In vitro expansion of human cardiac progenitor cells: exploring 'omics tools for characterization of cell-based allogeneic products.

Human cardiac stem/progenitor cells (hCPCs) have been shown to be capable to regenerate contractile myocardium. However, because of their relative low abundance in the heart, in vitro expansion of hCPC is mandatory to achieve necessary quantities for allogeneic or autologous cardiac regeneration therapy applications (10(6)-10(9) cells/patient). Up to now, cell number requirements of ongoing phase I/IIa trials have been fulfilled with production in static monolayer cultures. However, this manufacturing process poses critical limitations when moving to the following clinical phases where hundreds of patients will be enrolled. For this, increased process yield is required, while guaranteeing the quality of the cell-based products. In this work, we developed and validated a robust, scalable, and good manufacturing practice (GMP)-compatible bioprocess for the expansion of high-quality hCPC. We applied platforms extensively used by the biopharmaceutical industry, such as microcarrier technology and stirred systems, and assessed culture conditions' impact on hCPC's quality and potency, as required by regulatory agencies. Complementary analytical assays including gene expression microarrays and mass spectrometry-based approaches were explored to compare transcriptome, proteome, surface markers, and secretion profiles of hCPC cultured in static monolayers and in stirred microcarrier-based systems. Our results show that stirred microcarrier-based culture systems enabled achieving more than 3-fold increase in hCPC expansion, when compared with traditional static monolayers, while retaining cell's phenotype and similar "omics" profiles. These findings demonstrate that this change in the production process does not affect cell's identity and quality, with potential to be translated into a transversal production platform for clinical development of stem-cell therapies.

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells.

The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

Effects of a 12-month multi-component exercise program on the body composition of postmenopausal women.

OBJECTIVE: The aims of this study were to identify the effects of a 12-month exercise program on the body composition of postmenopausal women and to examine the interaction of menopause characteristics (nature and time since menopause, hormone therapy) with exercise. METHODS: A total of 158 postmenopausal Caucasian women were analyzed in this study (70 in the control and 88 in the exercise group). This subset is part of the 'Shape up during menopause' which is a program that aims to develop exercise and health promotion in postmenopausal women. Exercise and control groups were tested before and at the end of the program. Data related to menopause were obtained through medical history. Measurements of fat mass, visceral fat area, skeletal muscle mass, fat-free mass, soft lean mass, and basal metabolic rate were assessed by octopolar bioimpedance. RESULTS: Alongside basal metabolic rate, all the anthropometric and body composition variables were influenced by the exercise program. The major differences between groups were found in skeletal muscle mass, total soft lean mass, fat-free mass, and skeletal muscle mass index (effect sizes ranged from 0.89 to 6.64). There were no interactive effects found between exercise and menopause characteristics. CONCLUSIONS: There were positive changes in all measured variables and no significant interactive effects with menopause characteristics; therefore, our data suggest that exercise alone promoted improvements in the body composition of postmenopausal women.

Avaliação histomorfométrica do efeito do extrato aquoso de urucum (norbixina) no processo de cicatrização de feridas cutâneas em ratos / Histomorphometric evaluation of the effects of the aqueous extract of annatto (norbixin) on wound skin healing in rats

