Estudo comparativo entre as anastomoses cervicais esofagogástrica término-terminal com e sem invaginação após esofagectomia para câncer

OBJETIVO: Avaliar a incidência de fístula e estenose da anastomose esofagogástrica cervical com invaginação do coto esofágico no interior do estômago na esofagectomia para tratamento do carcinoma do esôfago. MÉTODOS: Foram estudados dois grupos de doentes com carcinoma do esôfago torácico ou abdominal submetidos à esofagectomia subtotal e esofagogastroplastia. O grupo I (estudo) foi constituído por 29 doentes operados no período de 1998 a 2007, no qual foi realizada a anastomose esofagogástrica cervical com invaginação de segmento do coto esofágico no interior do estômago. O grupo II (controle) foi constituído por 36 doentes operados no período de 1989 a 1997 submetidos à anastomose esfagogástrica cervical término-terminal sem invaginação. RESULTADOS: No grupo I, 3 (10,3%) doentes apresentaram fístula da anastomose esofagogástrica com repercussão clínica mínima. No grupo II observou-se fístula com franca saída de saliva em 11 (30,5%) doentes. A freqüência de fístula nos doentes do grupo I foi significantemente menor (p=0,04) do que nos do grupo II. No grupo I, estenose fibrótica da anastomose ocorreu em 7 (24,1%) enfermos, ao passo que no grupo II 10 (27,7%) evoluíram com estenose, não se constatando diferença significante (p=0,72) entre esses grupos. CONCLUSÃO: No tratamento do carcinoma do esôfago, a esofagectomia com anastomose esofagogástrica cervical com invaginação do coto esofágico no interior do estômago determina menor ocorrência de fístula esofagogástrica quando comparado à anastomose sem invaginação. A incidência de estenose da anastomose esofagogástrica não diferiu em ambos os grupos.

Aspectos endoscópicos no diagnóstico de doenças que acometem o íleo terminal

O íleo compreende cerca de 3/5 distais do intestino delgado, sendo responsável pela digestão e absorção de alimentos. O diagnóstico de doenças que afetam esse segmento pode ser feito por meio de avaliação clínica e exames complementares. A colonoscopia, além da possibilidade de análise macroscópica, permite realização de biópsias para avaliação histológica. Apenas três publicações sobre a descrição das características endoscópicas do íleo terminal foram encontradas na literatura. Ainda assim, não foram encontradas descrições ou classificação em publicações que mencionavam o aspecto endoscópico do íleo terminal, sendo reportados apenas como íleo normal. Isso reforça a idéia do desconhecimento ou não aceitação dessas pela comunidade científica. Os aspectos endoscópicos desse segmento, quando afetado por diversas doenças variam de íleo endoscopicamente normal a casos que o exame macroscópico demonstra características especificas dessas doenças. Nesse estudo, existem dúvidas quanto à necessidade de biópsias desse segmento em pacientes com ileoscopia normal. Além disso, foram encontrados poucos estudos com critérios para caracterização macro e microscópica do íleo.

Depressão e ansiedade nos cuidadores de mulheres em fase terminal de câncer de mama e ginecológico

OBJETIVO: avaliar a freqüencia de ansiedade e depressão em cuidadores principais de mulheres em fase terminal de câncer de mama ou genital. MÉTODOS: para este estudo de corte transversal foram incluídos 133 cuidadores de pacientes sem possibilidades curativas, internadas no Centro de Atenção Integral à Saúde da Mulher, entre agosto de 2002 e maio de 2004. Das pacientes incluídas, 71 apresentavam câncer de mama e 62, câncer ginecológico. Foi aplicada a Escala Hospitalar de Ansiedade e Depressão e realizada entrevista para obter outras informações como idade, sexo, religião, parentesco com a paciente, profissão, se cuidava de outras pessoas, se a rotina dele mudou e se outras pessoas ajudavam a cuidar da paciente. Utilizou-se a regressão logística para cálculo do odds ratio (OR) e seus respectivos intervalos de confiança (IC), para avaliar a relação entre os diagnósticos de ansiedade e depressão entre os cuidadores informais. Para a análise múltipla foi considerado o critério de seleção de variáveis passo a passo. RESULTADOS: observou-se que 43% das pacientes indicaram como cuidador principal a filha e 24% o marido. A maioria dos cuidadores tinha idade superior a 35 anos (63%), 68% eram do sexo feminino, 59% estavam desempregados ou aposentados, 47% cuidavam de outra pessoa e 84% referiram mudança na rotina pelo fato de cuidar. A ansiedade foi detectada em 99 cuidadores principais (74,4%) e a depressão em 71 (53,4%), sendo estes estados fortemente relacionados entre si (OR=5,6; IC 95%: 2,2 a 15,9). Na análise bivariada, o marido apresentou menos ansiedade e, após regressão logística, apenas o fato de ser homem esteve relacionado com menor ansiedade. CONCLUSÃO: o processo de cuidar de paciente na fase terminal levou a altas taxas de ansiedade e depressão. Os homens e maridos despontaram neste estudo como cuidadores menos ansiosos.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

