1α, 25 Dihydroxyvitamin D (1,25(OH)2D) inhibits the T cell suppressive function of myeloid derived suppressor cells (MDSC).

Myeloid derived suppressor cells (MDSC) suppress the ability of cytotoxic T cells to attack and clear tumor cells from the body. The active form of vitamin D, 1,25 dihydroxyvitamin D (1,25(OH)2D), regulates myeloid cell biology and previous research showed that in mouse models 1,25(OH)2D reduced the tumor level of CD34+ cells, an MDSC precursor, and reduced metastasis. We tested whether MDSC are vitamin D target cells by examining granulocytic- (G-MDSC) and monocytic (M-MDSC) MDSC from tumors, spleen, and bone marrow. Vitamin D receptor (VDR) mRNA levels are low in MDSC from bone marrow and spleen but are 20-fold higher in tumor MDSC. At all sites, M-MDSC have 4-fold higher VDR mRNA expression than G-MDSC. Bone marrow MDSC were induced to differentiate in vitro into tumor MDSC-like cells by treating with IFN-γ, IL-13, and GM-CSF for 48 h. This treatment significantly elevated Arg1 and Nos2 levels, activated the T cell-suppressive function of MDSC, increased VDR expression 50-fold, and made the MDSC responsive to 1,25(OH)2D treatment. Importantly, 1,25(OH)2D treatment reduced the T cell suppressive capacity of cytokine-induced total MDSC and M-MDSC by ≥70 % and tumor-derived M-MDSC by 30-50 %. Consistent with this finding, VDR deletion (KO) increased T cell suppressive function of in vitro M-MDSC by 30 % and of tumor-derived M-MDSC by 50 % and G-MDSC by 400 %. VDR KO did not alter Nos2 mRNA levels but significantly increased Arg1 mRNA levels in tumor M-MDSC by 100 %. In contrast, 1,25(OH)2D treatment reduced nitric oxide production in both in vitro derived M- and G- MDSC. The major finding of this study is that 1,25(OH)2D signaling through the VDR decreases the immunosuppressive capability of MDSC. Collectively, our data suggest that activation of vitamin D signaling could be used to suppress MDSC function and release a constraint on T-cell mediated clearance of tumor cells.

ATP release from the human ureter on distension and P2X(3) receptor expression on suburothelial sensory nerves.

It is not clear how the increase in intraluminal pressure behind an obstructing ureteric calculus causes an increase in action potential frequency in ureteric sensory nerves so the pain messages are transmitted to the brain. It has been proposed that ureteric distension causes urothelial release of ATP, which activates purinoceptors on suburothelial nociceptive sensory nerves. The purpose of this study was to determine whether distension of the human ureter results in the release of ATP and whether the nociceptive P2 receptor, P2X(3), is expressed on suburothelial sensory nerves in the human ureter. Human ureter segments were perfused with Krebs solution and intermittently distended to a range of pressures. Samples of perfusate were collected throughout and the ATP concentration ([ATP]) was determined using a luciferin-luciferase assay. Sections of ureter were stained using antibodies against P2X(3) and capsaicin receptors (TRPV1). [ATP] rose to more than 10 times baseline levels after distension beyond a threshold of 25-30 cmH(2)O. Immunofluorescence studies on consecutive frozen sections showed that suburothelial nerves stained positively for P2X(3) and capsaicin receptors, with no staining in controls. These findings are consistent with the hypothesis that purinergic signalling is involved in human ureteric mechanosensory transduction, leading to nociception.

Comparison of laparoscopic and open pyeloplasty in 100 patients with pelvi-ureteric junction obstruction.

