Risk factors in German twins with inflammatory bowel disease: results of a questionnaire-based survey.

INTRODUCTION: Environmental factors may play an important role in the pathogenesis of IBD. The history of patients of the German IBD twin study was analyzed by questionnaires and interviews. METHODS: Randomly selected German monozygotic (MZ) and dizygotic (DZ) twins with at least one sibling suffering from IBD (n=512) were characterized in detail including demography, medical history and concomitant medications. Controls comprised of non-twin IBD patients (n=392) and healthy subjects (n=207). RESULTS: The most significant variables that were associated with Crohn's disease (CD) or ulcerative colitis (UC) included living abroad before time of diagnosis (OR, 4.32; 95% CI, 1.57-13.69), high frequency of antibiotic use (MZ CD OR, 5.03; 95% CI 1.61-17.74, DZ CD OR, 7.66; 95% CI, 3.63-16.82, MZ UC OR, 3.82; 95% CI, 1.45-10.56, DZ UC OR, 3.08; CI, 1.63-5.92), high consumption of processed meat including sausage (MZ CD OR, 7.9; 95% CI, 2.15-38.12, DZ CD OR, 10.75; 95% CI, 4.82-25.55, MZ UC OR, 5.69; 95% CI, 1.89-19.48, DZ UC OR, 18.11; 95% CI, 7.34-50.85), and recall of bacterial gastrointestinal infections (MZ CD OR, 15.9; 95% CI, 4.33-77.14, DZ CD OR, 17.21; 95% CI, 4.47-112.5, MZ UC OR, 5.87; 95% CI, 1.61-28.0, DZ UC OR, 11.34; 95% CI, 4.81-29.67). CONCLUSIONS: This study reinforced the association of life style events, in particular a specific dietary and infections history, with IBD. Alteration of gut flora or alterations of the mucosal immune system in reactivity to the flora could be an important factor to explain the relationship between life-style and disease.

Natural occurrence and physiological role of a truncated eIF4E in the porcine endometrium during implantation.

The present study is the first report providing evidence for a physiological role of a truncated form of the mRNA cap-binding protein eIF4E1 (eukaryotic initiation factor 4E1). Our initial observation was that eIF4E, which mediates the mRNA cap function by recruiting the eIF4F complex (composed of eIF4E, 4G and 4A), occurs in two forms in porcine endometrial tissue in a strictly temporally restricted fashion. The ubiquitous prototypical 25 kDa form of eIF4E was found in ovariectomized and cyclic animals. A new stable 23 kDa variant, however, is predominant during early pregnancy at the time of implantation. Northern blotting, cDNA sequence analysis, in vitro protease assays and MS showed that the 23 kDa form does not belong to a new class of eIF4E proteins. It represents a proteolytically processed variant of eIF4E1, lacking not more than 21 amino acids at the N-terminus. Steroid replacements indicated that progesterone in combination with 17ß-oestradiol induced the formation of the 23 kDa eIF4E. Modified cell-free translation systems mimicking the situation in the endometrium revealed that, besides eIF4E, eIF4G was also truncated, but not eIF4A or PABP [poly(A)-binding protein]. The 23 kDa form of eIF4E reduced the repressive function of 4E-BP1 (eIF4E-binding protein 1) and the truncated eIF4G lacked the PABP-binding site. Thus we suggest that the truncated eIF4E provides an alternative regulation mechanism by an altered dynamic of eIF4E/4E-BP1 binding under conditions where 4E-BP1 is hypophosphorylated. Together with the impaired eIF4G-PABP interaction, the modified translational initiation might particularly regulate protein synthesis during conceptus attachment at the time of implantation.

On the effects of the Fusarium toxin deoxynivalenol (DON) administered per os or intraperitoneal infusion to sows during days 63 to 70 of gestation.

