In vitro exposure to Hymenoptera venom and constituents activates discrete ionotropic pathways in mast cells.

Calcium entry is central to the functional processes in mast cells and basophils that contribute to the induction and maintenance of inflammatory responses. Mast cells and basophils express an array of calcium channels, which mediate responses to diverse stimuli triggered by small bioactive molecules, physicochemical stimuli and immunological inputs including antigens and direct immune cell interactions. These cells are also highly responsive to certain venoms (such as Hymenoptera envenomations), which cause histamine secretion, cytokine release and an array of pro-inflammatory functional responses. There are gaps in our understanding of the coupling of venom exposure to specific signaling pathways such as activation of calcium channels. In the present study, we performed a current survey of a model mast cell line selected for its pleiotropic responsiveness to multiple pro-inflammatory inputs. As a heterogenous stimulus, Hymenoptera venom activates multiple classes of conductance at the population level but tend to lead to the measurement of only one type of conductance per cell, despite the cell co-expressing multiple channel types. The data show that ICRAC, IARC, and TRPV-like currents are present in the model mast cell populations and respond to venom exposure. We further assessed individual venom components, specifically secretagogues and arachidonic acid, and identified the conductances associated with these stimuli in mast cells. Single-cell calcium assays and immunofluorescence analysis show that there is heterogeneity of channel expression across the cell population, but this heterogeneity does not explain the apparent selectivity for specific channels in response to exposure to venom as a composite stimulus.

TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection.

Calcium signaling in phagocytes is essential for cellular activation, migration, and the potential resolution of infection or inflammation. The generation of reactive oxygen species (ROS) via activation of NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase activity in macrophages has been linked to altered intracellular calcium concentrations. Because of its role as an oxidative stress sensor in phagocytes, we investigated the function of the cation channel transient receptor potential melastatin 2 (TRPM2) in macrophages during oxidative stress responses induced by Helicobacter pylori infection. We show that Trpm2-/- mice, when chronically infected with H. pylori, exhibit increased gastric inflammation and decreased bacterial colonization compared with wild-type (WT) mice. The absence of TRPM2 triggers greater macrophage production of inflammatory mediators and promotes classically activated macrophage M1 polarization in response to H. pylori. TRPM2-deficient macrophages upon H. pylori stimulation are unable to control intracellular calcium levels, which results in calcium overloading. Furthermore, increased intracellular calcium in TRPM2-/- macrophages enhanced mitogen-activated protein kinase and NADPH-oxidase activities, compared with WT macrophages. Our data suggest that augmented production of ROS and inflammatory cytokines with TRPM2 deletion regulates oxidative stress in macrophages and consequently decreases H. pylori gastric colonization while increasing inflammation in the gastric mucosa.

Differential regulation of calcium signalling pathways by components of Piper methysticum ('Awa).

Kava is a soporific, anxiolytic and relaxant in widespread ritual and recreational use throughout the Pacific. Traditional uses of kava by indigenous Pacific Island peoples reflect a complex pharmacopeia, centered on GABA-ergic effects of the well-characterized kavalactones. However, peripheral effects of kava suggest active components other than the CNS-targeted kavalactones. We have previously shown that immunocytes exhibit calcium mobilization in response to traditionally prepared kava extracts, and that the kavalactones do not induce these calcium responses. Here, we characterize the complex calcium-mobilizing activity of traditionally prepared and partially HPLC-purified kava extracts, noting induction of both calcium entry and store release pathways. Kava components activate intracellular store depletion of thapsigargin-sensitive and -insensitive stores that are coupled to the calcium release activated (CRAC) current, and cause calcium entry through non-store-operated pathways. Together with the pepper-like potency reported by kava users, these studies lead us to hypothesize that kava extracts contain one or more ligands for the transient receptor potential (TRP) family of ion channels. Indeed, TRP-like conductances are observed in kava-treated cells under patch clamp. Thus TRP-mediated cellular effects may be responsible for some of the reported pharmacology of kava.

[An endodontic ultrasonic system for apical endodontic surgery]. / Ultrasone wortelpuntpreparatie bij een chirurgische apicale endodontische behandeling.