Diversity and structure of the tree community of a fragment of tropical secondary forest of the brazilian Atlantic Forest domain 15 and 40 years after logging

Two adjacent tracts of tropical secondary forest, situated in Itambé do Mato Dentro, south-eastern Brazil, which had been regenerating for 15 and 40 years after clearing, were compared with the purpose of detecting differences in species diversity and composition, species guild composition (regeneration, stratification and dispersion), and stand structure. Four and three 1,125 m² plots laid on the 15- and 40-year-old stands, respectively, sampled 2,430 trees with diameter at the base of the stem > 5 cm. The number of species (S = 199) was high for this forest type and significantly higher for the older stand. Tree density was significantly higher in the younger stand, particularly for smaller trees, whereas the two stands did not differ in both basal area and volume per hectare. Trees of shade-tolerant and understory species were significantly more abundant in the older stand. Though sharing a large proportion of species (49%), the two stands differed significantly in the abundance of many species. Live stumps probably contributed to the relatively quick restoration of some forest characteristics, particularly species diversity, basal area and volume.

Lognormal abundance distribution of woody species in a cerrado fragment (São Carlos, southeastern Brazil)

The lognormal distribution model is frequently found in communities, especially those which are rich in species and influenced by many environmental factors, as those of the cerrado. We tested the hypothesis that the abundance distribution of woody plant species in a cerrado fragment fits the lognormal model. We placed 20 lines in a cerrado fragment and sampled, with the point-quarter method, 800 individuals with stem perimeter equal or larger than 3 cm. We plotted the abundance-class histogram of the species, verified its normality with the Kolmogorov-Smirnov test, and estimated the expected number of woody species for this community. Of the 63 obtained species, Anadenanthera falcata (with 185 species), Eriotheca gracilipes (43), Stryphnodendron obovatum (37), and Miconia albicans (36) were the most abundant ones. Twelve species were represented by only one individual. We did not reject the null hypotheses that the distribution of woody component species was normal and, thus, their abundances fitted the lognormal model. Therefore, with our work, we can predict that cerrado plant communities fit the lognormal model. If this pattern is maintained in other cerrado communities, there would be implications for the conservation of this vegetation type, because rare species are susceptible of extinction, and implications to their structure, because the dominant species may act as keystone species.

Floristic composition and similarity analysis of an Atlantic rain forest fragment in Cananéia, São Paulo State, Brazil

The aim of this study is to investigate the floristic composition of an Atlantic rain forest fragment located in Cananéia, São Paulo, Brazil, and to contribute to the knowledge on Atlantic forest through the comparative analysis of this and other floristic surveys both on the southern and southeastern Brazil, in different soil and relief types. We surveyed 215 species in 132 genera and 51 families. Classification and ordination analysis were applied to a binary matrix in order to analyze the similarity among 24 surveys, including the present one, of Atlantic forest from the south and southeast coast of Brazil. Higher floristic similarity was observed among this area and the ones where there was marine influence and more rugged relief. The surveys in areas with greater marine influence (sandy soil) were separated from those in other conditions, possibly indicating a species replacement gradient from the steep slopes towards the lowland and were probably related to different edaphic conditions. A latitudinal gradient was found among the surveys apparently confirming a continuous species replacement along the Atlantic forest, related to a restricted distribution of the species. This suggests that it is essential to preserve areas from the whole Atlantic coast. Atlantic forest distribution is quite complex and its composition cannot be adequately represented by small localized areas.

pH titration of native and unfolded ß-trypsin: evaluation of the D D G0 titration and the carboxyl pK values

The stabilizing free energy of ß-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (D D G0 titration) was 9.51 ± 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in ß-trypsin. In fact, one class of carboxyl group with a pK = 3.91 ± 0.01 and another one with a pK = 4.63 ± 0.03 were also found by hydrogen ion titration of the protein in the folded state

Characterization of six rat strains (Rattus norvegicus) by mitochondrial DNA restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.

