Osteoporosis is accompanied by reduced CD274 expression in human bone marrow-derived mesenchymal stem cells.

Underlying pathomechanisms of osteoporosis are still not fully elucidated. Cell-based therapy approaches pose new possibilities to treat osteoporosis and its complications. The aim of this study was to quantify differences in human bone marrow-derived mesenchymal stem cells (hBMSCs) between healthy donors and those suffering from clinically manifest osteoporosis. Cell samples of seven donors for each group were selected retrospectively from the hBMSC cell bank of the Trauma Department of Hannover Medical School. Cells were evaluated for their adipogenic, osteogenic and chondrogenic differentiation potential, for their proliferation potential and expression of surface antigens. Furthermore, a RT2 Osteoporosis Profiler PCR array, as well as quantitative real-time PCR were carried out to evaluate changes in gene expression. Cultivated hBMSCs from osteoporotic donors showed significantly lower cell surface expression of CD274 (4.98 % ± 2.38 %) than those from the control group (26.03 % ± 13.39 %; p = 0.007), as assessed by flow cytometry. In osteoporotic patients, genes involved in inhibition of the anabolic WNT signalling pathway and those associated with stimulation of bone resorption were significantly upregulated. Apart from these changes, no significant differences were found for the other cell surface antigens, adipogenic, osteogenic and chondrogenic differentiation ability as well as proliferation potential. These findings supported the theory of an influence of CD274 on the regulation of bone metabolism. CD274 might be a promising target for further investigations of the pathogenesis of osteoporosis and of cell-based therapies involving MSCs.

Trehalose-6-phosphate synthase from the cat flea Ctenocephalides felis and Drosophila melanogaster: gene identification, cloning, heterologous functional expression and identification of inhibitors by high throughput screening.

Trehalose phosphate synthase (EC 2.4.1.15; TPS) is the crucial enzyme for the biosynthesis of trehalose, the main haemolymph sugar of insects, and therefore a potential insecticidal molecular target. In this study, we report the functional heterologous expression of Drosophila melanogaster TPS, the gene identification, full length cDNA cloning and functional expression of cat flea (Ctenocephalides felis) TPS, and the Michaelis-Menten constants for their specific substrates glucose-6-phosphate and uridinediphosphate-glucose. A novel high throughput screening-compatible TPS assay and its use for the identification of the first potent insect TPS inhibitors from a large synthetic compound collection (>115 000 compounds) is described. One compound class that emerged in this screening, the 4-substituted 2,6-diamino-3,5-dicyano-4H-thiopyrans, was further investigated by analysing preliminary structure-activity relationships. Here, compounds were identified that show low µM to high nM half maximal inhibitory concentrations on insect TPS and that may serve as lead compounds for the development of insecticides with a novel mode of action.

Two-stage late reconstruction with a fresh large osteochondral shell allograft transplantation (FLOCSAT) for a large ostechondral defect in a non-union after a lateral tibia plateau fracture 2-year follow up.

This is the description of a 58-year-old female patient presenting 8 months after a horse riding accident with significant pain and inability to walk independently. Imaging revealed a large osseous defect of the lateral tibia plateau which was not united posteriorly. The patient refused knee replacement and we developed a patient specific two-step procedure for her. Step 1: Filling of the defect with a large cortico-cancellous autograft from the posterior iliac crest; step 2: Transplantation of a fresh large osteochondral shell allograft (FLOCSAT). The postoperative protocol included continuous passive motion (CPM), partial weight bearing for three months, and physiotherapy. Based on the concept of immuno-privileged cartilage tissue, the patient did not get any immuno-suppressive therapy. Pain-, activity of daily living, Lysholm and Tegner scores were evaluated before defect filling surgery with autograft, before allograft transplantation, and at 12 and 24 months after allograft transplantation. There were no complications. Radiographic analyses with plain films and CT scans revealed solid osseous integration within 3 month. The patient regained excellent functionality in both, activities of daily living and sports (back to horse riding, trampolin jumping). Knee arthroscopy after 1year showed excellent condition of the lateral meniscus and the cartilage of the lateral tibia plateau. Chimerism/DNA analysis of a cartilage biopsy showed, that at 1year 32% of the donor cells have been already replaced by the patient's own cells. To our knowledge, this is the first case of a patient who sustained such a large defect during a tibia plateau fracture, and got successfully treated with a fresh large osteochondral shell allograft transplantation in a two-step procedure.

Age-dependent insulin secretion of the endocrine pancreas in vitro from fetuses of diabetic and nondiabetic patients.