Apical endodontic surgery is applied frequently following a failed conventional endodontic treatment. The apical preparation can be carried out conventionally using a round bur or using an endodontic ultrasonic system. The purpose of this study was to compare the outcome of the 2 treatment options by a randomized prospective clinical study. Patients (n=399) were at random allocated to treatment using a conventional round bur or using an ultrasonic system (P-max Newtron) according to a for the rest similar treatment protocol. One year post treatment, the treatment outcomes were determined by 2 oral and maxillofacial surgeons, blinded for the treatment option. Adequate follow-up data were obtained from 290 patients. The overall success rate was 71% in the patients treated conventionally and 81% in the patients treated using the ultrasonic system. In molar teeth, the difference in success rate was statistically significant.

Transition zone cancers undermine the predictive accuracy of Partin table stage predictions.

PURPOSE: The Partin tables represent the most widely used predictor of pathological stage in men with localized prostate cancer (PCa). The accuracy and performance of the tables have been tested across different populations. However, to our knowledge the potential limitations that may stem from differences between transition zone (TZ) and peripheral zone (PZ) prostate cancers has not been explored. We tested the predictive accuracy and performance of the Partin tables according to TZ vs PZ tumor predominance. MATERIALS AND METHODS: Preoperative serum prostate specific antigen, clinical stage and biopsy Gleason sum data on 1,990 patients treated with radical retropubic prostatectomy were used to define the 2001 Partin probabilities of organ confinement and seminal vesicle invasion (SVI). Data on 1,320 patients who underwent staging pelvic lymphadenectomy and radical retropubic prostatectomy were used to define the probabilities of lymph node invasion (LNI) and organ confined disease (OC). ROC area under the curve was used to assess the predictive accuracy of the 2001 Partin tables relative to observed extracapsular extension (ECE), SVI, LNI and OC. Performance characteristics for each prediction were explored graphically with local regression, nonparametric smoothing plots. Results were compared between 222 TZ cancers and 1,768 PZ cancers. RESULTS: The 1,990 radical retropubic prostatectomy specimens demonstrated ECE in 689 cases (34.6%) (TZ in 58 or 27.1% and PZ in 631 or 35.8%) and SVI in 224 (TZ in 13 or 6.1% and PZ in 211 or 11.9%). The 1,320 lymphadenectomy specimens demonstrated LNI in 56 cases (TZ in 2 or 0.9% and PZ in 54 or 4.6%). OC was found in 784 cases (59.4%) (TZ in 95 or 69.9% and PZ in 689 or 58.2%). Predictive accuracy was for ECE 76.4% (TZ 69.0% and PZ 77.2%), 78.0% for SVI (TZ 73.5% and PZ 78.3%), 78.6% for LNI (TZ 44.5% and PZ 79.9%) and 79.4% for OC (TZ 73.8% and PZ 80.0%). CONCLUSIONS: The biological tumor characteristics of TZ PCa differ from those of PZ PCa. These differences appear to undermine the accuracy of pathological stage predictions.

The gastrointestinal tract as target of steroid hormone action: quantification of steroid receptor mRNA expression (AR, ERalpha, ERbeta and PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR.

We have examined the tissue-specific mRNA expression pattern of androgen receptor (AR), both estrogen receptor (ER) subtypes ERalpha and ERbeta and progestin receptor (PR) in 10 bovine gastrointestinal compartments. Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation Ralgro on the compartment-specific expression regulation. Ralgro contains Zeranol which shows strong estrogenic and anabolic effects. Eight heifers were treated for 8 weeks with Ralgro at different dosages (0, 1, 3, and 10 times). To quantify the very low abundant steroid receptor mRNA transcripts sensitive and reliable real-time (kinetic) reverse transcription (RT)-PCR quantification methods were validated on the LightCycler. Expression results indicate the existence of AR and both ER subtypes in all 10 gastrointestinal compartments. PR receptor was expressed at very low abundancy. Gastrointestinal tissues exhibit a specific ERalpha and ERbeta expression pattern with high expression levels for both subtypes in rectum, colon and ileum. With increasing Zeranol concentrations a significant down-regulation for ERalpha and ERbeta was observed in jejunum (P<0.001 and <0.05, respectively). Significant up-regulations under estrogen treatment could be shown in abomasum for ERalpha (P<0.05) and in rectum for ERbeta (P<0.001). The authors conclude, that especially estrogens and the expression of their corresponding receptor subtypes may play an important role in the modulation and regulation in gastric as well as gut functions, cell proliferation and possibly in the pathophysiology of cell cancer. The different expression patterns of ERalpha and ERbeta can be regarded as support of the hypothesis that the subtype proteins may have different biological functions in the gastrointestinal tract. AR and PR seem to be not estrogen dependent.

