Isolation and characterization of farm pig adipose tissue-derived mesenchymal stromal/stem cells

Adipose tissue-derived mesenchymal stromal/stem cells (ASCs) are considered important tools in regenerative medicine and are being tested in several clinical studies. Porcine models are frequently used to obtain adipose tissue, due to the abundance of material and because they have immunological and physiological similarities with humans. However, it is essential to understand the effects and safe application of ASCs from pigs (pASCs) as an alternative therapy for diseases. Although minipigs are easy-to-handle animals that require less food and space, acquiring and maintaining them in a bioterium can be costly. Thus, we present a protocol for the isolation and proliferation of ASCs isolated from adipose tissue of farm pigs. Adipose tissue samples were extracted from the abdominal region of the animals. Because the pigs were not raised in a controlled environment, such as a bioterium, it was necessary to carry out rigorous procedures for disinfection. After this procedure, cells were isolated by mechanical dissociation and enzymatic digestion. A proliferation curve was performed and used to calculate the doubling time of the population. The characterization of pASCs was performed by immunophenotyping and cell differentiation in osteogenic and adipogenic lineages. The described method was efficient for the isolation and cultivation of pASCs, maintaining cellular attributes, such as surface antigens and multipotential differentiation during in vitro proliferation. This protocol presents the isolation and cultivation of ASCs from farm pig as an alternative for the isolation and cultivation of ASCs from minipigs, which require strictly controlled maintenance conditions and a more expensive process.

Effect of maternal hydration on the increase of amniotic fluid index

The objective of this study was to determine the effect of maternal hydration with oral isotonic solution and water on the amniotic fluid (AF) index of women with normohydramnios. Women with a normal AF index and gestational age between 33 and 36 weeks without maternal complications were randomized into three groups [isotonic solution (Gatorade®), water, control]. The isotonic solution and water groups were instructed to drink 1.5 L of the respective solution and the control group was instructed to drink 200 mL water over a period of 2 to 4 h. AF index was measured before and after hydration by Doppler ultrasonography. The investigator performing the AF index measurement was blind to the subject’s group. Ninety-nine women completed the study without any adverse maternal effects. The median increase in AF index after hydration was significantly greater for the isotonic solution and water groups than for the control group. There was no significant difference between the isotonic solution and water groups. Hydration with isotonic solution and water caused a 10-fold (95 percentCI: 2.09-49.89) and 6-fold (95 percentCI: 1.16-30.95) increase in the chance of a 20 percent increase of AF index, respectively. Maternal hydration with isotonic solution or water increased the AF index in women with normohydramnios.

Plasma concentrations and placental immunostaining of interleukin-10 and tumornecrosis factor-α as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

Interleukin-10 (IL-10) appears to be the key cytokine for the maintenance of pregnancy and inhibits the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α). However, there are no studies evaluating the profile of these cytokines in diabetic rat models. Thus, our aim was to analyze IL-10 and TNF-α immunostaining in placental tissue and their respective concentrations in maternal plasma during pregnancy in diabetic rats in order to determine whether these cytokines can be used as predictors of alterations in the embryo-fetal organism and in placental development. These parameters were evaluated in non-diabetic (control; N = 15) and Wistar rats with streptozotocin (STZ)-induced diabetes (N = 15). At term, the dams (100 days of life) were killed under anesthesia and plasma and placental samples were collected for IL-10 and TNF-α determinations by ELISA and immunohistochemistry, respectively. The reproductive performance was analyzed. Plasma IL-10 concentrations were reduced in STZ rats compared to controls (7.6 ± 4.5 vs 20.9 ± 8.1 pg/mL). The placental scores of immunostaining intensity did not differ between groups (P > 0.05). Prevalence analysis showed that the IL-10 expression followed TNF-α expression, showing a balance between them. STZ rats also presented impaired reproductive performance and reduced plasma IL-10 levels related to damage during early embryonic development. However, the increased placental IL-10 as a compensatory mechanism for the deficit of maternal regulation permitted embryo development. Therefore, the data suggest that IL-10 can be used as a predictor of changes in the embryo-fetal organism and in placental development in pregnant diabetic rats.