BACKGROUND: This study aimed to determine whether the morbidity and outcome rates for laparoscopic transperitoneal dismembered pyeloplasty are different from those for dismembered pyeloplasty, to analyze the learning curve of laparoscopic pyeloplasty, and to determine whether preoperative stent placement affects outcome. METHODS: For this study, 49 laparoscopic pyeloplasties (period 2000-2005) and 51 open pyeloplasties (period 1992-2003) were reviewed. RESULTS: Compared with open procedures, laparoscopic procedures were associated with a longer mean operating time (159 vs 91 min; p < 0.001), a shorter mean time to normal diet (38 vs 72 h; p < 0.001), and a similar mean hospital stay (5 days; p = 0.6). The operative complication rates were 17% for primary laparoscopic pyeloplasties and 24% for primary open pyeloplasties. The rates were higher for secondary procedures. The success rates for primary and secondary procedures were, respectively, 98% (41/42) and 57% (4/7) for laparoscopy and 96% (46/48) and 67% (2/3) for open surgery. Failed procedures showed no improvement in loin pain or obstruction. At the 6-month follow-up evaluation, 29% of the open surgery patients but none of the laparoscopic surgery patients reported wound pain. CONCLUSIONS: The efficacy of laparoscopic pyeloplasty is equivalent to that of open pyeloplasty, with less wound pain at 6 months. The outcome for secondary procedures is inferior. There was a trend toward a reduction in complications and the conversion rates with time, suggesting that there may be a learning curve of approximately 30 laparoscopic pyeloplasty cases. Preoperative stent insertion did not seem to affect any objective measures of outcome for laparoscopic pyeloplasty.

Life-threatening gastrointestinal haemorrhage in Von Recklinghausen's disease.

Neurofibromatosis Type I (NF-1), also known as Von Recklinghausen's disease, is a common disorder, but gastrointestinal manifestations are rare and can be associated with severe complications and malignancy. We describe a case of multiple intestinal tumours, which presented as major per-rectal bleeding and was diagnosed by laparotomy. Presenting symptoms of this condition are usually non-specific, but the risk of malignancy and perforation should allow for a high index of suspicion in patients with NF-1 presenting with gastrointestinal symptoms. We present this case as a reminder that blood loss from the bowel in Von Recklinghausen's disease may be life-threatening, requiring immediate surgery to control haemorrhage.

The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells.

BACKGROUND: Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. METHODS: Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. RESULTS: DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5-75.8), n = 18 and 21% (11.9-29.8), n = 65, respectively, p <.001]. Intermediate levels were found in reactive gastritis [55.5% (41.3-71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3-60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3-51.0), p =.007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = -.92 and -.88 for normal mucosa and H. pylori gastritis, respectively). CONCLUSIONS: Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.

Alterations in cholinergic and purinergic signaling in a model of the obstructed bladder.

PURPOSE: There is increasing evidence that purinergic signaling may have a role in the generation of detrusor contractions in the pathologically unstable human bladder. However, study of the rabbit model of partial bladder outlet obstruction showed a loss in cholinergic and purinergic innervation after 3 months. We examined changes in the cholinergic and purinergic components contributing to nerve mediated detrusor contraction in a rabbit model of detrusor instability secondary to bladder outlet obstruction during the early hypertrophic stage. MATERIALS AND METHODS: Partial bladder outlet obstruction was surgically induced in adult male rabbits. At 3 weeks detrusor strips were obtained and contractions were produced by electrical field stimulation in the presence of 1 microM. atropine and/or 30 microM. of the P2-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid, and after adding 1 microM. tetrodotoxin. Purinergic and cholinergic components were calculated and compared with those from sham operated controls. RESULTS: The cholinergic or atropine sensitive component was frequency dependent, that is smaller at lower frequencies. The cholinergic component was decreased in the early obstructed bladder. The pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid sensitive purinergic component was frequency dependent, that is larger at lower frequencies. The purinergic component was increased in the early obstructed bladder. The overall electrical field stimulation response or the response to KCl was unaltered in the obstructed group. There was no difference in the response in strips from the bladder neck and dome. CONCLUSIONS: The purinergic component of nerve mediated detrusor contraction is increased and the cholinergic component is decreased in early stages of bladder obstruction in this rabbit model.

Competition performance and basal nocturnal catecholamine excretion in cross-country skiers.

