Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

Estimativa do consumo de energia e de macronutrientes no domicílio e na escola em pré-escolares

OBJETIVO: Estimar o consumo de energia e de macronutrientes no domicílio e na escola em tempo integral em crianças de 2 a 6 anos e pesquisar diferenças no consumo entre as crianças de escolas públicas e particulares. MÉTODOS: Estudo transversal realizado com 362 pré-escolares em Caxias do Sul (RS). O estado nutricional foi avaliado pela razão peso para estatura. O consumo na escola foi avaliado por meio do método de pesagem direta individual dos alimentos consumidos pelas crianças e, no domicílio, por meio do método de registro alimentar realizado pelos pais ou responsáveis. Para as análises estatísticas utilizou-se o teste U de Mann-Whitney (p < 0,05). RESULTADOS: Observou-se que 28 crianças (7,7 por cento) apresentaram excesso de peso, 92 (25,4 por cento), risco para excesso de peso e sete (1,9 por cento), baixo peso para a estatura. A avaliação da ingestão alimentar em 24 horas mostrou que 51,3 por cento da energia, 60,3 por cento dos lipídios e 51,6 por cento das proteínas foramconsumidos nos domicílios, apesar de as crianças permanecerem em período integral nas escolas. Observou-se maior ingestão de energia (p = 0,001), carboidratos (p < 0,001) e lipídios (p = 0,04) nos pré-escolares de escolas particulares em relação aos de escolas públicas, porém o consumo total diário se mostrou similar nas diferentes instituições. CONCLUSÕES: Os achados sugerem que as crianças consomem proporcionalmente mais energia, proteínas e lipídios nas refeições complementares dos domicílios em relação às refeições diárias nas escolas infantis. Apesar das diferenças de consumo entre as escolas públicas e particulares, a ingestão diária mostrou-se similar entre as crianças

Women's satisfaction with childbirth's experience in different models of care: a descriptive study

Com o objetivo de comparar a satisfação das mulheres com a experiência do parto em três modelos assistenciais, foi realizada pesquisa descritiva, com abordagem quantitativa, em dois hospitais públicos de São Paulo, um promovendo o modelo "Típico" e o outro com um centro de parto intra-hospitalar (modelo "CPNIH") e um peri-hospitalar (modelo "CPNPH"). A amostra foi constituída por 90 puérperas, 30 de cada modelo. A comparação entre os resultados referentes à satisfação das mulheres com o atendimento prestado pelos profissionais de saúde, com a qualidade da assistência e os motivos de satisfação e insatisfação, com a indicação ou recomendação dos serviços recebidos, com a sensação de segurança no processo e com as sugestões de melhorias, mostrou que o modelo CPHPH foi o melhor avaliado, vindo em seguida o CPNIH e por último o Típico. Conclui-se que o modelo peri-hospitalar de assistência ao parto deveria receber maior apoio do SUS, por se constituir em serviço em que as mulheres se mostram satisfeitas com a atenção recebida

Pharmacogenetics of OATP Transporters Reveals That SLCO1B1 c.388A > G Variant Is Determinant of Increased Atorvastatin Response

Aims: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated. Material and Methods: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot (R) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan (R) Real-time PCR. Results: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%: 1.3-8.0, p < 0.05). Conclusion: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers

A series of photosensitizers (PS), which are meso-substituted tetra-cationic porphyrins, was synthesized in order to study the role of amphiphilicity and zinc insertion in photodynamic therapy (PDT) efficacy. Several properties of the PS were evaluated and compared within the series including photophysical properties (absorption spectra, fluorescence quantum yield Phi(f), and singlet oxygen quantum yield Phi(Delta)), uptake by vesicles, mitochondria and HeLa cells, dark and phototoxicity in HeLa cells. The photophysical properties of all compounds are quite similar (Phi(f) <= 0.02; Phi(Delta) similar to 0.8). An increase in lipophilicity and the presence of zinc in the porphyrin ring result in higher vesicle and cell uptake. Binding in mitochondria is dependent on the PS lipophilicity and on the electrochemical membrane potential, i.e., in uncoupled mitochondria PS binding decreases by up to 53%. The porphyrin substituted with octyl groups (TC8PyP) is the compound that is most enriched in mitochondria, and its zinc derivative (ZnTC8PyP) has the highest global uptake. The stronger membrane interaction of the zinc-substituted porphyrins is attributed to a complexing effect with phosphate groups of the phospholipids. Zinc insertion was also shown to decrease the interaction with isolated mitochondria and with the mitochondria of HeLa cells, an effect that has been explained by the particular characteristics of the mitochondrial internal membrane. Phototoxicity was shown to increase proportionally with membrane binding efficiency, which is attributed to favorable membrane interactions which allow more efficient membrane photooxidation. For this series of compounds, photodynamic efficiency is directly proportional to the membrane binding and cell uptake, but it is not totally related to mitochondrial targeting.