A norbixina é um carotenóide dicarboxílico hidrofílico presente no pericarpo das sementes do urucuzeiro (Bixa orellana L.). O urucum é comumente utilizado na indústria alimentícia e cosmética como corante natural. O objetivo deste trabalho foi avaliar o processo de reparo cicatricial de feridas cutâneas abertas de ratos tratadas com extrato aquoso de urucum contendo 2,5% de norbixina através de análise histomorfométrica. Para tanto, realizou-se feridas cutâneas no dorso de 32 ratos Wistar machos. Estas foram tratadas com extrato de urucum (grupo experimental, n = 16) e solução salina 0,9% (grupo controle, n = 16). Aos 3, 7, 14 e 21 dias após realização do procedimento cirúrgico, os animais foram sacrificados. Os preparados histológicos obtidos foram submetidos à técnica de coloração pela Hematoxilina-Eosina (H.E.) para contagem de células inflamatórias e de fibroblastos; corados pelo azul de toluidina 1% para contagem dos mastócitos e com picrossirius-red para avaliação das fibras colágenas totais. As imagens histológicas destas lâminas foram capturadas por câmera digital acoplada ao microscópio óptico, sob foco fixo e clareza de campo, obtendo-se 10 campos por lâmina com aumento final de 400X. As fotomicrografias foram avaliadas através do software ImageJ. Os resultados obtidos foram submetidos ao teste t de student sendo o valor de p considerado significativo para p< 0,05. O tratamento tópico com extrato de urucum utilizado aumentou o infiltrado inflamatório durante o 3º, 14º e 21º dia pós-operatório. O grupo tratado apresentou maior densidade vascular quando comparado ao controle a partir do 7º e menor quantidade de fibroblastos até o 14º dia pós-cirúrgico. Conclui-se que o extrato de urucum contendo 2,5% de norbixina não é inócuo aos tecidos cutâneos e possui efeitos pró-inflamatórios e pró-angiogênicos durante o processo de reparo tecidual cutâneo em ratos, interferindo no processo fisiológico de cicatrização.

Norbixin is a dicarboxylic water-soluble carotenoid present in the pericarp of the achiote (Bixa orellana L.) seed. Annatto is commonly used by the food and cosmetic industries as a natural pigment. The aim of this study was to evaluate the healing process of open dermal wounds of rats treated with an aqueous solution of annatto containing 2.5% of norbixin carried out by histomorphometric analysis. For this end, cutaneous wounds were made on the back of 32 male Wistar rats. The wounds were treated with annatto solution (experimental group, n = 16) and saline solution 0.9% (control group, n = 16). At 3, 7, 14 and 21 days after the surgical procedure, the animals were sacrificed. After histological preparation, the histological material was submitted to the staining technique with hematoxylin-eosin for the counting of inflammatory cells and fibroblasts, with toluidine blue 1% for mast cell counting and with picrosirius red for the total collagen fibers. The images of those histological slides were captured by a digital camera connected to an optical microscope, with fixed focal length and clear field; 10 fields were captured from each slide with final zoom of 400X. The photomicrographs were analyzed by the ImageJ software. The results were submitted to the Student's t-test and the value of p < 0.05 was considered as relevant. The topical treatment with the annatto solution increased the inflammatory infiltrate during the 3rd, 14th and 21st days after surgery. The experimental group presented increased vascular density compared to the control group after the 7th day and a smaller amount of fibroblasts up to the 14th day after surgery. We concluded that the annatto solution containing 2.5% of norbixin is not innocuous to skin tissues and has proinflammatory and proangiogenic effects during the process of skin wound healing in rats, interfering in the physiological healing process.

Evaluation of alternative schemes for recording body weights in meat-type quails by using random regression.

Data from 8759 meat-type quails from the UFV1 strain and 9128 from the UFV2 strain were used to assess the possibility of reducing the number of body weight records in genetic evaluations. The evaluated animals were weighed weekly since hatching to the 6th week of life, with up to 7 records of body weight for each bird. The data were evaluated by random regression models, with 9 alternative schemes of data recording, which included 4 records for each scheme and their covariance functions for additive and permanent environmental effects of order 3, fitting 4 intervals for residual variance, and a complete scheme, with 7 records, order of fit 6 for additive and permanent environmental effects and 7 intervals for residual variance. Estimates of heritability for body weight at the 6th week varied from 0.45 to 0.53 for the UFV1 strain and from 0.28 to 0.54 for UFV2 strain. The schemes that had more records in points at the final extreme of the age range showed better estimates, which was likely due to certain properties of polynomial regression that led to biased results in the final extreme of the age range when data are unbalanced. The reduction of the number of body weight records taken during the growth phase is feasible, with little change to breeding value estimates, when 4 body weight records are used in random regression models.

Temporal trends in diarrhea-related hospitalizations and deaths in children under age 5 before and after the introduction of the rotavirus vaccine in four Latin American countries.