Effect of obesity on rat reproduction and on the development of their adult offspring

The aim of the present study was to assess the reproductive parameters of obese Wistar rats and to determine the frequency of their obese adult offspring. Neonatal rats were divided into two groups: F1 generation, induced to obesity by monosodium glutamate (MSG; F1MSG, N = 30), and rats given saline (F1CON, N = 13). At 90 days of age all animals were mated, producing the F2 offspring (F2CON, N = 28; F2MSG, N = 15). Reproductive parameters (fertility, pregnancy, and delivery indexes) were evaluated in F1 rats. F2 newborns were weighed, and the obesity parameter for F1 and F2 generations was determined from months 5 to 7 of life. At month 7, periovarian fat was weighed and no differences were found. Mean newborn weight also did not differ. The F1 and F2MSG groups presented approximately 90 percent of obese rats since month 5 of life, whereas F1 and F2CON groups presented only 33 percent. There was no difference in periovarian weight among groups. Although obesity did not affect reproductive parameters, obese dams (F1MSG) were responsible for the appearance of obesity in the subsequent generation. Thus, obesity induced by neonatal MSG administration did not interfere with reproduction, but did provide a viable model for obesity in second-generation adult Wistar rats. This model might contribute to a better understanding of the pathophysiological mechanisms involved in transgenerational obesity.

Effect of Ginkgo biloba on the reproductive outcome and oxidative stress biomarkers of streptozotocin-induced diabetic rats

The aim of the present study was to evaluate the effect of Ginkgo biloba treatment (EGb 761, 200 mg kg-1 day-1) administered from day 0 to 20 of pregnancy on maternal reproductive performance and on the maternal and fetal liver antioxidant systems of streptozotocin-induced diabetic Wistar rats. On day 21 of pregnancy, the adult rats (weighing approximately 250 ± 50 g, minimum number = 13/group) were anesthetized to obtain maternal and fetal liver samples for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and total glutathione (GSH-t) determinations. The uterus was weighed with its contents. The diabetic (G3) and treated diabetic (G4) groups of rats presented significant maternal hyperglycemia, reduced term pregnancy rate, impaired maternal reproductive outcome and fetal-placental development, decreased GSH-Px (G3 = G4 = 0.6 ± 0.2) and SOD (G3 = 223.0 ± 84.7; G4 = 146.1 ± 40.8), and decreased fetal CAT activity (G3 = 22.4 ± 10.6; G4 = 34.4 ± 14.1) and GSH-t (G3 = G4 = 0.3 ± 0.2), compared to the non-diabetic groups (G1, untreated control; G2, treated). For G1, maternal GSH-Px = 0.9 ± 0.2 and SOD = 274.1 ± 80.3; fetal CAT = 92.6 ± 82.7 and GSH-t = 0.6 ± 0.5. For G2, G. biloba treatment caused no toxicity and did not modify maternal or fetal-placental data. EGb 761 at the nontoxic dose used (200 mg kg-1 day-1), failed to modify the diabetes-associated increase in maternal glycemia, decrease in pregnancy rate, decrease in antioxidant enzymes, and impaired fetal development when the rats were treated throughout pregnancy (21 days).

Studies of natural killer cells in pregnancy-induced hypertension

The number and activity of natural killer (NK) cells were studied in 20 patients with pregnancy-induced hypertension (PIH), 15 uncomplicated pregnant women and 16 healthy non-pregnant women. All the pregnant women were evaluated during the third trimester of gestation. Peripheral blood NK cells were detected with monoclonal antibodies by indirect immunofluorescence and cytotoxic activity was measured using a single-cell assau against K562 target cells, Hypertensive pregnant women had an increased number of circulating NK cells associated with a significant decrease of NK activity. The cytotoxic activity was significantly lower in normal pregnant and PIH women when compared with non-pregnant controls. The onset of immune NK cells in peripheral blood and the impairment of their cytotoxic activity in PIH patients may be associated with hormones and immunosuppresive substances produced by tissues occurring at the maternal-fetal interface

Diabetes and experimental pregnancy in rats: course of maternal blood glucose levels and its repercussions on the blood glucose levels and pancreas of newborn pups

