor de transcrição PAX5 regula negativamente a expressão do gene FLT3, que desempenha uma função pró-proliferativa, é expectável que a haplo-insuficiência de PAX5 no caso com dic(9;12) terá tido como consequência uma elevação dos níveis de expressão de FLT3, contribuindo deste modo para uma proliferação celular aumentada. A t(X;20;16) foi identificada num doente com trombocitémia essencial (TE), uma doença que está intimamente relacionada com alterações de vias intracelulares reguladas por citocinas. Neste caso, através da utilização de um array genómico, identificámos a presença de pequenas delecções associadas com os pontos de quebra nos cromossomas 16 e 20. No cromossoma 16 apenas um gene, MAF, estava deleccionado, enquanto no cromossoma 20 a delecção tinha abrangido 3 genes. Dos genes deleccionados, dois deles, NFATC2 (20q13) e MAF (16q23), codificam proteínas que operam como reguladores transcricionais de citocinas hematopoiéticas. Dado que NFATC2 se localiza numa região que constitui um alvo frequente de delecções em neoplasmas ieloproliferativos, incluindo a trombocitémia essencial,efectuámos um estudo detalhado do papel deste gene na proliferação megacariocítica e na regulação da expressão de uma citocina hematopoiética (GM-CSF), implicada na maturação das diferentes linhagens mielóides. Utilizando um modelo de linha celular de trombocitémia essencial, verificámos que a supressão do transcrito do gene NFATC2 in vitro, por interferência de RNA, estava associada com um aumento da proliferação celular. Em concordância, o bloqueio da activação da proteína NFATC2 através de um inibidor específico da sua interacção com a calcineurina, conduziu a um aumento da proliferação celular in vitro. Utilizando a PCR quantitativa em tempo real, detectou-se um aumento da produção do RNA de GM-CSF em ambos os ensaios celulares, indicando que o factor de transcrição NFATC2 pode regular negativamente a expressão de GM-CSF em células de trombocitémia essencial. No geral, estes resultados mostram que a redução dos níveis fisiológicos do transcrito NFATC2, ou a redução da respectiva actividade proteica, estão relacionados com a proliferação de megacariocitos através do aumento da produção de GM-CSF. De acordo com estes resultados, verificámos que as células dos doentes com TE apresentam níveis mais baixos do transcrito NFATC2 do que a população normal. Dado que o factor de transcrição MAF desempenha igualmente um papel como regular transcricional de citocinas, é plausível que a haplo-insuficiência dos genes NFATC2 e MAF, resultante do rearranjo cromossómico complexo t(X;20;16), teve um efeito cooperativo importante na patogénese da trombocitémia essencial através da alteração do padrão normal de expressão das citocinas hematopoiéticas. Em síntese, efectuámos nesta dissertação um estudo citogenético de 4 translocações cromossómicas complexas incluindo t(12;21;5), t(12;6;15), t(9;11;19) e t(X;20;16), e de uma translocação dicêntrica dic(9;12), associadas com diferentes neoplasmas hematológicos. Em casos seleccionados efectuámos também um estudo molecular detalhado das regiões dos pontos de quebra. Esta análise permitiu-nos identificar 2 genes, CCDC94 no cromossoma 19 e NFATC2 no cromossoma 20, cuja haplo-insuficiência pode promover o aumento da proliferação celular das células leucémicas. A partir destes estudos podem ser retiradas 2 noções principais: (i) Os pontos de quebra adicionais, que ocorrem em translocações complexas associadas com a formação de genes de fusão, podem ter como consequência a desregulação de genes controladores da proliferação celular (e.g., CCDC94); (ii) As translocações complexas caracterizadas pela ausência de genes de fusão recorrentes poderão estar preferencialmente associadas com a presença de delecções, envolvendo um ou mais genes, nos pontos de quebra; nestas situações, serão necessários pelo menos 2 genes com funções celulares semelhantes (e.g., NFATC2 e MAF) ou complementares (e.g., ETV6 e CDKN1B) para, quando deleccionados, promoverem de forma cooperativa a leucemogénese. Nestes termos, o modelo de alterações genéticas sequenciais que caracteriza o desenvolvimento do cancro pode ser substituído por um modelo em que vários genes-alvo são simultaneamente desregulados pela formação de uma translocação cromossómica complexa, evitando deste modo a necessidade de ocorrência de alterações genéticas subsequentes.----------------------ABSTRACT: Tumourigenesis is a multistep process which results from the accumulation of successive genetic mutations in a normal cell. In leukemia for instance, recurrent translocations play a part in this process by generating fusion genes which lead to the production of hybrid proteins with an oncogenic role. However, a minor subset of chromosomal translocations referred to as complex or variant involves extra breakpoints at variable genome locations in addition to those implicated in the formation of fusion genes. We aimed to describe in this work the role, if any, of genes located at extra breakpoint locations or which are affected by breakpoint-adjacent deletions through the study of 5 leukemia patients.Two of the patients presented with TV6(12p13)-RUNX1(21q22) and MLL(11q23)- MLLT3(9p22) fusion genes as a result of a t(12;21;5) and a t(9;11;19), respectively. Detailed molecular characterization of the extra breakpoint at chromosome 19 in the latter case revealed that a novel ubiquitously expressed gene, CCDC94, with a potential role in cell cycle regulation, was disrupted by the breakpoint. We demonstrated using in vitro cellular assays that this gene codifies for a nuclear protein which negatively regulates cell cycle progression. These data shows that extra breakpoint locations of complex translocations may result in haplo-insufficiency of critical proliferation genes, thereby cooperating with the generation of hybrid proteins to provide unrestrained cell proliferation. In the other 3 patients there were reakpoint-associated deletions which precluded the formation of putative fusion genes. In a case with a t(12;6;15) we characterized a deletion at 12p13 which eliminated ETV6 and 8 other genes including CDKN1B. These findings indicate that concomitant loss of ETV6 and CDKN1B, which encodes a cyclin-dependent kinase inhibitor responsible for blocking entry of cells into the G1 phase of the cell cycle, acted cooperatively to promote leukemogenic proliferation. The same notion applied to a case with a dic(9;12) in which 2 genes encoding hematopoietic transcription factors - ETV6 and PAX5 (9p13)- were deleted as a result of breakpoint-adjacent deletions. Similarly, we found that 2 transcription factor genes involved in the regulation of cytokine expression, NFATC2 (20q13) and MAF (16q23), were involved in deletions contiguous to the breakpoints in a patient with a t(X;20;16). In vitro suppression of NFATC2 mRNA or inhibiton of NFATC2 protein activity enhanced cell proliferation as a result of an increase in the production of a myeloid-lineage stimulating hematopoietic cytokine, GM-CSF. These results suggest that haplo-insufficiency of NFATC2 and MAF genes had a cooperative effect in inducing cell proliferation as a result of a disregulation of cytokine production. Two main conclusions may be drawn from our studies: (i) In complex translocations associated with the production of fusion genes, additional breakpoints may cooperate in tumourigenesis by targeting genes that control cell proliferation; (ii) In complex translocations associated with small breakpoint-adjacent deletions, at least 2 genes with similar or complementary functions need to be deregulated to promote tumourigenesis.

