Progression of valve heart disease in a cohort of patients undergoing renal replacement therapy / Progressão da doença cardíaca valvar em coorte de doentes em terapia renal substitutiva

Abstract Introduction: Cardiovascular disease is an important cause of death among patients with chronic kidney disease (CKD). Valve calcification is a predictor of cardiovascular mortality and coronary artery disease. Objective: To assess heart valve disease frequency, associated factors, and progression in CKD patients. Methods: We conducted a retrospective study on 291 CKD patients at Hospital das Clínicas de Pernambuco. Inclusion criteria were age ≥ 18 with CKD and valve disease, while those on conservative management or with missing data were excluded. Clinical and laboratory variables were compared, and patients were categorized by dialysis duration (<5 years; 5-10 years; >10 years). Statistical tests, including chi-square, Fisher's exact, ANOVA, and Kruskal-Wallis, were employed as needed. Simple and multivariate binary regression models were used to analyze valve disease associations with dialysis duration. Significance was defined as p < 0.05. Results: Mitral valve disease was present in 82.5% (240) of patients, followed by aortic valve disease (65.6%; 86). Over time, 106 (36.4%) patients developed valve disease. No significant association was found between aortic, pulmonary, mitral, or tricuspid valve disease and dialysis duration. Secondary hyperparathyroidism was the sole statistically significant factor for mitral valve disease in the regression model (OR 2.59 [95% CI: 1.09-6.18]; p = 0.031). Conclusion: CKD patients on renal replacement therapy exhibit a high frequency of valve disease, particularly mitral and aortic valve disease. However, no link was established between dialysis duration and valve disease occurrence or progression.

Resumo Introdução: Doenças cardiovasculares são uma causa significativa de morte em pacientes com Doença Renal Crônica (DRC). A calcificação valvar é preditor de mortalidade cardiovascular e doença arterial coronariana. Objetivo: Avaliar a frequência, fatores associados e progressão de valvopatias em pacientes com DRC. Métodos: Coorte retrospectiva com 291 pacientes ambulatoriais no Hospital das Clínicas de Pernambuco. Inclusão: ≥18 anos com DRC e valvopatia; exclusão: tratamento conservador ou dados incompletos. Variáveis clínicas e laboratoriais foram comparadas e categorizadas por tempo de terapia dialítica (TTD): <5 anos, 5-10 anos, >10 anos. Foram aplicados os testes Qui-quadrado, exato de Fisher, ANOVA, Kruskal-Wallis. Associação entre valvopatia e TTD foi avaliada por regressão binária. Significância foi definida como p < 0,05. Resultados: A valvopatia mitral foi encontrada em 82,5% (240) dos casos, seguida da aórtica (65,6%; 86). Houve progressão da doença valvar em 106 (36,4%) pacientes. Não houve associação entre valvopatias aórtica, pulmonar, mitral ou tricúspide e TTD. Hiperparatireoidismo secundário foi a única variável explicativa significativa na regressão para valvopatia mitral (OR 2,59 [IC95%: 1,09-6,18]; p = 0,031). Conclusão: Encontramos alta frequência de valvopatias, especialmente mitral e aórtica, aem pacientes com DRC. Não houve associação entre TTD e valvopatia.

Progression of valve heart disease in a cohort of patients undergoing renal replacement therapy.

INTRODUCTION: Cardiovascular disease is an important cause of death among patients with chronic kidney disease (CKD). Valve calcification is a predictor of cardiovascular mortality and coronary artery disease. OBJECTIVE: To assess heart valve disease frequency, associated factors, and progression in CKD patients. METHODS: We conducted a retrospective study on 291 CKD patients at Hospital das Clínicas de Pernambuco. Inclusion criteria were age ≥ 18 with CKD and valve disease, while those on conservative management or with missing data were excluded. Clinical and laboratory variables were compared, and patients were categorized by dialysis duration (<5 years; 5-10 years; >10 years). Statistical tests, including chi-square, Fisher's exact, ANOVA, and Kruskal-Wallis, were employed as needed. Simple and multivariate binary regression models were used to analyze valve disease associations with dialysis duration. Significance was defined as p < 0.05. RESULTS: Mitral valve disease was present in 82.5% (240) of patients, followed by aortic valve disease (65.6%; 86). Over time, 106 (36.4%) patients developed valve disease. No significant association was found between aortic, pulmonary, mitral, or tricuspid valve disease and dialysis duration. Secondary hyperparathyroidism was the sole statistically significant factor for mitral valve disease in the regression model (OR 2.59 [95% CI: 1.09-6.18]; p = 0.031). CONCLUSION: CKD patients on renal replacement therapy exhibit a high frequency of valve disease, particularly mitral and aortic valve disease. However, no link was established between dialysis duration and valve disease occurrence or progression.