Phenotypic variation among four family members with congenital hypothyroidism caused by two distinct thyroglobulin gene mutations

Background: Thyroglobulin (Tg) is a large glycoprotein that is intimately involved in the biosynthesis of thyroxine and triiodothyronine. At least 38 mutations have been described in the Tg gene that are associated with varying degrees of hypothyroidism. We studied the Tg gene in four related subjects with congenital hypothyroidism. Summary: We found a novel compound heterozygous constellation (IVS30 + 1G>T/A2215D) in a brother and sister and one previously described related mutation (IVS30+1G>T) in their two sibling second degree cousins. The brother with the IVS30 + 1G>T/A2215D mutation and the two siblings with the IVS30+1G>T mutation had fetal or neonatal goiter and all had hypothyroidism. Conclusions: This study further confirms the association of the IVS30+1G>T mutation of the Tg gene with hypothyroidism. Computer analysis predicts that the A2215D mutation, first reported here, should cause structural instability of Tg but when present as a compound heterozygous mutation with IVS30+G>T/A its effect is unclear but is likely to be influenced by iodine intake.

Infliximab Induces Increase in Triglyceride Levels in Psoriatic Arthritis Patients

Objectives. To evaluate lipid profile changes after anti-TNF therapy in patients with psoriatic arthritis (PsA). Methods. Fifteen PsA patients (eight polyarticular, four oligoarticular, two axial, and one mutilating) under infliximab were included. None had dyslipoproteinemia or previous statin use. Total cholesterol (TC) and its fractions, inflammatory markers, and prednisone use were evaluated. Results. The comparisons of lipid levels between baseline and after three months (3M) of anti-TNF therapy showed that there was a significant increase in mean triglycerides (117.8 +/- 49.7 versus 140.1 +/- 64.1 mg/dL, P = 0.028) and VLDL-c (23.6 +/- 10.5 versus 28.4 +/- 13.7 mg/dL, P = 0.019) levels. In contrast, there were no differences in the mean TC (P = 0.28), LDL-c (P = 0.42), and HDL-c (P = 0.26) levels. Analysis of the frequencies of each lipid alteration at baseline and at 3M were alike (P > 0.05). Positive correlations were found between VLDL-c and CRP (r = 0.647, P = 0.009) and between triglycerides and CRP (r = 0.604, P = 0.017) levels at 3M. ESR reduction was observed after 3M (P = 0.04). Mean prednisone dose remained stable at beginning and at 3M (P = 0.37). Conclusion. This study demonstrated that anti-TNF may increase TG and VLDL-c levels in PsA patients after three months.

Comparison of the Effects of Electrical Field Stimulation and Low-Level Laser Therapy on Bone Loss in Spinal Cord-Injured Rats

Objective: This study investigated the effects of low-level laser therapy (LLLT) and electrical stimulation (ES) on bone loss in spinal cord-injured rats. Materials and Methods: Thirty-seven male Wistar rats were divided into four groups: standard control group (CG); spinal cord-injured control (SC); spinal cord-injured treated with laser (SCL; GaAlAs, 830 nm, CW, 30mW/cm, 250 J/cm(2)); and spinal cord-injured treated with electrical field stimulation (SCE; 1.5 MHz, 1: 4 duty cycles, 30 mW, 20 min). Biomechanical, densitometric, and morphometric analyses were performed. Results: SC rats showed a significant decrease in bone mass, biomechanical properties, and morphometric parameters (versus CG). SCE rats showed significantly higher values of inner diameter and internal and external areas of tibia diaphyses; and the SCL group showed a trend toward the same result (versus SC). No increase was found in either mechanical or densitometric parameters. Conclusion: We conclude that the mentioned treatments were able to initiate a positive bone-tissue response, maybe through stimulation of osteoblasts, which was able to determine the observed morphometric modifications. However, the evoked tissue response could not determine either biomechanical or densitometric modifications.