UNLABELLED: Objective techniques to determine whether an athlete is optimally prepared for a competition are virtually nonexistent. Preparedness for sports competition is commonly judged through the experience of the athletes and their coaches. Evidence from overtraining studies suggests that catecholamine (Cat) excretion rates may correlate with performance in athletes. PURPOSE: We therefore attempted to determine whether a relationship existed between performances of world-class cross-country skiers and basal nocturnal Cat excretion (BNCE). METHOD: During the Cross-Country Skiing World Championships, we determined BNCE in nine cross-country skiers of the Swiss national team by measuring free Cat concentration (dopamine = D, norepinephrine = NE, epinephrine = E) in morning urine samples, using high performance liquid chromatography. Expert judgments of competition performance (ECP) were assessed by two experienced professional coaches of the national team by using an 11-step scale. RESULTS: The BNCE correlated significantly with ECP in cross-country skiers (r2 = 0.84 and P < for NE; r2 = 0.86 and P < 0.001 for D). Athletes who had their best absolute competition results (ACR) showed the highest NE and D concentrations. CONCLUSION: These data suggest that competitive cross-country skiers with higher D and NE excretion may reach better competition levels compared with those with lower levels. Measures of BNCE provide objective information about competition performance, which may benefit athletes in their precompetition preparation.

Health status of children with moderate to severe cerebral palsy.

The aim of the study was to evaluate the health of children with cerebral palsy (CP) using a global assessment of quality of life, condition-specific measures, and assessments of health care use. A multicenter population-based cross-sectional survey of 235 children, aged 2 to 18 years, with moderate to severe impairment, was carried out using Gross Motor Function Classification System (GMFCS) levels III (n = 56), IV (n = 55), and V (n = 122). This study group scored significantly below the mean on the Child Health Questionnaire (CHQ) for Pain, General Health, Physical Functioning, and Impact on Parents. These children used more medications than children without CP from a national sample. Fifty-nine children used feeding tubes. Children in GMFCS level V who used a feeding tube had the lowest estimate of mental age, required the most health care resources, used the most medications, had the most respiratory problems, and had the lowest Global Health scores. Children with the most severe motor disability who have feeding tubes are an especially frail group who require numerous health-related resources and treatments. Also, there is a relationship among measures of health status such as the CHQ, functional abilities, use of resources, and mental age, but each appears to measure different aspects of health and well-being and should be used in combination to reflect children's overall health status.

The structure of the IgE Cepsilon2 domain and its role in stabilizing the complex with its high-affinity receptor FcepsilonRIalpha.

The stability of the complex between IgE and its high-affinity receptor, FcepsilonRI, on mast cells is a critical factor in the allergic response. The long half-life of the complex of IgE bound to this receptor in situ ( approximately 2 weeks, compared with only hours for the comparable IgG complex) contributes to the permanent sensitization of these cells and, hence, to the immediate response to allergens. Here we show that the second constant domain of IgE, Cepsilon2, which takes the place of the flexible hinge in IgG, contributes to this long half-life. When the Cepsilon2 domain is deleted from the IgE Fc fragment, leaving only the Cepsilon3 and Cepsilon4 domains (Cepsilon3-4 fragment), the rate of dissociation from the receptor is increased by greater than 1 order of magnitude. We report the structure of the Cepsilon2 domain by heteronuclear NMR spectroscopy and show by chemical shift perturbation that it interacts with FcepsilonRIalpha. By sedimentation equilibrium we show that the Cepsilon2 domain binds to the Cepsilon3-4 fragment of IgE. These interactions of Cepsilon2 with both FcepsilonRIalpha and Cepsilon3-4 provide a structural explanation for the exceptionally slow dissociation of the IgE-FcepsilonRIalpha complex.

Validating a practical approach to determine weight control in obese children and adolescents.