Occult Renal Calcifications in Patients with Normocalcemic Primary Hyperparathyroidism and Their Association with the Parathyroid Hormone-Vitamin D Axis.

Normocalcemic primary hyperparathyroidism (NPHPT) is characterized by elevated serum levels of parathyroid hormone (PTH) with persistently normal serum calcium concentrations after excluding secondary causes of hyperparathyroidism. Urolithiasis and/or nephrocalcinosis may occur in hypercalcemic PHPT, but little is known about these complications in NPHPT. Objectives. To identify occult urolithiasis and nephrocalcinosis in asymptomatic patients with NPHPT and evaluate biochemical markers as risk predictors for the development of renal calcification (RC). Methods. Cross-sectional analysis of 34 patients with no history of urolithiasis and/or nephrocalcinosis. The diagnosis of NPHPT was as follows: elevated serum PTH (reference range: 15-65 pg/mL), normal albumin-corrected serum calcium, normal urinary calcium excretion, serum 25(OH)D >30 ng/mL, eGFR (CKD-EPI) > 60 mL/min/1.73 m2, without intestinal disease, and not on medications such as thiazide diuretics, lithium, bisphosphonates, or denosumab. Patients were categorized according to the presence or absence of RC identified by renal imaging. Their clinical and biochemical characteristics were then compared. Results. The patients had a mean age of 67.97 ± 10.45 years, predominantly postmenopausal women (88.2%); serum PTH, 119.67 ± 64.44 pg/mL; 25(OH)D, 39.00 ± 8.88 ng/dL; 1.25(OH))2D, 74.53 ± 26.37 pg/mL; corrected serum calcium, 9.34 ± 0.62 mg/dL; and 24-hour urinary calcium, 134.87 ± 79.68 mg/day. RC was identified in 26.5% of the patients. There was no difference in anthropometric and clinical parameters, renal function, 25(OH)D, and urinary pH in patients with or without RC. Patients with RC had higher PTH values (176.22 vs. 99.32 pg/mL, P = 0.001), 1.25(OH) 2D (96.83 vs. 62.36 pg/mL, P = 0.005), and 24-hour urinary calcium (181.9 vs. 117.94 mg/day, P = 0.037). Conclusion. Occult renal calcifications are common in NPHPT and are associated with increased serum PTH, 1.25(OH))2D, and 24 h urinary calcium.

Decompensated Heart Failure with Mid-Range Ejection Fraction: Epidemiology and In-Hospital Mortality Risk Factors

Abstract Background: Recently, a new HF entity, with LVEF between 40-49%, was presented to comprehend and seek better therapy for HF with preserved LVEF (HFpEF) and borderline, in the means that HF with reduced LVEF (HFrEF) already has well-defined therapy in the literature. Objective: To compare the clinical-therapeutic profile of patients with HF with mid-range LVEF (HFmrEF) with HFpEF and HFrEF and to verify predictors of hospital mortality. Method: Historical cohort of patients admitted with decompensated HF at a supplementary hospital in Recife/PE between April/2007 - August/2017, stratified by LVEF (< 40%/40 - 49/≥ 50%), based on the guideline of the European Society of Cardiology (ESC) 2016. The groups were compared and Logistic Regression was used to identify predictors of independent risk for in-hospital death. Results: A sample of 493 patients, most with HFrEF (43%), HFpEF (30%) and HFmrEF (26%). Average age of 73 (± 14) years, 59% men. Hospital mortality 14%, readmission within 30 days 19%. In therapeutics, it presented statistical significance among the 3 groups, spironolactone, in HFrEF patients. Hospital death and readmission within 30 days did not make difference. In the HFmrEF group, factors independently associated with death were: valve disease (OR: 4.17, CI: 1.01-9.13), altered urea at admission (OR: 6.18, CI: 1.78-11.45) and beta-blocker hospitalization (OR: 0.29, CI: 0.08-0.97). In HFrEF, predictors were: prior renal disease (OR: 2.84, CI: 1.19-6.79), beta-blocker at admission (OR: 0.29, CI: 0.12-0.72) and ACEI/ ARB (OR: 0.21, CI: 0.09-0.49). In HFpEF, only valve disease (OR: 4.61, CI: 1.33-15.96) and kidney disease (OR: 5.18, CI: 1.68-11.98) were relevant. Conclusion: In general, HFmrEF presented intermediate characteristics between HFrEF and HFpEF. Independent predictors of mortality may support risk stratification and management of this group.