Association between EcoRI fragment-length polymorphism of the immunoglobulin lambda variable 8 (IGLV8) gene family with rheumatoid arthritis and systemic lupus erythematosus

The human immunoglobulin lambda variable 8 (IGLV8) subgroup is a gene family containing three members, one of them included in a monomorphic 3.7-kb EcoRI genomic fragment located at the major lambda variable locus on chromosome 22q11.1 (gene IGLV8a, EMBL accession No. Z73650) at 100% frequency in the normal urban population. The second is a polymorphic RFLP allele included in a 6.0-kb EcoRI fragment at 10% frequency, and the third is located in a monomorphic 8.0-kb EcoRI fragment at 100% frequency, the last being translocated to chromosome 8q11.2 and considered to be an orphan gene. Our Southern blot-EcoRI-RFLP studies in normal individuals and in patients with rheumatoid arthritis (RA) or with systemic lupus erythematosus (SLE), using a specific probe for the IGLV8 gene family (probe pVL8, EMBL accession No. X75424), have revealed the two monomorphic genomic fragments containing the IGLV8 genes, i.e., the 3.7-kb fragment from chromosome 22q11.1 and the 8.0-kb fragment from 8q11.2, both occurring at 100% frequency (103 normal individuals, 48 RA and 28 SLE patients analyzed), but absence of the 6.0-kb IGLV8 polymorphic RFLP allele in all RA or SLE patients. As expected, the frequency of the 6.0-kb allele among the normal individuals was 10%. These findings suggest an association between the absence of the 6.0-kb EcoRI fragment and rheumatoid arthritis and systemic lupus erythematosus.

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

IS1245 restriction fragment length polymorphism typing of Mycobacterium avium from patients admitted to a reference hospital in Campinas, Brazil

Mycobacterium avium is an important pathogen among immunodeficient patients, especially patients with AIDS. The natural history of this disease is unclear. Several environmental sources have been implicated as the origin of this infection. Polyclonal infection with this species is observed, challenging the understanding of its pathogenesis and treatment. In the present study 45 M. avium strains were recovered from 39 patients admitted to a reference hospital between 1996 and 1998. Species identification was performed using a species-specific nucleic acid hybridization test (AccuProbe®) from Gen-Probe®. Strains were genotyped using IS1245 restriction fragment length polymorphism typing. Blood was the main source of the organism. In one patient with disseminated disease, M. avium could be recovered more than once from potentially sterile sites. Strains isolated from this patient had different genotypes, indicating that the infection was polyclonal. Four patient clones were characterized in this population, the largest clone being detected in eight patients. This finding points to a common-source transmission of the organism.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

N-terminal-pro-brain natriuretic peptide, but not brain natriuretic peptide, is increased in patients with severe obesity

Elevated body mass index (BMI) has been reported as a risk factor for heart failure. Prevention of heart failure through identification and management of risk factors and preclinical phases of the disease is a priority. Levels of natriuretic peptides as well as activity of their receptors have been found altered in obese persons with some conflicting results. We investigated cardiac involvement in severely obese patients by determining N-terminal-pro-brain natriuretic peptide (NT-proBNP) and brain natriuretic peptide (BNP) and attempting to correlate the levels of these peptides in serum and plasma, respectively, with BMI, duration of obesity, waist circumference, and echocardiographic parameters. Thirty-three patients with severe obesity (mean BMI: 46.39 kg/m², mean age: 39 years) were studied. The control group contained 30 healthy age-matched individuals (BMI: <25 kg/m², mean age: 43 years). The t-test and Spearman correlation were used for statistical analysis. Log-NT-proBNP was significantly higher (P = 0.003) in obese patients (mean 1.67, 95% CI: 1.50-1.83 log pg/mL) compared to controls (mean: 1.32, 95% CI: 1.17-1.47 log pg/mL). The Log-NT-proBNP concentration correlated with duration of obesity (r = 0.339, P < 0.004). No difference was detected in the Log-BNP concentration (P = 0.63) of obese patients (mean: 0.73, 95% CI: 0.46-1.00 log pg/mL) compared to controls (mean: 0.66, 95% CI: 0.51-0.81 log pg/mL). NT-proBNP, but not BNP, is increased in severely obese patients and its concentration in serum is correlated with duration of obesity. NT-proBNP may be useful as an early diagnostic tool for the detection of cardiac burden due to severe obesity.