INTRODUCTION: Rotavirus infection mainly affects children under 5 years of age and causes 453,000 deaths annually throughout the world. Several countries in Latin America have introduced the rotavirus vaccine and the majority have epidemiological data to measure impact following vaccine introduction. OBJECTIVE: To assess the impact of rotavirus immunization on the number of all-cause diarrhea-related deaths and hospitalizations in children under 1 and 5 years of age in Bolivia, El Salvador, Honduras and Venezuela. METHODS: Interrupted time-series analyzed with the integral method and the projection method to evaluate the pre and post-vaccine introduction trend in diarrheal disease compared to Argentina as the control country. The analysis period was from 2002 to 2010, including 2 to 4 post-vaccine years depending on the country. Information sources included records from PAHO, the Ministry of Health, public hospitals, social security, the private health system, the Expanded Programme on Immunization and UNPop 2008. RESULTS: Over the period studied, reductions were observed in trends of diarrhea-related deaths and hospitalizations in children under five. In diarrhea-related deaths, under the integral method, the range of reduction was between 15.7% (13.5-17.9) and 56.8% (56.0-57.5) while with the projection method was between 19.9% (4.9-34.8) and 63.7%(56.1-71.4). In diarrhea-related hospitalizations, under the integral method was 5.6% (4.1-6.7) and 17.9% (16.7-19.1)) while with the projection method was between 5.1%(1.7-8.7) and 11.1% (5.8-16.3) CONCLUSIONS: A decrease was observed in the number of diarrhea related deaths and hospitalizations in all countries under study following introduction of the rotavirus vaccine as opposed to the control country. The impact on reduction of deaths was greater than hospitalization.

Downstream processing of triple layered rotavirus like particles.

Rotavirus like particles (RLPs) constitute a potential vaccine for the prevention of rotavirus disease, responsible for the death of more than half a million children each year. Increasing demands for pre-clinical trials material require the development of reproducible, scaleable and cost-effective purification strategies as alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation methods commonly used for the purification of these complex particles. Self-assembled virus like particles (VLPs) composed by VP2, VP6 and VP7 rotavirus proteins (VLPs 2/6/7) were produced in 5l scale using the insect cells/baculovirus expression system. A purification process using depth filtration, ultrafiltration and size exclusion chromatography as stepwise unit operations was developed. Removal of non-assembled rotavirus proteins, concurrently formed particles (RLP 2/6), particle aggregates and products of particle degradation due to shear was achieved. Particle stability during storage was studied and assessed using size exclusion chromatography as an analytical tool. Formulations containing either glycerol (10% v/v) or trehalose (0.5 M) were able to maintain 75% of intact triple layered VLPs, at 4 degrees C, up to 4 months. The overall recovery yield was 37% with removal of 95% of host cell proteins and 99% of the host cell DNA, constituting a promising strategy for the downstream processing of other VLPs.

Anticonvulsant properties of N-salicyloyltryptamine in mice.

A new tryptamine analogue, N-salicyloyltryptamine (STP), a potential central nervous system (CNS) depressant, was tested in the pentylenetetrazol (PTZ) and maximal electroshock (MES) models of epilepsy in mice. When administered concurrently, STP (100 mg/kg ip) significantly reduced the number of animals that exhibited PTZ-induced seizures and eliminated the extensor reflex of maximal electric-induced seizures test in 50% of the experimental animals. In addition, it showed protection in the PTZ test by diminishing the death rate.

Rapid weight gain, at least in some women, is an expression of a neuroendocrine state characterized by reduced hypothalamic dopaminergic tone.