Estradiol-17beta is produced in bovine corpus luteum.

The aim of this study was to investigate the expression of cytochrome P450 aromatase (aromatase) mRNA, its activity, and estradiol-17beta (estradiol) secretion in bovine corpus luteum (CL) during the estrous cycle. Expression of aromatase mRNA was examined in CL at the early, mid, late, and regressed luteal stages by using a reverse transcription-polymerase chain reaction. Aromatase mRNA was detected in all luteal stages examined, although aromatase expression was significantly lower during the early and regressed luteal phases compared to the mid and late luteal phases. Moreover, cultured midluteal cells clearly converted exogenous [(3)H]androstenedione into estradiol, and an aromatase inhibitor significantly inhibited this conversion. To characterize the local release of estradiol within the CL during the estrous cycle, an in vitro microdialysis system (MDS) of CL was conducted. Estradiol in MDS perfusate was confirmed by a reverse-phase high-performance liquid chromatography in combination with enzyme immunoassays. Basal release of estradiol from microdialyzed CL did not change during the estrous cycle. Additionally, when freshly prepared midluteal cells were exposed to estradiol (10(-14) to 10(-9) M), estradiol stimulated prostaglandin (PG) F(2alpha) secretion (P < 0.05), although it did not affect progesterone and oxytocin secretion. The overall results indicate that estradiol is produced locally in bovine CL throughout the estrous cycle, and they suggest that estradiol plays a role in regulating PGF(2alpha) production in CL as an autocrine/paracrine factor.

Tissue-specific expression pattern of estrogen receptors (ER): quantification of ER alpha and ER beta mRNA with real-time RT-PCR.

We have examined the tissue-specific mRNA expression of ER alpha and ER beta in various bovine tissues using real-time RT-PCR. The goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation RALGRO on the tissue-specific expression and regulation of both ER subtypes. RALGRO contains Zeranol (alpha-Zearalanol), a derivative of the mycotoxin Zearalenon, shows strong estrogenic and anabolic effects, and exhibits all symptoms of hyperestrogenism, in particular reproductive and developmental disorders. Eight heifers were treated over 8 weeks with multiple-dose implantations (0x, 1x, 3x, 10x) of Zeranol. Plasma Zeranol concentration, measured by enzyme immunoassay, of multiple treated heifers was elevated. To quantify ER alpha and ER beta transcripts also in low-abundant tissues, sensitive and reliable real-time RT-PCR quantification methods were developed and validated on the LightCycler. Expression results indicate the existence of both ER subtypes in all 15 investigated tissues. All tissues exhibited a specific ER alpha and ER beta expression pattern and regulation. With increasing Zeranol concentrations, a significant downregulation of ER alpha mRNA expression could be observed in jejunum (p<0.001) and kidney medulla (p<0.05). These data support the hypothesis that ER beta may have different biological functions than ER alpha, especially in kidney and jejunum.

Hormone contents in peripheral tissues after correct and off-label use of growth promoting hormones in cattle: effect of the implant preparations Filaplix-H, Raglo, Synovex-H and Synovex Plus.