OBJECTIVE: To study the usefulness of a mathematical index for assessing changes in body composition of obese children and adolescents who undergo a weight control program. DESIGN: A short-term longitudinal (mean of 19 months) cohort study. SUBJECTS: Sixty-seven obese children and adolescents (38 M, 29 F, age 6-16 (mean 11)y) who took part in a clinic-based weight control program. MEASUREMENTS: Percentage body fat was assessed at the start of the program by underwater weighing (UWW) and by bioelectrical impedance (BIA). Response to the program was assessed by a mathematical index (MI), based on observed and expected changes in height and weight, and by changes in percentage fat as measured by BIA. RESULTS: Adiposity, as assessed by BIA at the start of the program, was highly correlated to that obtained by UWW (r = 0.96 for fat-free mass). Changes in the MI over the program were correlated fairly well (r = -0.81, SEE = 3.57 kg) with changes in percentage fat as assessed by BIA. CONCLUSION: Using change in BIA as criterion, the MI is valid for assessing changes in percentage body fat of obese children and adolescents over time. This index is of use to clinicians who lack body composition equipment and need a quick method to analyze the effectiveness of a weight control program in obese children and adolescents.

A potential mechanism for fumonisin B(1)-mediated hepatocarcinogenesis: cyclin D1 stabilization associated with activation of Akt and inhibition of GSK-3beta activity.

Fumonisin B(1) (FB(1)) is a worldwide corn contaminant and has been epidemiologically linked to the high incidence of human esophageal cancer in South Africa and China. FB(1) is hepatocarcinogenic in rats by an unknown mechanism. Inhibition of ceramide synthase and disruption of membrane phospholipids have been shown to be mechanisms of toxicity. Here we show overexpression of cyclin D1 protein in both preneoplastic and neoplastic liver specimens obtained from a long-term feeding study of FB(1) in rats. In rats fed FB(1) short-term, cyclin D1 protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in mRNA levels of cyclin D1. 2D electrophoresis of cyclin D1 protein in FB(1)-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3beta (GSK-3beta) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D1. In FB(1)-treated samples we detected GSK-3beta phosphorylated on serine 9; activated protein kinase B (Akt) appears to be responsible for this activity-inhibiting phosphorylation. These findings suggest that overexpression of cyclin D1 results from stabilization due to a lack of phosphorylation mediated by GSK-3beta. We also observed an increase in cyclin dependent kinase 4 (Cdk4) complexes with cyclin D1 in FB(1)-treated samples; additionally, elevated Cdk4 activity was shown by increased phosphorylation of the retinoblastoma protein. In summary, the activation of Akt leads to increased survival, inhibition of GSK-3beta activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle G(1)/S restriction point in hepatocytes. This is the first report suggesting the mechanism by which FB(1) acts as a carcinogen.

In vivo DNA damage in gastric epithelial cells.

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.

Caged ATP - an internal calibration method for ATP bioluminescence assays.

ATP bioluminescence, based on the firefly luciferase system, is used for the rapid determination of hygienic practices in the food industry. This study has demonstrated the use of caged ATP as an internal ATP standard and quantified the effects of industrial cleansing solutions, alcoholic beverages and pH on firefly luciferase activity. The light signal was quenched 6-47% by five cleansing solutions at standard working concentrations. Ethanol at 1% (v/v) inhibited bioluminescence by 15% (w/v) whereas concentrations above 4% enhanced the light output. The light signal was quenched by 20-25% at pH values below pH 4 and above pH 10.

Normal and pathological erectile function: the potential clinical role of endothelin-1 antagonists.

Erectile dysfunction (ED) is a common problem, particularly in older men. The production of penile erection involves an interplay between autonomic nerves and locally released vasoactive mediators. Endothelin-1 (ET-1) is a peptide released from endothelium in the corpus cavernosum, which causes smooth muscle contraction. Recent studies have investigated the physiological significance of ET-1 in the control of erectile function and it may play a role in detumescence. There is also much evidence to link ET-1 to risk factors for ED. ET-1 antagonists may prove beneficial in the treatment of ED and also in prevention of long term deterioration of erectile function. These antagonists may also find a role when used in combination with agents, which are established for the treatment of ED.