Abnormal collagen deposition in synovia after collagen type V immunization in rabbits

Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N= 10) were immunized with collagen V plus Freund's adjuvant and compared with animals inoculated with adjuvant only (N= 10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69 +/- 80.31; 24.46 +/- 2.58; 70.51 +/- 7.66, respectively) in immunized rabbits when compared with animals from control group (164.91 +/- 15.67; 12.89 +/- 1.05; 32 +/- 3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients.

Prospective and Randomized Comparison of Two Techniques of Staple Line Reinforcement During Open Roux-en-Y Gastric Bypass: Oversewing and Bioabsorbable Seamguard (R)

Aims: Surgical staple line dehiscence usually leads to severe complications. Several techniques and materials have been used to reinforce this stapling and thus reduce the related complications. The objective was to compare safety of two types of anastomotic reinforcement in open gastric bypass. Methods: A prospective, randomized study comparing an extraluminal suture, fibrin glue, and a nonpermanent buttressing material, Seamguard (R), for staple line reinforcement. Fibrin glue was excluded from the study and analysis after two leaks, requiring surgical reintervention, antibiotic therapy, and prolonged patient hospitalization. Results: Twenty patients were assigned to the suture and Seamguard reinforcement groups. The groups were similar in terms of preoperative characteristics. No staple line dehiscence occurred in the two groups, whereas two cases of dehiscence occurred in the fibrin glue group. No mortality occurred and surgical time was statistically similar for both techniques. Seamguard made the surgery more expensive. Conclusion: In our service, staple line reinforcement in open bariatric surgery with oversewing or Seamguard was considered to be safe. Seamguard application was considered to be easier than oversewing, but more expensive.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Hantavirus Pulmonary Syndrome, Central Plateau, Southeastern, and Southern Brazil

Hantavirus pulmonary syndrome (HPS) is an increasing health problem in Brazil because of encroachment of sprawling urban, agricultural, and cattle-raising areas into habitats of subfamily Sigmodontinae rodents, which serve as hantavirus reservoirs. From 1993 through June 2007, a total of 884 cases of HIPS were reported in Brazil (case-fatality rate 39%). To better understand this emerging disease, we collected 89 human serum samples and 68 rodent lung samples containing antibodies to hantavirus from a 2,500-km-wide area in Brazil. RNA was isolated from human samples and rodent lung tissues and subjected to reverse transcription-PCR. Partial sequences of nucleocapsid protein and glycoprotein genes from 22 human and 16 rodent sources indicated only Araraquara virus and Juquitiba virus lineages. The case-fatality rate of HPS was higher in the area with Araraquara virus. This virus, which may be the most virulent hantavirus in Brazil, was associated with areas that have had greater anthropogenic changes.

Vascular Endothelial Growth Factor Haplotypes Associated with Childhood Obesity

Expansion of adipose tissue in obesity is associated with angiogenesis and adipose tissue mass depends on neovascularization. Vascular endothelial growth factor (VEGF) is the main angiogenic factor in the adipose tissue, and VEGF expression is tightly regulated at both transcriptional and translational levels. However, no previous study has tested the hypothesis that genetic polymorphisms in the VEGF gene could affect susceptibility to obesity. To test this hypothesis, we compared the distribution of genotypes and haplotypes including three VEGF genetic polymorphisms in obese children and adolescents with those found in healthy controls. We studied 172 healthy children and adolescents and 113 obese children and adolescents. Genotypes of three clinically relevant VEGF polymorphisms in the promoter region (C-2578A, G-1154A, and G-634C) of the VEGF gene were determined by TaqMan allele discrimination assay and real-time polymerase chain reaction. VEGF haplotypes were inferred using Haplo. stats and PHASE 2.1 programs. We found no differences in the distributions of VEGF genotypes and alleles (p > 0.05). However, the CAG haplotype was more frequent in the obese group than in the control group (4% versus 0%, respectively, in white subjects; p = 0.008; odds ratio 10.148 (95% confidence interval: 1.098-93.788). Our findings suggest that VEGF haplotypes affect susceptibility to obesity in children and adolescents.