Assessment of the usage of biodegradable polymeric matrix in vaginal devices to sustain progesterone release in cows.

The usage of timed artificial insemination (TAI) at a low cost leading to better reproductive rates has been the aim of several research groups in the field. Usually during TAI protocols, sustained progesterone (P4 ) release devices are employed. Most devices are constituted of a nylon skeleton covered with a silicon layer with P4 . A device based on biopolymers was developed in order to reduce costs and decrease its environmental impact. In this study, we compared the kinetics of sustained progesterone release among devices manufactured with a polymeric blend made of polyhydroxybutyrate-valerate (PHBV) and poly-ε-caprolactone (PCL) (DISP) which were compared with DIB® (Internal Bovine Device) used as the control. In the in vitro and in vivo progesterone release tests, two types of biopolymer-based devices with a superficial area of 147 cm2 were used: DISP8 (46% PHBV, 46% PCL and 8% P4 ; n = 4), DISP10 (45% PHBV, 45% PCL, 10% P4 ; n = 4) and DIB® (1 g P4 , 120 cm2 area; n = 3). The in vitro tests were carried out according to USP XXIII specifications and were performed in a dissolutor sink using an alcohol/water mixture (60/40 v/v) as a release media and samples were collected at 2 min, 2, 4, 8, 12, 24, 48, 60, 72, 84 and 96 h. P4 concentrations were measured through spectrophotometry in a 244 nm long wave. Three to 3 comparisons of angular coefficients of the straight lines obtained by regression analysis of accumulated P4 concentrations as a function of square root of time were carried out. Furthermore, the diffusion coefficient values of P4 were also determined for DISP8 and DISP10. The results showed that the concentrations of P4 were higher in the DISP10 (774.63 ± 45.26 µg/cm2 /t1/2 ) compared to DISP8 (566.17 ± 3.68 µg/cm2 /t1/2 ) (P < 0.05). However, both DISP10 and DISP8 P4 concentrations did not differ from DIB® (677.39 ± 16.13 µg/cm2 /t1/2 ). For the analysis of released quantities per day of the in vitro test, four periods were considered: 0-24, 24-48, 48-72 and 72-96 h. In the first 24 h, DISP8 released significantly less P4 than DISP10 or DIB®, which did not differ among them. Between 24 and 48 h, DISP10 released significantly more P4 than DIB®. DISP8 released an intermediate P4 amount and did not differ significantly from DIB® or DISP10. Between 48 and 72 h, P4 quantity released by DISP10 was significant higher (P < 0.01) than that of DIB® and DISP8, which did not differ among themselves. Between 72 and 96 h, DISP10 released significantly more P4 than DIB®, and DISP8 released an intermediate amount which did not differ from DIB® or DISP10 (P < 0.01). There was interaction between treatment and time (P = 0.0024). The diffusion coefficient values were: 1.36 × 10-8 (cm2 /s) for DISP10 and 1.12 × 10-8 (cm2 /s) for DISP8. For the in vivo test, ovariectomized crossbred cows received DIB® (n = 4) or DISP8 (n = 8) in an alternate design with a non-balanced sequence (cross-over) added of measures repeated in time referring to 16 days of blood samples collection. Samples were analyzed through radioimmunoassay in solid phase using the commercial kit of DPC (Diagnostics Products Corporation). Plasma concentrations of P4 peaked at 4 h after the placement of the device, this being the only time in which plasma P4 concentrations differed between DIB® (11.45 ± 1.96) compared with DISP8 (9.23 ± 1.15 ng/mL) (P = 0.027). On day 8, plasma P4 concentrations were similar for DIB® (2.44 ± 0.09) and DISP8 (1.89 ± 0.13 ng/mL) (P = 0.58) showing that both devices were able to keep P4 concentrations above 1 ng/mL in the plasma of the cow during the 16 day in vivo test. In conclusion, devices manufactured with the blend of PHBV/PCL biopolymers can sustain the release of P4 in a similar manner as silicon.

