Update of the Gene Discovery Program in Schistosoma mansoni with the Expressed Sequence Tag Approach

Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of these genes (81%) had not previously been described in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor

Transtorno de ansiedade generalizada em idosos com oitenta anos ou mais

OBJETIVOS: Descrever a prevalência de transtorno de ansiedade generalizada (TAG) em uma população de idosos residentes em uma comunidade e com idade acima de 80 anos e comparar os padrões de sono, a função cognitiva e a taxa de prevalência de outros diagnósticos psiquiátricos entre controles normais e sujeitos com TAG. MÉTODOS: Para o diagnóstico de TAG, foram utilizados os critérios do "Diagnostic and Statistical Manual of Mental Disorders" (DSM-IV). Selecionou-se uma amostra randômica e representativa de 77 sujeitos (35%), residentes em uma comunidade, entre todos os idosos com idade acima de 80 anos do município Veranópolis, RS. Os padrões de sono foram aferidos pelo índice de qualidade de sono de Pittsburgh e pelo diário sobre sono/vigília a ser preenchido ao longo de duas semanas. Cinco testes neuropsicológicos foram usados na avaliação cognitiva: teste das lembranças seletivas de Buschke-Fuld; lista de palavras da bateria CERAD (Consortium to Establish a Registry for Alzheimer's Disease); teste de fluência verbal e dois subtestes da escala de memória Wechsler. RESULTADOS: A prevalência estimada de TAG foi de 10,6%, cuja presença estava associada a uma maior ocorrência de depressão clinicamente diagnosticável, com um significativo maior número de sintomas depressivos, quando medidos pela escala de depressão geriátrica, e com uma maior ocorrência de depressão menor. Os padrões de sono e o funcionamento cognitivo, entre sujeitos com TAG, não estavam afetados. A gravidade das doenças físicas não variava entre sujeitos com TAG e os controles normais. A presença de TAG estava associada a um significativo pior padrão de qualidade de vida relativa à saúde. CONCLUSÃO: Em comparação com os estudos prévios, a prevalência de TAG é alta entre a população de idosos mais velhos. Esse transtorno ocorre em freqüente associação com a sintomatologia depressiva e também está associado a um pior padrão de qualidade de vida relativa à saúde.

Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

Transtornos de ansiedade: um estudo de prevalência e comorbidade com tabagismo em um ambulatório de psiquiatria

OBJETIVOS: Este estudo tem por objetivo investigar a presença de transtornos de ansiedade e tabagismo entre pacientes atendidos em um ambulatório de psiquiatria. MÉTODOS: Realizou-se um estudo transversal em um ambulatório de psiquiatria em Porto Alegre, no qual se aplicou SCID-I em 84 pacientes, para examinar a ocorrência de transtornos de ansiedade e dependência à nicotina; também se utilizou o Teste de Fagerström para identificar o grau de dependência nicotínica. Excluíram-se pacientes com esquizofrenia, outros transtornos psicóticos e retardo mental. RESULTADOS: Verificou-se freqüência de 75% para os transtornos de ansiedade, sendo mais freqüentes fobia específica e transtorno de ansiedade generalizada (TAG) com 26,2% cada um. Tabagistas representaram 21,43% da amostra, e a maior parte destes obteve escore leve para o grau de dependência. Constatou-se associação entre ter TAG e ser tabagista, e a chance dos pacientes com TAG fumarem é 5,2 vezes em relação aos que não têm esse transtorno de ansiedade. CONCLUSÕES: Os transtornos de ansiedade têm uma freqüência elevada entre pacientes ambulatoriais, sendo importante sua identificação. A freqüência de tabagismo entre pacientes com transtorno de ansiedade é alta, apresentando importante associação com TAG, e por isso deve também ser foco de atenção no tratamento desses pacientes.