Fetal hyperinsulinemia is assumed to play a key role in the pathogenesis of diabetic fetopathy. To investigate the role of enhanced fetal B-cell mass as one cause of fetal hyperinsulinemia during diabetic pregnancy, we studied human fetal pancreatic slices from diabetic women (FDW) with poor metabolic control and nondiabetic women (FNDW) between 11 and 26 wk of pregnancy, morphometrically and by in vitro incubation experiments. Abortions had been performed due to different medical indications. We found a good correlation between the calculated B-cell mass and the gestational age in both FDW and FNDW, but the increase in FDW was much more pronounced. Such a correlation was also found in vitro regarding the insulin response to glucose and IBMX. The FDW had significantly higher values than FNDW of the same age range. In contrast to this, we found in two diabetic patients with tight metabolic control during the whole pregnancy results similar to those in FNDW. Therefore, we assume that it could be possible to prevent fetal hyperinsulinemia and perhaps even diabetic fetopathy in diabetic women by tight metabolic control during the whole pregnancy, but further investigations are necessary.

Developmental delay of astrocytes in hippocampus of rhesus monkeys reflects the effect of pre- and postnatal chronic low level lead exposure.

Rhesus monkeys were pre- and postnatally exposed to lead-acetate at 0, 350, or 600 ppm in diet for nine years, followed by a period of lead-free diet for 32 months. During this time blood lead levels declined to normal, but still showed dose-related differences. In behavioral and neurophysiological studies the rhesus monkeys exhibited dose-related cognitive and functional deficits. After sacrifice hippocampal sections were processed for immunohistological staining. GFAP, introduced as a marker of neurotoxicity and Vimentin, which is expressed by immature or reactive astrocytes were investigated. A dose-dependent increase of GFAP due to prenatal and chronic low level lead exposure was not observed. We found a dose-related increase of GFAP-positive radial glia and star-shaped Vimentin-positive astrocytes in the high lead group. We consider these findings as indication of immature astrocytes, which are not able to react with gliosis in respond to pre- and postnatal low level lead exposure. The lack of pronounced glial response due to low level lead exposure may result in a delay of astrocytic differentiation, shown by persistence of radial glia.

Immunohistochemical localization of neuronal and glial calcium-binding proteins in hippocampus of chronically low level lead exposed rhesus monkeys.

The purpose of this study was to investigate the distribution of the neuronal calcium-binding proteins parvalbumin, calbindin D28k, calretinin and the glial protein S100 in the hippocampus of lead exposed rhesus monkeys. It has been suggested that lead may exert its toxic effects by perturbing the intracellular calcium homeostasis. Lead is able to increase the intracellular Ca2+ concentration and can serve as a calcium substitute. It has been shown that some calcium-binding proteins are capable of binding lead. We tried to find a putative dose-depending relation between long-term low level lead exposure and the expression of the proteins investigated. Rhesus monkeys were pre- and postnatally exposed to 600 mg-350 mg-0 mg lead-acetate in diet for nine years, as described by Lilienthal et al. (1986). After a lead-free period of 32 months animals were sacrificed. Hippocampal paraffin sections were stained for parvalbumin (PV), calbindin D28k (CB), calretinin (CR), and S100 with immunohistochemical methods. The distribution of the neuronal calcium-binding proteins was almost identical for the different exposure groups. The most striking observation was a marked decrease of S100 immunoreactivity in astrocytes in the high lead group. Considering a protective role against high Ca2+ concentration and Pb2+ accumulation respectively the unchanged expression of PV, CB, and CR remains to be clarified. The apparent difference in S100 expression supports the hypothesis that glial cells are the main target of lead toxicity. The reduced expression may indicate a developmental retardation of astroglia.

Long-term effects of hysterectomy and bilateral oophorectomy on lymphoid tissue in female Lewis rats.

The effects of ovariectomy, ovariohysterectomy and hysterectomy on morphologically demonstrable characteristics of lymphoid organs, peripheral white blood cell counts and antibody production against sheep red blood cells (SRBC) were studied in female Lewis rats. Removal of ovaries induced enlarged thymus weight and cellularity. No differences were observed between the groups in spleen weight, while hysterectomy together with ovariectomy influenced relative uterus draining lymph node (UDLN) weight. The percent numbers of pan T, helper T lymphocytes, cytotoxic/suppressor T cells and IgG bearing cells (B lymphocytes) of all investigated organs showed only moderate changes caused by the surgical procedures. In contrast, removal of ovaries generated elevated total leukocyte and lymphocyte counts in peripheral blood. The changes in absolute blood lymphocyte counts were accompanied by similar variations in absolute T and B lymphocyte numbers. Ovariohysterectomy had slightly greater effects on these parameters than ovariectomy alone. In addition, ovaries and uterus had only moderate influences on systemic immune responses toward SRBC. In conclusion, the present results indicate that the removal of ovaries and uterus can modify morphological characteristics of lymphoid organs and peripheral blood but antibody production showed only moderate changes caused by the surgical procedures.

The porcine lymphotropic herpesvirus 1 encodes functional regulators of gene expression.

The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1(h), ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta, (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1(h) and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.

[PH3 residues in hazelnuts, soybeans and wheat following phosphine fumigation with non-constant concentrations]. / PH3-Rückstände in Haselnüssen, Sojabohnen und Weizen nach Phosphin-Begasungen mit zeitlich nicht konstanter Konzentration.