Selenium Status in Patients with Turner Syndrome: a Biochemical Assessment Related with Body Composition.

Studies about selenium status in patients with Turner syndrome (TS) are non-existent in the literature. The aim of this study was to evaluate selenium status in patients with TS, while considering the different ages of the studied population and the relation with body composition. In total, 33 patients with TS were evaluated and grouped according to their developmental stages (children, adolescents, and adults). Selenium concentrations in their plasma, erythrocytes, urine, and nails were determined by using hydride generation atomic absorption spectrometry and erythrocyte glutathione peroxidase activity were measured by using Randox commercial kits. Additionally, height, weight, body fat percentage, waist circumference, and waist-height ratio were measured to characterize the patients. No differences in the selenium concentrations in the plasma, erythrocyte, urine, and nails or in the glutathione peroxidase activity were observed among the age groups (p > 0.05). The evaluated selenium levels were less than the established normal ones. The patients with larger waist circumference, body fat percentage, body mass index, and waist-height ratio showed lower glutathione peroxidase enzyme activity (p = 0.023). The present study shows that most patients with TS are deficient in selenium and that those with a greater accumulation of body fat have a lower GPx activity.

Integração lavoura pecuária floresta na região nordeste do Brasil / Integrated systems for the Brazilian northeast

A região Nordeste do Brasil compreende 1,56 milhões de km2, dos quais o Semiárido ocupa 0,98 milhão, sendo o 0,58 milhão restante ocupado pelo Meio Norte, Zona da Mata e Agreste. Áreas de cerrado são encontradas no Nordeste nos estados do Maranhão, Piauí e Bahia, que juntamente com parte do Tocantins constituem o MaToPiBa. Nessas áreas é praticada uma lavoura intensiva. A forma de integração mais adaptada para a sub-região é a ILP. Em localidades do Meio Norte fora da área do MaToPiBa existem exemplos de integração pecuária/floresta, constituídos por pastagens sobre cajueiros, coqueiros e babaçuais. Resultados de pesquisa comprovam as vantagens desses sistemas integrados em relação aos sistemas de monocultura. Para o Semiárido o foco é o manejo racional da vegetação nativa da caatinga e o desenvolvimento de modelos produtivos. Além das nativas, espécies exóticas fazem parte dos estudos. Quanto aos modelos produtivos o foco tem sido a integração dos elementos nativos ou exóticos adaptados. As pesquisas com ILPF para as Zonas da Mata e Agreste estão em sua maioria baseadas no uso da gliricídia ou da leucena como componente arbóreo em consórcio com lavouras, gramíneas e palma forrageira. Especificamente para a condição das áreas costeiras o consórcio da gliricídia com o coqueiro tem mostrado resultados bastante promissores. Na parte do agreste Sul de Sergipe e Norte da Bahia desponta uma forte atividade lavoureira representada principalmente pelo cultivo do milho. Nessas áreas a ILP é a mais indicada para melhoria da sustentabilidade da cultura na região.

The northeastern Brazilian region comprises 1.56 million km2, of which the semi-arid occupies 0.98 million, with 0.58 million rest occupied by the Meio Norte, Mata and Agreste Zone. Savanna areas are found in the Northeast in the states of Maranhão, Piauí and Bahia, which together with part of the Tocantins comprises the MaToPiBa. By intensive cropping feature practiced in MaToPiBa the more tailored integration form to the sub-region is the CLI. In locations of the Meio Norte outside the MaToPiBa area there are examples of livestock/forest integration consisting of pastures under cashew, coconut and babaçu. Research results show the advantages of these systems integrated in relation to monoculture systems. For the Semi-Arid the focus is the rational management of native vegetation of the caatinga and the development of production models. In addition to the native, exotic species are part of the studies. For the production models the focus has been the integration of native or exotic elements adapted. Research on LFI to the zones of Mata and Agreste are mostly based on the use of gliricidia or leucena as tree component in consortium with crops, grasses and prickly pea. Specifically for the condition of coastal areas gliricidia in consortium with the coconut has shown very promising results. In part of the Southern Agreste of Sergipe and North of Bahia emerges a strong crop activity represented mainly by corn cultivation. In these areas the CLI is the most suitable for improving the sustainability of corn culture in the region.