A group of 17 consecutive regularly menstruating women who gained at least 5 kg the previous year (Group 1) was compared with a control group of similar age, parity and social class (Group 2). Galactorrhea was observed in 6/17 women from group 1 and in 1/16 women from group 2 (chi 2 4.571; p < .05). Average morning prolactin levels were higher in group 1 (8.15 +/- 4.92 micrograms/l) than in group 2 (5.29 +/- 2.48 micrograms/l; p < .05). The two groups were similar in their morning thyroxin, triiodothyronine, TSH, estradiol, cortisol, gastrin, cholecystokinin, somatostatin, oxytocin, insulin and IGF-1 levels. Leptin levels were significantly higher in group 1 than in group 2 (18.85 +/- 10.63 micrograms/l vs. 10.15 +/- 6.38 micrograms/l; p < .02) but this difference could be attributed exclusively to the higher body mass index (BMI) of group 1 (MANCOVA). Analysis of the distribution of basal prolactin levels in group 1 revealed a skewed distribution due to the presence of six outliers (Barnett and Lewis test associated with Mahalanobis distance) whose values were higher than the highest value found in group 2. These outliers were henceforth considered as subgroup 1a, and the remnant patients in group 1 as subgroup 1b. Besides the expected difference in basal prolactin levels between subgroups 1a and 1b (13.72 +/- 3.69 and 5.12 +/- 1.81 micrograms/l, respectively) and the higher frequency of galactorrhea in group 1a (4/6 vs. 2/11; p < .05) no other differences were observed in clinical or basal biochemical parameters. Following domperidone (10 mg, i.v.) the percentual increase in prolactin (delta Prl 20'/Prl 0') was significantly lower in group 1 than in group 2 (23.9 + 15.2 vs. 37.0 +/- 21.2; p < .05). In absolute values, the prolactin rise in subgroup 1a (100.7 +/- 45.5 micrograms/l) was significantly lower (p < .02) than that of subgroup 1b (157.3 +/- 50.3 micrograms/l) and group 2 (152.7 +/- 34.5 micrograms/l). Group 1 (and each one of its two sub-groups) also differed from group 2 in a higher incidence of meaningful life-events the year preceding the study. This study confirms previous observations that recent weight gain in women is preceded by important life-events and is associated with galactorrhea and increased prolactin levels in a number of them. Besides, it provides evidence that the increased prolactin levels are due to reduced hypothalamic dopaminergic tone.

Calcium beta-hydroxy-beta-methylbutyrate. 1. Potential role as a phosphate binder in uremia: in vitro study.

The binding capacity of calcium beta-hydroxy-beta-methylbutyrate (calcium HMB), compared to other binders, was investigated in an in vitro study. Fifty milliequivalents of either calcium HMB, calcium acetate, calcium carbonate, aluminum hydroxide gel or non-gel aluminum hydroxide was added to a phosphate solution, titrated (HCl or NaOH), shaken and centrifuged to four different pH levels at 37 degrees C (simulating the gastrointestinal milieu). The difference in phosphate concentration between that of the initial and that of the supernatant represented from the bound phosphate in the precipitate. After 4 h at a pH of 6 (representing the intestinal condition after a meal), the binding percentage was: calcium acetate = 95.6%, calcium HMB = 92.6%, calcium carbonate = 46.4%, aluminum hydroxide gel = 33.4% and non-gel aluminum hydroxide = 17.8%. There was no significant difference (p > 0.05) between calcium HMB and calcium acetate. These results suggest that calcium HMB is an efficient phosphate binder in vitro, which may predict its effective role in vivo.

Endocrine and psychological evaluation of women with recent weight gain.

A group of 13 consecutive regularly menstruating women who gained at least 5 kg the previous year (Group I) was compared to a control group of similar age, parity, and social class (Group II). The two groups were similar in estimated and observed food intakes; pre- and postprandial gastrin levels; hourly 24-h profiles of cortisol and insulin; urinary cortisol and 17-hydroxycorticosteroids. Group I had higher serum prolactin concentrations at all times than Group II (mean values 14.60 micrograms/l vs. 8.84 micrograms/l; p = .0121). Galactorrhea was observed in 5 women from Group I and in none of the women from Group II (p < .05). Group I also differed from Group II in a higher incidence of meaningful life-events the year preceding the study, higher prevalence of sexual dysfunction (9/13 vs. 4/13; p < .01) and higher indexes (p < .05) of several parameters in the MMPI and SCL 90. Median serum cortisol and prolactin concentrations were negatively correlated, both in Group I (R = -.669; p = .012) and in the whole sample (R = -.453; p = .0298). It is suggested that the rapid weight gain is part of a neuroendocrine response to environmental stimuli also characterized by hyperprolactinemia. The significant negative correlation between serum prolactin and cortisol indicates that this response differs from, and is possibly an alternative to, the sympathoadrenal "stress" response.