Risco de suicídio e comorbidades psiquiátricas no transtorno de ansiedade generalizada

RESUMO Objetivo Investigar o risco de suicídio nos pacientes com transtorno de ansiedade generalizada (TAG). Métodos Estudo transversal com 253 pacientes dos ambulatórios de Psiquiatria, Nefrologia e Cardiologia do Hospital Universitário da Universidade Federal de Alagoas. O instrumento utilizado foi o MINI 5.0.0 (Mini-International Neuropsychiatric Interview). Resultados Os indivíduos com TAG corresponderam a 16,6% da amostra, sendo 30,8% nos pacientes entrevistados no ambulatório de psiquiatria e 11,3% nos pacientes entrevistados nos demais ambulatórios. A maioria é do sexo feminino (83,3%), casada (53,8%), sendo a média de idade de 44 (±12,2) anos. Houve comorbidade psiquiátrica em 88,1% dos entrevistados; 54,8% possuíam duas ou mais e 33,3% tinham apenas uma. A mais prevalente foi depressão maior (53,7%), seguida de distimia (21,4%) e depressão recorrente (14,6%). O risco de suicídio foi observado em 54,8% dos indivíduos com TAG. Conclusão A prevalência de TAG foi significativamente maior que na população geral, principalmente no ambulatório de psiquiatria. A maioria dos pacientes apresentou comorbidade psiquiátrica, especialmente transtornos de humor, além de risco de suicídio. O TAG mostrou elevada correlação com transtornos de humor e significativa associação com o risco de suicídio.

Prevalência de transtornos ansiosos e algumas comorbidades em idosos: um estudo de base populacional

RESUMO Objetivo Avaliar a prevalência de transtornos ansiosos e fatores associados em uma amostra populacional de idosos do Sul de Santa Catarina. Métodos Estudo transversal com base em dados populacionais, que avaliou 1.021 indivíduos idosos entre 60 e 79 anos. Foram realizadas entrevistas domiciliares para aferição de variáveis sobre transtornos ansiosos, por meio do questionário MINI, dados sociodemográficos, hipertensão arterial sistêmica (HAS), infarto agudo do miocárdio (IAM) e dosagem de colesterol. Resultados As prevalências entre os transtornos ansiosos foram de 22,0% para o transtorno de ansiedade generalizada (TAG); 14,8% para fobia social (FS); 10,5% para transtorno do pânico (TP); e 8,5% para o transtorno obsessivo-compulsivo (TOC). Além disso, 40,5% dos indivíduos apresentaram pelo menos um transtorno de ansiedade. A distribuição dos transtornos foi semelhante nos dois gêneros; TAG foi mais prevalente nos indivíduos de menor escolaridade; TOC foi mais presente em indivíduos casados ou em união estável. Em relação às variáveis clínicas, HAS foi associada à presença de TOC; FS foi associada com IAM; TOC e FS foram associados com HDL > 40 mg/dL. Conclusão Os dados demonstram que os quadros de ansiedade são muito frequentes em idosos da comunidade, se sobrepõem de forma significativa e estão associados a algumas variáveis clínicas cardiovasculares.

Perfil glicometabólico inicial em pacientes com síndrome coronariana aguda e síndrome metabólica

FUNDAMENTO: Pacientes com síndrome metabólica (SM) têm alto risco coronariano e a disfunção da célula beta ou resistência à insulina pode prever um risco adicional de eventos cardiovasculares precoces. OBJETIVO: Avaliar as alterações glicometabólicas precoces em pacientes com SM, mas sem diagnóstico de diabete tipo 2, após síndrome coronariana aguda. MÉTODOS: Um total de 114 pacientes foi submetido ao teste oral de tolerância à glicose (TOTG), 1-3 dias da alta hospitalar, após infarto agudo do miocárdio ou angina instável. Baseado no TOTG, definimos três grupos de pacientes: tolerância normal à glicose (TNG; n=26), tolerância alterada à glicose (TAG; n=39) ou diabetes mellitus (DM; n=49). O Modelo de Avaliação da Homeostase (HOMA-IR) foi usado para estimar a resistência à insulina; a responsividade da célula beta foi avaliada através do índice insulinogênico de 30 minutos (ΔI30/ΔG30). RESULTADOS: Baseado no HOMA-IR, os pacientes com DM eram mais insulino-resistentes do que aqueles com TNG ou TAG (p<0,001). De acordo com o índice insulinogênico, a responsividade da célula beta também estava alterada em indivíduos com DM (p<0,001 vs TNG ou TAG). CONCLUSÃO: Altas taxas de alterações glicometabólicas foram encontradas após síndrome coronariana aguda em pacientes com SM. Como essas anormalidades acentuadamente aumentam o risco de desfechos adversos, o TOTG precoce pode ser utilizado em pacientes com SM para identificar aqueles que apresentam maior risco coronariano.