Certain hormonal growth promoters are licensed in several beef producing countries outside the European Union (EU). Use in compliance with Good Veterinary Practice is mandatory. As risk assessment of hormone residues in animal tissues up to now has neglected potential off-label use, the present study dealt with two topics: 1) multiple treatment with the implant preparations Finaplix-H (200 mg trenbolone acetate), Ralgro (36 mg zeranol) and Synovex-H (200 mg testosterone propionate plus 20 mg estradiol benzoate) in heifers (1-fold, 3-fold and 10-fold dose), and 2) non-approved treatment of female veal calves (1-fold dose of Synovex-H or Synovex Plus with 200 mg trenbolone acetate plus 28 mg estradiol benzoate). Residues of estradiol-17beta, estradiol-17alpha, estrone and testosterone, trenbolone-17beta, trenbolone-17alpha and trendione or zeranol, respectively, were measured in loin, liver, kidney and peri-renal fat by high performance liquid chromatography/enzyme immunoassay (HPLC/EIA) after liquid-liquid extraction and solid-phase clean-up. The hormone residues in the multiple-dose experiments were dose-dependent and partially exceeded the threshold values: in the liver in one animal after 3-fold dose and in two animals after 10-fold dose of Finaplix-H, and in the liver and kidney after 3-fold and 10-fold dose of Synovex-H. Mean hormone residues in calves were mainly below those of heifers and did not infringe threshold values.

Detection of anabolic residues in misplaced implantation sites in cattle.

Eight weeks before slaughter, 26 heifers, 2 calves, and 1 steer were implanted with licensed anabolic preparations at off-label injection sites. After slaughter, 24 of 31 implantation sites (77%) were detected. Residual pellets of Revalor H contained a mean of 42.9 mg trenbolone acetate (range 19.8-57.7 mg) and 4.6 mg (1.96-6.45 mg) estradiol, corresponding to 30% (19.8-57.7%) and 32.7% (14.0-46.6%) of the originally applied dose, respectively. In the tissue areas containing residual Revalor H pellets, total residues ranged from 14.8 microg to 12.6 mg trenbolone acetate, 41.7 microg to 1.45 mg trenbolone, and 11.1 microg to 3.39 mg estradiol. The outer tissue areas of the injection sites contained <2 microg hormones. The preparations Synovex H, Finaplix H, Implus S, and Component EC behaved similarly to Revalor H. Residues of Synovex Plus were low, whereas the Compudose silicone rubber contained 58.8% of the implanted dose, but left no significant tissue residues. If implantation sites are processed in meat manufacturing, international threshold levels of the respective substances will be exceeded in tons of meat products.

The wide spectrum of clinical expression in Adams-Oliver syndrome: a report of two cases.

Two children are described with the combination of aplasia cutis congenita (ACC) and transverse limb defects known as Adams-Oliver syndrome. Whereas in the first child the typical features of ACC, syndactyly and transverse nail dystrophy were only mildly expressed and associated defects of the central nervous system and cardiac malformations were absent, the second child suffered from a very severe expression of the syndrome, with a combination of ACC, syndactyly, cutis marmorata telangiectatica congenita and multiple cardiac and central nervous system malformations which resulted in fatal central respiratory insufficiency.

[Plasma TGF-beta1 concentrations in patients with prostate carcinoma or benign prostatic hyperplasia]. / Plasma-TGF-beta1-Konzentrationen bei Patienten mit einem Prostatakarzinom oder einer benignen Prostatahyperplasie.

Transforming growth factor-beta 1 (TGF-beta 1) is a pleiotrophic growth factor in carcinogenesis and regulars multiple cell functions. We wanted to evaluate the diagnostic meaning of TGF-beta 1 plasma levels in patients with a biopsy proven prostate cancer. The TGF-beta 1 blood level was analysed in 394 patients. In 242 patients (group I) the blood was taken before any prostate manipulation and biopsy. The TGF-beta 1 plasma concentrations were almost similar in the group of patients with a prostate cancer (n = 157) and patients with a benign prostate hyperplasia (n = 85; 14,258 pg/ml versus 14,658 pg/ml, SD 10,516 pg/ml). In 152 patients the blood was taken 6-12 months after radical prostatectomy (group II). There was no significant difference between the patients with a PSA-progress and without PSA-progress after. Our results suggest that TGF-beta 1 plasma levels can not be used to distinguish between patients with prostate cancer and benign prostate hyperplasia.

A case of varix dilatation of the umbilical vein and review of the literature.

Umbilical cord cysts diagnosed antenatally present a challenge to the clinician as they may be associated with adverse perinatal outcomes including abnormal karyotypes and stillbirths. We present a case of an umbilical cord cyst diagnosed by routine ultrasound at 30 weeks of gestation. Findings on sonography included unidirectional movement of echogenic particles suggesting a large varix of the umbilical vein. The patient delivered vaginally at 39 weeks and histopathology confirmed the diagnosis.