Integração lavoura pecuária floresta na região nordeste do Brasil / Integrated systems for the Brazilian northeast

A região Nordeste do Brasil compreende 1,56 milhões de km2, dos quais o Semiárido ocupa 0,98 milhão, sendo o 0,58 milhão restante ocupado pelo Meio Norte, Zona da Mata e Agreste. Áreas de cerrado são encontradas no Nordeste nos estados do Maranhão, Piauí e Bahia, que juntamente com parte do Tocantins constituem o MaToPiBa. Nessas áreas é praticada uma lavoura intensiva. A forma de integração mais adaptada para a sub-região é a ILP. Em localidades do Meio Norte fora da área do MaToPiBa existem exemplos de integração pecuária/floresta, constituídos por pastagens sobre cajueiros, coqueiros e babaçuais. Resultados de pesquisa comprovam as vantagens desses sistemas integrados em relação aos sistemas de monocultura. Para o Semiárido o foco é o manejo racional da vegetação nativa da caatinga e o desenvolvimento de modelos produtivos. Além das nativas, espécies exóticas fazem parte dos estudos. Quanto aos modelos produtivos o foco tem sido a integração dos elementos nativos ou exóticos adaptados. As pesquisas com ILPF para as Zonas da Mata e Agreste estão em sua maioria baseadas no uso da gliricídia ou da leucena como componente arbóreo em consórcio com lavouras, gramíneas e palma forrageira. Especificamente para a condição das áreas costeiras o consórcio da gliricídia com o coqueiro tem mostrado resultados bastante promissores. Na parte do agreste Sul de Sergipe e Norte da Bahia desponta uma forte atividade lavoureira representada principalmente pelo cultivo do milho. Nessas áreas a ILP é a mais indicada para melhoria da sustentabilidade da cultura na região.(AU)

The northeastern Brazilian region comprises 1.56 million km2, of which the semi-arid occupies 0.98 million, with 0.58 million rest occupied by the Meio Norte, Mata and Agreste Zone. Savanna areas are found in the Northeast in the states of Maranhão, Piauí and Bahia, which together with part of the Tocantins comprises the MaToPiBa. By intensive cropping feature practiced in MaToPiBa the more tailored integration form to the sub-region is the CLI. In locations of the Meio Norte outside the MaToPiBa area there are examples of livestock/forest integration consisting of pastures under cashew, coconut and babaçu. Research results show the advantages of these systems integrated in relation to monoculture systems. For the Semi-Arid the focus is the rational management of native vegetation of the caatinga and the development of production models. In addition to the native, exotic species are part of the studies. For the production models the focus has been the integration of native or exotic elements adapted. Research on LFI to the zones of Mata and Agreste are mostly based on the use of gliricidia or leucena as tree component in consortium with crops, grasses and prickly pea. Specifically for the condition of coastal areas gliricidia in consortium with the coconut has shown very promising results. In part of the Southern Agreste of Sergipe and North of Bahia emerges a strong crop activity represented mainly by corn cultivation. In these areas the CLI is the most suitable for improving the sustainability of corn culture in the region.(AU)

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana córioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação ...(AU)

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2) are part of the caveolae...(AU)

Serum thiamine concentration and oxidative stress as predictors of mortality in patients with septic shock.

PURPOSE: The purpose of the study is to determine the influence of serum thiamine, glutathione peroxidase (GPx) activity, and serum protein carbonyl concentrations in hospital mortality in patients with septic shock. MATERIALS AND METHODS: This prospective study included all patients with septic shock on admission or during intensive care unit (ICU) stay, older than 18 years, admitted to 1 of the 3 ICUs of the Botucatu Medical School, from January to August 2012. Demographic information, clinical evaluation, and blood sample were taken within the first 72 hours of the patient's admission or within 72 hours after septic shock diagnosis for serum thiamine, GPx activity, and protein carbonyl determination. RESULTS: One hundred eight consecutive patients were evaluated. The mean age was 57.5 ± 16.0 years, 63% were male, 54.6% died in the ICU, and 71.3% had thiamine deficiency. Thiamine was not associated with oxidative stress. Neither vitamin B1 levels nor the GPx activity was associated with outcomes in these patients. However, protein carbonyl concentration was associated with increased mortality. CONCLUSIONS: In patients with septic shock, oxidative stress was associated with mortality. On the other hand, thiamine was not associated with oxidative stress or mortality in these patients.