In model tests hazelnuts, soy beans and wheat were fumigated with phosphine (PH3) at non constant concentrations. The influence of different concentration characteristics on the fumigation and the decomposition of phosphine residues was investigated in accordance with the fumigation technique. At the beginning the concentration increases, and after attaining the maximum gradually decreases to zero. The level of residues during the fumigation as well as the behaviour of residues during the storage of the fumigated products was monitored with a gas chromatographic method. The residues correlate with the concentration of phosphine, they also pass through a peak. The rate of decomposition of residues which had been formed in the phase of increasing concentration is greater than the rate of residues of equal magnitude which had been formed during the decreasing phase. When the concentration is even the maximum residue occurs later than the maximum concentration; when there is a steep trend both maximums coincide. This behaviour can be explained by the sorption and diffusion of phosphine. A comparison is made with the phosphine concentration which occurs during fumigation in practice. The parameters which produce a constant concentration trend with only one maximum and a non constant trend with an often increasing and decreasing concentration are discussed. The different behaviour of residues in these cases is described. Conclusions are drawn for the practice of fumigation.

[A semiempirical mathematical model for assessment of PH3 residues as exemplified by phosphine-fumigated hazelnuts]. / Ein halbempirisches mathematisches Modell zur Abschätzung von PH3-Rückständen am Beispiel phosphinbegaster Haselnüsse.

A mathematical model for the calculation of residues of PH3 in fumigated hazelnuts in dependence on dosage, fumigation- and storage time is discussed. The model is based on the physical process of the diffusion of a gas out of a sphere. An empirical dependence of the diffusion coefficient on the residue levels can be observed. Adequate agreement is obtained between calculated and experimental values.

The effect of litter size on the development of the endocrine rat pancreas.

The influence of litter size on the development of the endocrine rat pancreas in the fetal period has been investigated. Rats from 3 spontaneously different litter sizes were chosen to estimate sex, body weight, food uptake, pancreatic wet weight, insulin and glucagon concentrations, plasma glucose and insulin. With increasing litter size we could observe an enhancement of the pancreatic insulin concentration whereas the glucagon content was decreased. Food uptake caused an increase of insulin and glucagon in the pancreas. The observed changes were not sex dependent. Our results show that the litter size has an obvious effect on the endocrine pancreas already in the fetal and early neonatal phase of development.

[Gastroscopy before cholecystectomy]. / Die Gastroskopie vor der Cholecystektomie.

In a prospective study the routine endoscopy of the upper gastrointestinal tract was carried out in 100 patients before they underwent elective cholecystectomy for gallstones. In 31/100 patients we detected abnormalities which changed the plan of therapy. In 18/100 patients the cholecystectomy was performed 4 to 8 weeks later, after additional medical treatment. 7 patients were discharged from the cholecystectomy. The preoperative endoscopy of the upper gastrointestinal tract should be used in patients undergoing cholecystectomy to exclude other gastrointestinal disorders.

Effects of 3-isobutyl-1-methylxanthine on secretory response, cAMP accumulation and DNA synthesis of islets from postnatal and adult Wistar rats.

A possible role for cyclic adenosine-3'-5'-monophosphate (cAMP) in islet cell replication was examined in collagenase-isolated pancreatic islets from Wistar rats of different age and different metabolic state (non-pregnant, pregnant, days 15.5-17.5). Islets obtained from pregnant rats released significantly more insulin in response to 10 mmol/l glucose (culture for 24 h) and their DNA synthesis (incorporation of [3H]thymidine into islet DNA) was doubled compared to islets from non-pregnant controls. Islets obtained from 4-6 days old rats showed a maximal stimulation of DNA synthesis after exposure to 0.1 mmol/l IBMX (3-isobutyl-1-methylxanthine) whereas the cAMP accumulation and the insulin biosynthesis measured in a subsequent short-term incubation were dose-dependent stimulated up to 1.0 mmol/l IBMX. In islets of 12 days old rats or 3 months old rats, however, IBMX did not stimulate DNA synthesis or insulin release measured during culture, although the cAMP content per islet was significantly enhanced after culture in the presence of IBMX.

The in vitro insulin secretion of human fetal pancreatic slices from diabetic and non-diabetic women--a methodical study.

The in vitro insulin secretion of pancreatic slices between the 11th and 15th week of pregnancy of fetuses from non diabetic ( FNDW ) and diabetic women ( FDW ) after incubation in media supplemented with different secretagogues was investigated in order to study the development of diabetic fetopathy during human pregnancy in diabetic women. There was a stimulatory effect on the insulin secretion in FNDW even if glucose alone was used, which became more pronounced if IBMX was added to the incubation medium. The insulin secretion was significantly enhanced in FDW compared to FNDW . This incubation model using fetal pancreatic slices seems to be appropriate for studying the ontogenesis of the human fetal pancreas.