Six pregnant sows of 180.6 ± 5.6 kg were fed either a Fusarium-contaminated (4.42 mg DON and 48.3 µg ZON per kg, DON per os, n = 3) or a control diet (0.15 mg DON and 5 µg ZON/kg) in the period of days 63 and 70 of gestation. On day 63 of gestation, sows fed the control diet were implanted with an intraperitoneal osmotic minipump (delivery rate of 10 µL/h, for 7 days) containing 50 mg pure (98%) DON in 2 ml 50% DMSO (DON ip, n = 3). Frequent plasma samples were taken to estimate the kinetics after oral and ip DON exposure. The intended continuous delivery of DON by the intraperitoneal minipump could not be shown, as there was a plasma peak (Cmax) of 4.2-6.4 ng DON/mL either immediately (sow IP-2+3) or 2.5 h (sow IP-1) after implantation of the pump followed by a one-exponential decline with a mean half-time (t1/2) of 1.75-4.0 h and only negligible DON plasma concentrations after 12 h. Therefore, the DON ip exposure has to be regarded as one single dose 1 week before termination of experiment. The DON per os sows showed a mean basis level (after achieving a steady state) of DON plasma concentration of about 6-8 ng/mL, as also indicated by the plasma DON concentration at the termination of the experiment. On day 70, caesarean section was carried out, the fetuses were killed immediately after birth, and samples of plasma, urine, and bile were taken to analyze the concentration of DON and its metabolite de-epoxy-DON. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of sows liver and spleen revealed no alterations. The proliferation rate of peripheral blood mononuclear cells (PBMC) with or without stimulation was not affected by the kind of DON treatment. The exposure of pregnant sows at mid-gestation (days 63-70, period of organogenesis) to a Fusarium toxin-contaminated diet (4.42 mg DON and 0.048 mg ZON per kg) or pure DON via intraperitoneal osmotic minipump did not cause adverse effects on health, fertility, maintenance of pregnancy, and performance of sows and their fetuses. However, DON was detected in fetus plasma, indicating that this toxin can pass the placental barrier and may cause changes in the proportion of white blood cells (lower monocyte and neutrophil and higher lymphocyte proportion in DON per os fetuses).

Practically relevant concentrations of deoxynivalenol in diets for growing-finishing pigs offered as mash or pellets.

A complete 2 x 3 two factorial design was applied to investigate the effects of Fusarium-infected wheat (2.5 mg DON/kg, 0, 25 and 50% of the diets), feed processing (mash and pellets) and the interactions thereof on fattening pigs (96, n= 16/group). Feed-to-gain ratio was significantly increased by contaminated wheat (2.65; 2.62 and 2.73 kg/kg for diets containing 0, 25 and 50% Fusarium-infected wheat, respectively) while digestibility of nutrients and metabolizable energy were not affected by the wheat batch. The feed processing also resulted in significant differences in feed-to-gain ratio but was accompanied by significant effects on the digestibility of organic matter and crude fat and on the metabolizable energy. Clinical chemical parameters were not significantly altered by the inclusion of the infected wheat. The lymphocyte proliferation capacity was not significantly affected by any of the experimental factors. A contribution of the feed processing to the variation of the deoxynivalenol (DON) effect may not be deduced from the present results.

Comparison of the molecular effects of the mycotoxins beta-zearalenol and deoxynivalenol in porcine endometrial cells--a review.

The mycotoxins beta-zearalenol (beta-ZOL) and deoxynivalenol (DON) produce toxic effects that result in diseases in humans and animals. The molecular mechanisms that control the mycotoxin-mediated effects are far from being completely understood. Various results show that these mycotoxins could inhibit cell proliferation. In the present short communication, the influence of beta-ZOL and DON on the abundance and phosphorylation state of kinases that are included in regulation of the initiation of mRNA translation (which is correlated with cell proliferation) was compared in porcine endometrial cells (PEC). Our results indicate that these mycotoxins modulate the expression and phosphorylation of these factors in a different manner. Whereas beta-ZOL mainly had an impact on the biological activity of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (Akt), eukaryotic initiation factor 4E (eIF4E) and its repressor 4E binding protein 1 (4E-BP1), DON reduced the abundance of p38 MAPk, Akt and specific 4E-BP1 bands. In summary, these results indicate that beta-ZOL influences molecular events that are included in the initiation of mRNA translation in the porcine endometrium but DON does not alter such processes clearly.

Platelet-activating factor receptor (PAF-R) and acetylhydrolase (PAF-AH) are co-expressed in immature bovine trophoblast giant cells throughout gestation, but not at parturition.

Platelet-activating factor (PAF) was associated with successful implantation in the cow, trophoblast invasiveness and angiogenesis. Bovine placentation is characterized by the limited invasion of trophoblast giant cells (TGC) into the maternal caruncular epithelium. TGC exhibit both endocrine activity and properties of tumor cells and may thus be targets of and mediators for the action of PAF. We examined PAF-receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) gene expression and localized mRNA and corresponding proteins in bovine placentomes throughout gestation and at parturition. PAF-R and PAF-AH protein and mRNA were highly expressed and colocalized in immature TGC from early gestation until near term, while mature TGC were negative. After the onset of parturition both PAF-R and PAF-AH were expressed in the maternal stroma, predominantly endothelial cells. The expression of PAF-R and PAF-AH in immature but not mature TGC during gestation implicates a role for PAF in the differentiation, maturation and function of bovine placentomal TGC. Placentomal angiogenesis could be mediated by binding of PAF to PAF-R present in endothelial cells. The parturition-related "switch" of PAF-R and PAF-AH from TGC to the maternal stroma suggests that PAF may participate in the regulation of parturition and in prepartum tissue programming.