Anabolic effects of insulin and amino acids in promoting nitrogen accretion in postoperative patients.

The present study evaluates the effect of insulin and amino acids on nitrogen balance in the immediate postoperative period in moderately stressed patients who have undergone major abdominal surgical operations. Patients were randomly assigned into two large groups (n = 16 each) and four subgroups (n = 8 each). Groups ICON and IINS received an infusion of total parenteral nutrition solution containing 0.25 g of nitrogen per kilogram per day with a calorie:nitrogen (C:N) ratio of 150:1. Groups IICON and IIINS received twice the load of nitrogen with a C:N ratio of 75:1. Groups IINS AND IIINS received an additional continuous infusion of insulin at a rate of 1.0 mU/kg per minute for 7 days. During the total parenteral nutrition period, the patients were kept NPO, and 24-hour urine output was collected for estimation of total nitrogen excretion and nitrogen balance. Net nitrogen excretion (grams per kilogram per day) averaged 0.143 +/- 0.06 in IINS and 0.23 +/- 0.08 in ICON (p < .05) and 0.178 +/- 0.6 in IIINS and 0.25 +/- 0.10 in IICON (p < .05). Nitrogen balance (grams per day) was positive in the four groups: +0.65 +/- 3.8 in IINS and +6.74 +/- 2.94 in ICON (p < .05), and +14.4 +/- 2.61 in IIINS and +11.63 +/- 6.44 in IICON (p = not significant). The average nitrogen incorporation (percent per day) was: 41.3 +/- 6.2 in IINS and 14.6 +/- 20.1 in ICON (p < .05), and 58.3 +/- 4.5 in IIINS and 38.7 +/- 26.2 in IICON (p = not significant).(ABSTRACT TRUNCATED AT 250 WORDS)

Is idiopathic hyperprolactinemia a transitional stage toward prolactinoma?

The basal prolactin (PRL) levels on five different days, the PRL responses to thyrotropin-releasing hormone and to domperidone, and the thyroid-stimulating hormone (TSH) responses to domperidone were studied in 75 regularly menstruating women and 44 patients with moderate hyperprolactinemia. It was found that, for the entire sample, the responses to each of the stimuli could be described by a continuous function of the basal PRL levels. The present work provides evidence for the following conclusions: 1) The PRL responses to thyrotropin-releasing hormone and to domperidone merely bring additional diagnostic information relative to basal PRL levels in the occasional patients with macroprolactinemia; 2) there is a continuous spectrum of lactotroph activities in women, ranging from normal secretors through an intermediate group of hypersecretors (with progressively increased serum PRL levels and decreased responsiveness to stimuli) to full-blown prolactinomas; and 3) idiopathic hyperprolactinemia is a heterogeneous entity that includes the above intermediate group of patients, women with macroprolactinemia, and patients with undiagnosed prolactinomas.

Psychosomatic factors in patients with hyperprolactinemia and/or galactorrhea.

A biographic and clinical investigation of 101 patients with hyperprolactinemia and/or galactorrhea is reported. Fifty-one patients were reared without their fathers and 18 with an alcoholic, violent one. These situations were uncommon in the control population, and the differences were statistically significant. There was a high frequency of complaints of obesity, headaches, frigidity, lightheadedness, and fullness of the abdomen, limbs, or face. There was a significant temporal correlation of external events in the natural history with onset or worsening of the symptoms. It is concluded that exposure during childhood to an environment characterized by an absent or alcoholic, violent father conditions some women to develop hyperprolactinemia and/or galactorrhea later in life as a response to specific environmental changes. These conclusions apply similarly to patients with prolactinoma, idiopathic hyperprolactinemia, and euprolactinemic galactorrhea, suggesting a close relationship among the 3 entities.