Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions.

UNLABELLED: Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs. INTRODUCTION: Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation. METHODS: cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, ß-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies. RESULTS: In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition. CONCLUSIONS: The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.

Biocatalytically active silCoat-composites entrapping viable Escherichia coli.

Application of whole cells in industrial processes requires high catalytic activity, manageability, and viability under technical conditions, which can in principle be accomplished by appropriate immobilization. Here, we report the identification of carrier material allowing exceptionally efficient adsorptive binding of Escherichia coli whole cells hosting catalytically active carbonyl reductase from Candida parapsilosis (CPCR2). With the immobilizates, composite formation with both hydrophobic and hydrophilized silicone was achieved, yielding advanced silCoat-material and HYsilCoat-material, respectively. HYsilCoat-whole cells were viable preparations with a cell loading up to 400 mg(E. coli) · g(-1)(carrier) and considerably lower leaching than native immobilizates. SilCoat-whole cells performed particularly well in neat substrate exhibiting distinctly increased catalytic activity.

Acupuncture for treatment of acute vomiting in children with gastroenteritis and pneumonia.

OBJECTIVE: Acupuncture is successfully used to alleviate vomiting in children after general anesthesia. However there is no data on treatment of vomiting in children with gastroenteritis (GE) and pneumonia (PM). METHODS: Descriptive analysis of 18 cases, where acupuncture was used as an individual therapy attempt to treat vomiting in children with GE or PM before starting the conventional antiemetic therapy. Feasibility and acceptance by patients and parents as well as the incidence of vomiting and use of antiemetic drugs after acupuncture were recorded. RESULTS: Acupuncture was feasible in all children and application of the indwelling needles was tolerated without fear. Side effects were not observed. 13 patients stopped vomiting immediately after the insertion of acupuncture needles, none of the patients required conventional antiemetic medication. CONCLUSION: Acupuncture for the treatment of vomiting is feasible and acceptable. Suggested antiemetic effect should be examined in a randomized multicenter controlled trial.

Loss of enteroendocrine cells in autoimmune-polyendocrine-candidiasis-ectodermal-dystrophy (APECED) syndrome with gastrointestinal dysfunction.

BACKGROUND: Enteroendocrine (EE) cells are necessary for the regulation of gastrointestinal function. The lack of intestinal enteroendocrine cells in enteroendocrine cell dysgenesis causes severe malabsorptive diarrhea. Autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED) is often accompanied by gastrointestinal (GI) symptoms. AIMS: We hypothesized that an autoimmune attack against the cells of the GI-associated diffuse endocrine system may be a specific feature of GI dysfunction in APECED disorders. METHODS: Biopsies were obtained during routine diagnostic endoscopy from 35 pediatric patients with gastrointestinal symptoms as well as from five healthy controls; biopsies were immunostained for chromogranin A and serotonin. Four patients were classified as APECED syndrome on molecular and clinical grounds. RESULTS: Immunohistological analysis of biopsies along the GI tract (stomach, duodenum, colon) immunostained with chromogranin A and serotonin revealed a widespread reduction or complete loss of EE cells in all four patients with APECED syndrome suffering from severe diarrhea, vomiting, malabsorption, or constipation. In contrast, EE cells were present in pediatric patients with similar gastrointestinal symptoms caused by inflammatory bowel disease, celiac disease, lymphocytic colitis, and autoimmune disorders without endocrinopathy or graft vs. host disease of the gut. CONCLUSIONS: The reduction of EE cells is a specific and important early event in the pathogenesis of APECED with GI dysfunction. We propose a diagnostic algorithm integrating clinics, genetics and immunohistology.

Evaluation of a rapid whole blood test to detect Helicobacter pylori infection in children.

BACKGROUND: Rapid, patient near tests for detecting antibodies against Helicobacter pylori are offered for clinical use. We evaluated the BM-Test (Boehringer Mannheim, Germany; identical to the Helisal Rapid Blood Test) in children with recurrent abdominal pain. METHODS: The BM-Test and 13C-urea breath test (UBT) were performed in 195 children (4-18 years). Symptoms were assessed using a 4-week diary. Upper endoscopy was performed in all UBT-positive children and in UBT-negative children with symptoms suggestive of organic disease. H. pylori status was considered positive if at least two of three methods (UBT, histology or rapid urease test) or culture were positive. RESULTS: After exclusion of children with previous H. pylori therapy (n = 8) and undetermined H. pylori status (n = 1), 61/186 (33%) children were H. pylori positive. The BM-Test in relation to H. pylori status revealed a sensitivity of 54%, specificity of 90%, a likelihood ratio of 5.2 for a positive, and of 0.4 for a negative test result. Accuracy of the test was independent of ethnicity, gender, age, family history for ulcer disease, frequency or severity of abdominal symptoms, epigastric tenderness, type of blood sampling (capillary versus venous) and DOB values of the UBT. In eight previously treated children, the test gave one false-positive and three false-negative results. CONCLUSIONS: Almost half of H. pylori-infected children and 10% of non-infected children were misclassified by the BM-Test. False-negative results are not related to young age or certain ethnic groups. The poor performance makes the test unsuitable for epidemiological and clinical use in children.

Circadian rhythms in the renin-angiotensin system and adrenal steroids may contribute to the inverse blood pressure rhythm in hypertensive TGR(mREN-2)27 rats.

The transgenic TGR(mREN-2)27 rat is not only characterized by fulminant hypertension, but also by a disturbance in circadian blood pressure regulation, resulting in inverse circadian blood pressure profiles. The reasons for these alterations are not very well understood at present. We therefore investigated the circadian rhythms in several hormones participating in blood pressure regulation. From TGR and Sprague-Dawley (SPRD) control rats synchronized to 12h light and 12h dark (LD 12:12) blood was collected at different circadian times (07, 11, 15, 19, 23, 03, and 07 again, 5 rats per strain and time). The activities of plasma renin and converting enzyme, as well as plasma concentrations of corticosterone and aldosterone, were determined by radioimmunoassay (RIA). SPRD rats showed significant circadian rhythms in all variables except plasma renin activity, with maxima occurring during the day. TGR rats showed significant circadian rhythmicity in plasma renin activity and corticosterone and daily variation in aldosterone; angiotensin-converting enzyme (ACE) activity did not reach statistical significance. In TGR rats, 24h means in plasma renin activity and aldosterone were approximately sevenfold and fourfold higher, respectively, than in SPRD rats. Peak concentrations in corticosterone around 15h were more than two times higher in TGR rats than in SPRD rats, whereas no differences were observed during the night. It is concluded that, in TGR rats, the overall increase in plasma renin activity and aldosterone may contribute to the elevated blood pressure. The comparatively high levels in corticosterone and plasma renin activity during daytime may be involved in the inverse circadian blood pressure profiles in the transgenic animals.