Fetal-maternal interactions in the synepitheliochorial placenta using the eGFP cloned cattle model.

BACKGROUND: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. METHODOLOGY/PRINCIPAL FINDINGS: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. CONCLUSIONS/SIGNIFICANCE: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse.

Treatment of nuclear-donor cells or cloned zygotes with chromatin-modifying agents increases histone acetylation but does not improve full-term development of cloned cattle.

Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.

Viable calves produced by somatic cell nuclear transfer using meiotic-blocked oocytes.

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Clinical disorders observed during the first 30 days of life of cloned Zebu calf

Background: The advent of animal cloning from embryos reconstructed with nuclei from cells at different stages of differentiation has been responsible for the appearance of several anomalies in calves, with some alterations diagnosed during pregnancy and others after the birth of cloned animals. Despite efforts over the past five years, the rate of mortality after the birth of cloned calves is still high, at about 50%. Below is reported the research experience gained at the University of São Paulo in the period from 2005 to 2010, related to the birth of cloned calves from Nellore. Review: Research showed that cloned calves present cardiopulmonary disorders characterized by tachycardia with episodes of arrhythmia with bradycardia, increase in the 1st heart sound, presence or absence of heart murmurs in the 1st and 2nd heart sound associated with dyspnea, breathing harsh and crackling dry, moist and crackling. During the first days of life of cloned calves that died, ultrasound examination demonstrated the presence of concentric hypertrophy congenital cardiac and turbulent flow at the foramen of botal towards the right heart to left heart, indicating reversal of blood flow to the pattern observed in fetal life. As a result, mixture of arterial and venous blood occurs, compromising the ability of oxygen to the blood of cloned calves. The occurrence of macrosomia was observed in 20.9% of Nellore calves. It is believed that this syndrome is associated with disorders of carbohydrate metabolism in the placenta and/or fetus, leading to fetal gigantism, which mimicks excessive fetal growth seen in pregnant women with diabetes. We observed the occurrence of moderate to severe normocytic and normochromic anemia, with the anemia gradually appearing from 12 h of life onwards, reaching its maximum intensity at the end of the first week, to start a gradual recovery to normal values from the 15th day of life. The anemia observed in cloned calves was ferropriva, as such animals showed a significant decrease in serum iron levels associated with decreased transferrin saturation index (STI). Umbilical anomalies occurred in most clones. There was an increase in the thickness of the umbilical cord, which hindered its spontaneous rupture at birth. The umbilical arteries did not undergo retraction into the abdominal cavity, leaving them exposed in the remnant of the umbilical cord. In the first three days after birth, strong pulse of these arteries was noted in umbilical cord, making it necessary to use clamps on the arteries in order to prevent bleeding. There were also intra-abdominal hematomas involving the arteries and the urachus. Between 15 and 20 days of life, it was observed the occurrence of alopecia in about 75.0% of the calves, whose origin may be related to disturbances in the synthesis and absorption of vitamins, since the supplementation of calves with ADE vitamin complex decreased symptoms. Conclusion: The results confirm the occurrence of serious cardiopulmonary disorders characterized by tachycardia, hyperphonesis, presence of heart murmurs in the 1st and 2nd heart sounds, episodes of arrhythmia and bradycardia associated with dyspnea, rales, and coarse breath. As a result of the non-closure of the foramen of Botal and the Ductus Arteriosus, arterial and venous blood a mix, compromising the oxygen carrying capacity in the blood of cloned calves. There was also the occurrence of macrosomia, hypoglycemia, hypothermia, abnormal umbilical structures, anemia and alopecia

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus).

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such 'labour-intensive technology'.

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus)

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal–fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are manydescriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such ‘labour-intensive technology’.

Serum-starved apoptotic fibroblasts reduce blastocyst production but enable development to term after SCNT in cattle.

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.