Intrauterine thoracentesis of fetal cystic lung malformations.

Fetal pulmonary malformations comprise a rare but often lethal group of congenital anomalies. Until recently, diagnosis and therapy were directed postnatally and therefore some cases of fetal compromise were inevitably missed. We present 2 cases in which intermittent thoracentesis of fetal cystic lung malformations resulted in a successful outcome. Intrauterine thoracentesis should be considered in the second and third trimester of pregnancy in cases which demonstrate early fetal compromise.

Fetoscopy in the assessment of unexplained fetal hydrops.

Pure fetal blood samples, obtained fetoscopically from 30 patients with unexplained fetal hydrops at 16 to 32 weeks gestation were investigated for cytogenetic, haematological, biochemical and virological properties. In two patients with oligohydramnios, the fetoscope was introduced transabdominally into the fetal peritoneal cavity and sampling was undertaken from the intra-abdominal portion of the umbilical vein; in all the other patients an umbilical cord vessel was sampled. Ten (33%) of the fetuses had chromosomal abnormalities, one an erythroblastic process, possibly erythroleukaemia, one alpha-thalassaemia and one cytomegalovirus infection. Blood-film abnormalities were seen in 23 (88%) of 26 fetuses that had this examination. Biochemical analysis of fetal plasma was undertaken in 18 fetuses and hypoproteinaemia was found in all cases. One fetus was subsequently found to have a paroxysmal tachyarrhythmia that responded to digitilization. Three (10%) of the fetuses survived.

Fetal biophysical profile score and the nonstress test: a comparative trial.

In this prospective blind study, 735 patients with high-risk pregnancies referred for antepartum testing of fetal well-being were randomly assigned to either a fetal biophysical profile scoring (375 patients) or a nonstress testing scheme (360 patients). Management was based on the results of antepartum tests, but the method of testing used was not disclosed. Fetal biophysical profile scoring resulted in a significantly higher positive predictive value in regards to low Apgar scores. Sensitivity, specificity, and accuracy, although higher with fetal biophysical profile scoring, did not demonstrate significant differences when compared with the nonstress test. The negative predictive value between the two methods was similar. All major anomalies were detected during ultrasound scanning, whereas none of these anomalies were detected by heart rate testing alone.

The transfer of cephradine across the placenta.

The transfer of maternally administered cephradine (1 g given intravenously) to the fetal circulation and amniotic fluid was investigated in 2 pregnant patients undergoing fetoscopy. Cephradine was detected in fetal serum between 23 and 72 minutes after injection and was present in therapeutic concentrations. Fetal serum levels appeared to peak at approximately 40-50 min. Amniotic fluid levels were rising during the sampling period of 97 min. Cephradine is transferred across the placenta and appears to be a suitable antibiotic for use in selected obstetric patients when penetration of the fetal compartment is desirable.

Treatment of the fetus in utero: evolving concepts.

The rapidly evolving ability to detect the presence of major anomalies in the developing fetus and the emerging prospect of intrauterine treatment of some of these disorders are coupled to create an exciting new frontier in care of the high-risk pregnancy. Preliminary reports suggest that in some highly selected instances perinatal death and morbidity may be prevented by intrauterine surgical procedures. The technical ability to treat the disorder as described is at hand and is unlikely to be the limiting factor in the advance of this area of fetal treatment. However, the very fact that treatment is possible cannot be considered as proof of efficacy. The physician faced with the knowledge that a potentially treatable condition is present in a given fetus may feel the necessity of instituting treatment, but the validity of such an approach is far from established. It is our opinion that efforts should be made to set at the earliest point proper controlled scientific studies from which the benefit, if any, of this surgical approach to fetal disease may be established. Such clinical studies should be conducted in concert with studies in appropriate animal models. It should be noted that fetal surgical treatment is not without potentially lethal fetal and maternal complications. Before such risks, however small, are taken, it seems reasonable to determine the long-term benefits of the surgical procedure. To this end, participation in the International Fetal Surgery Registry is encouraged.