The objective of the present investigation was to determine the course of maternal blood glucose levels in pregnant rats and its repercussions on the glucose levels an pancreas of their newborn pups. Diabetes was induced by alloxan (42mg/Kg body weight) and steptozotocin (40mg/Kg). Sixty-two pregnant Wistar rats weighing 180 to 250 g were divided into a control group and two groups with moderate (120 to 200 mg/dl glucose) and severe diabetes (greater than 200 mg/dl glucose), respectively. Blood glucose levels were measured in the dams on the 1st, 14th, and 21st days of pregnancy and in the pups at birth. The results were pooled for each litter. The fetal pancrases were removed after cesarian section performed on the 21st day of pregnancy, pooled for each litter and processed for histopathologic examination by light microscopy. Maternal blood glucose levels were significantly increased compared with the first day of pregnancy in both normal and diabetic ratsd starting on the 14 th day of pregnancy. Fetal blood glucose levels correlated with maternal levels. The histopathologic changes characterized by vacuolization and basophilia of the cytoplasm of endocrine pancreas of newborn pups from dams with moderate or severe diabetes suggested pancreatic hyperactivity

Consideraçöes sobre o câncer localmente avançado de mama (CLAM) acrescidas de identificaçäo dos receptores de estrogênios usando anticorpos monoclonais / Considerations of locally advanced cancer of the breast (LACB) increased of indentification of estrogen receptors by using monoclonal

Para investigar os resultados do tratamento do câncer localmente avançado de mama nós estudamos 49 pacientes, que foram submetidas à associaçäo de telecobaltoterapia convencional e quimioterapia dupla com ciclofosfamida e 5-fluoracil, combinadas com mastectomia radical nos casos operáveis. Obteve-se controle local do tumor em 86,7% dos casos. Näo houve recidivas logorregionais nas pacientes submetidas à cirurgia, índice que alcançou 21,7% nas inoperáveis que receberam só radioterapia e quimioterapia. O seguimento médio das pacientes falecidas foi de 29,5 meses e das sobreviventes, de 79,3 meses. O índice de respostas completas foi de 24,5%. O período médio livre de doença de 22,9 meses e índice global de sobrevida, entre três e cinco anos, de 32,7%. Os receptores de estrogênios foram identificados mediante método imuno-histoquímico (ER-ICA) usando anticorpo monoclonal antiestrogênio (RE, 22-SPy, Abbot). Näo houve diferenças de resultados entre as pacientes RE-positivas e RE-negativas, o que explica pelo adiantado estágio evolutivo dos tumores - no que tange aos índices de respostas completas, período livre de doença e sobrevida global. A presença de receptores de estrogênios correlacionou-se, significativamente, com alguns sinais histopatológicos das neoplasias: grau de diferenciaçäo, quantidade de elastose, ausência de infiltraçäo linfocitária e ausência de necrose

The oral glucose tolerance test is a poor predictor of hyperglycemia during pregnancy

1. In the order to assess the efficacy of the use of the diurnal plasma glucose profile rather than that of the glucose tolerance tes (GTT) to predict hyperglycemia during pregnancy, we compared the results of the two tests. A total of 192 pregnant women seen at the Prenatal Clinic of the Faculty of Medicine of Botucatu were submitted to the glucose tolerance test (GTT) and determination of diurnal plasma glucose profile. 2. On the basis of two blood tests (GTT) and diurnal plasma glucose profile). the subjects were divided into four groups: Group I-A, normal GTT and profile (79 patients, 41.2%); Group I-B, normal GTT and altered profile (63 patients, 32.8%); Group II-A, altered GTT and normal profile (18 patients, 9.4%) Group II-B, altered Gtt and profile (32 patients, 16,7%). 3. Large babies were delivered by 25.6% of Group I-A, 53.8% of GroupI-B28.6% of Group II-A and 51.9% of Group II-B patients. Group I-A patients are normoglycemic, Group I-B patients have intolerance to carbohydrates, protein and lipides, Group II-A patients have intolerance to high carbohydrate amounts, especially in the form of glucose, and Group II-B patients are diabetic. 4. We propose that Group I-A patients should receive no treatment, Group II-A patients should be adivised to avoid excess carbohydrate intake and Groups I-B and II-B patients should be places on a low-calorie diet and treated with insulin if necessary to obtain normal blood glucose levels. 5. Routine determination of blod glucose levels under fasting conditions represents a screening method for diabetes and values of > ou = 90 mg/dl identify a population at risk that should be submitted to GTT and determination of plasma glucose profile