Protection against Toxoplasmosis in Mice Immunized with Different Antigens of Toxoplasma gondii Incorporated into Liposomes

Different toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoite plus tissue cyst (L/TCAg) or purified antigen of tachyzoite (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kD in SDS PAGE and silver stain using reduced condition. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by Elisa. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigens entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts.

r-Sm14 - pRSETA efficacy in experimental animals

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

Clustering of Schistosoma mansoni mRNA sequences and analysis of the most transcribed genes: implications in metabolism and biology of different developmental stages

The study of the Schistosoma mansoni genome, one of the etiologic agents of human schistosomiasis, is essential for a better understanding of the biology and development of this parasite. In order to get an overview of all S. mansoni catalogued gene sequences, we performed a clustering analysis of the parasite mRNA sequences available in public databases. This was made using softwares PHRAP and CAP3. The consensus sequences, generated after the alignment of cluster constituent sequences, allowed the identification by database homology searches of the most expressed genes in the worm. We analyzed these genes and looked for a correlation between their high expression and parasite metabolism and biology. We observed that the majority of these genes is related to the maintenance of basic cell functions, encoding genes whose products are related to the cytoskeleton, intracellular transport and energy metabolism. Evidences are presented here that genes for aerobic energy metabolism are expressed in all the developmental stages analyzed. Some of the most expressed genes could not be identified by homology searches and may have some specific functions in the parasite.

Adjuvant requirement for successful immunization with recombinant derivatives of Plasmodium vivax merozoite surface protein-1 delivered via the intranasal route

Recently, we generated two bacterial recombinant proteins expressing 89 amino acids of the C-terminal domain of the Plasmodium vivax merozoite surface protein-1 and the hexa-histidine tag (His6MSP1(19)). One of these recombinant proteins contained also the amino acid sequence of the universal pan allelic T-cell epitope (His6MSP1(19)-PADRE). In the present study, we evaluated the immunogenic properties of these antigens when administered via the intra-nasal route in the presence of distinct adjuvant formulations. We found that C57BL/6 mice immunized with either recombinant proteins in the presence of the adjuvants cholera toxin (CT) or the Escherichia coli heat labile toxin (LT) developed high and long lasting titers of specific serum antibodies. The induced immune responses reached maximum levels after three immunizing doses with a prevailing IgG1 subclass response. In contrast, mice immunized by intranasal route with His6MSP1(19)-PADRE in the presence of the synthetic oligonucleotides adjuvant CpG ODN 1826 developed lower antibody titers but when combined to CT, CpG addition resulted in enhanced IgG responses characterized by lower IgG1 levels. Considering the limitations of antigens formulations that can be used in humans, mucosal adjuvants can be a reliable alternative for the development of new strategies of immunization using recombinant proteins of P. vivax.

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.

Examining ERBB2 as a candidate gene for susceptibility to leprosy (Hansen’s disease) in Brazil

Leprosy remains prevalent in Brazil. ErbB2 is a receptor for leprosy bacilli entering Schwann cells, which mediates Mycobacterium leprae-induced demyelination and the ERBB2 gene lies within a leprosy susceptibility locus on chromosome 17q11-q21. To determine whether polymorphisms at the ERBB2 locus contribute to this linkage peak, three haplotype tagging single nucleotide polymorphisms (tag-SNPs) (rs2517956, rs2952156, rs1058808) were genotyped in 72 families (208 cases; 372 individuals) from the state of Pará (PA). All three tag-SNPs were associated with leprosy per se [best SNP rs2517959 odds ratio (OR) = 2.22; 95% confidence interval (CI) 1.37-3.59; p = 0.001]. Lepromatous (LL) (OR = 3.25; 95% CI 1.37-7.70; p = 0.007) and tuberculoid (TT) (OR = 1.79; 95% CI 1.04-3.05; p = 0.034) leprosy both contributed to the association, which is consistent with the previous linkage to chromosome 17q11-q21 in the population from PA and supports the functional role of ErbB2 in disease pathogenesis. To attempt to replicate these findings, six SNPs (rs2517955, rs2517956, rs1810132, rs2952156, rs1801200, rs1058808) were genotyped in a population-based sample of 570 leprosy cases and 370 controls from the state of Rio Grande do Norte (RN) and the results were analysed using logistic regression analysis. However, none of the associations were replicated in the RN sample, whether analysed for leprosy per se, LL leprosy, TT leprosy, erythema nodosum leprosum or reversal reaction conditions. The role of polymorphisms at ERBB2 in controlling susceptibility to leprosy in Brazil therefore remains unclear.

Construction of three new Gateway&#174; expression plasmids for Trypanosoma cruzi

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).