High prevalence of GB Virus C/Hepatitis G Virus RNA among Brazilian blood donors

The aim of this study was to investigate the presence of the Hepatitis G Virus on a population of blood donors from São Paulo, Brazil and to evaluate its association to sociodemographic variables. Two RT-PCR systems targeting the putative 5'NCR and NS3 regions were employed and the former has shown a higher sensitivity. The observed prevalence of HGV-RNA on 545 blood donors was 9.7% (CI 95% 7.4;12.5). Statistical analysis depicted an association with race/ethnicity, black and mulatto donors being more frequently infected; and also with years of education, less educated donors presenting higher prevalences. No association was observed with other sociodemographic parameters as age, gender, place of birth and of residence. DNA sequencing of nine randomly chosen isolates demonstrated the presence of genotypes 1, 2 and 3 among our population but clustering of these Brazilian isolates was not detected upon phylogenetic analysis.

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing

INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

Cytokine profile in Montenegro skin test of patients with localized cutaneous and mucocutaneous leishmaniasis

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL.

Impact of Donor and Recipient Cytokine Genotypes on Renal Allograft Outcome

Allelic differences in gene promoter or codifying regions have been described to affect regulation of gene expression, consequently increasing or decreasing cytokine production and signal transduction responses to a given stimulus. This observation has been reported for interleukin (IL)-10 (-1082 A/G; -819/-592 CT/CA), transforming growth factor (TGF)-beta (codon 10 C/T, codon 25 G/C), tumor necrosis factor (TNF)-alpha (-308 G/A), TNF-beta (+252 A/G), interferon (IFN)-gamma (+874 T/A), IL-6 (-174 G/C), and IL-4R alpha (+1902 G/A). To evaluate the influence of these cytokine genotypes on the development of acute or chronic rejection, we correlated the genotypes of both kidney graft recipients and cadaver donors with the clinical outcome. Kidney recipients had 5 years follow-up, at least 2 HLA-DRB compatibilities, and a maximum of 25% anti-HLA pretransplantation sensitization. The clinical outcomes were grouped as follows: stable functioning graft (NR, n = 35); acute rejection episodes (AR, n = 31); and chronic rejection (CR, n = 31). The cytokine genotype polymorphisms were defined using PCR-SSP typing. A statistical analysis showed a significant prevalence of recipient IL-10 -819/-592 genotype among CR individuals; whereas among donors, the TGF-beta codon 10 CT genotype was significantly associated with the AR cohort and the IL-6 -174 CC genotype with CR. Other albeit not significant observations included a strong predisposition of recipient TGF-beta codon 10 CT genotype with CR, and TNF-beta 252 AA with AR. A low frequency of TNF-alpha -308 AA genotype also was observed among recipients and donors who showed poor allograft outcomes.