Effect of the Fusarium toxin deoxynivalenol (DON) on IgA, IgM and IgG concentrations and proliferation of porcine blood lymphocytes.

An important effect of the trichothecene mycotoxins is the impairment of the immune function, but immunotoxicity studies have mainly been conducted on the mouse model. In the present study, the effect of deoxynivalenol (DON) on the proliferation of ConA stimulated porcine peripheral blood lymphocytes (PBL) was assessed in vitro after adding of 70-560 ng DON per ml medium, and in vivo after chronic and acute (one single dose) dietary DON exposure (5.7 mg/kg). Immunoglobulin (IgA, IgG, IgM) concentrations were measured by ELISA in supernatants and serum of pigs. The proliferation rate was estimated with two different assays (BrdU incorporation and MTT cleavage). In vitro the ConA stimulated proliferation was inhibited to 50% (IC50) at 200 and 309 ng DON/ml for the BrdU and MTT assay, respectively, indicating a higher sensitivity of DNA synthesis to DON. Immunoglobulin concentrations in the supernatant after in vitro proliferation of PBL with increasing DON concentrations for 72 h were significantly decreased, with IC50 values of 120.6, 84.1 and 71.7 ng DON/ml for IgA, IgM and IgG, respectively. In vivo significant inhibition of lymphocyte proliferation was observed only in the DON acute group using the MTT assay, but values tended to be decreased in the BrdU assay and after chronic DON exposure. Immunoglobulins (IgA, IgM and IgG) in the supernatant of cultured lymphocytes were not significantly affected after dietary DON exposure. Serum IgA of pigs showed no significant differences between the groups, whereas IgM and IgG were significant increased in the DON acute group.

On the effects of graded levels of Fusarium toxin contaminated wheat in diets for gilts on feed intake, growth performance and metabolism of deoxynivalenol and zearalenone.

A total of 36 gilts (103 +/- 6 kg) were divided into four groups and fed diets with increasing proportions of a Fusarium toxin contaminated wheat over a period of 35 days. The concentrations of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON) which were analyzed by HPLC methods were 210 and 4, 3070 and 88, 6100 and 235 and 9570 and 358 mug.kg(-1) diet fed to groups 1-4 respectively. Feed was partially refused during the first 21 days of the experiment by groups 2, 3 and 4 where two, three and six out of nine gilts were affected. No signs of hyperestrogenism or uterotrophic effects were observed due to dietary treatments. Blood serum, urine, bile and liver were analyzed for residues of DON, ZON and their metabolites. DON and its de-epoxidized metabolite (de-epoxy-DON) were detected in all analyzed specimens and increased in a significantly linearly related fashion. Alpha-zearalenol (alpha-ZOL) and beta-ZOL could be detected besides the parent toxin ZON, but only in bile and urine. In conclusion, the impact of dietary treatments on the performance parameters was most pronounced in the highest exposed group. The maximum ratio between DON concentration in liver and diet was 0.0013, and suggests that a possible contamination of pig liver with DON is negligible and does not contribute significantly to human DON exposure.

Effects of galectin-1 on regulation of progesterone production in granulosa cells from pig ovaries in vitro.

The detection of galectin-1 (gal-1) in pig granulosa cell lysates by immunoblotting and its cytosolic as well as membrane-associated localization prompted us to study its effects on cell proliferation and regulation of progesterone synthesis. The lectin stimulated the proliferation of granulosa cells from pig ovaries cultured in serum-free medium. Gal-1 inhibited the FSH-stimulated progesterone synthesis of granulosa cells. This inhibitory effect was strongly reduced by the disaccharidic competitor lactose at 30 mM. The absence of inhibitory effects on dibutyryl-cAMP (db-cAMP), forskolin, and pregnenolone-enhanced cellular progesterone synthesis suggests that gal-1interferes with the receptor-dependent mechanism of FSH-stimulated progesterone production. In FSH-stimulated granulosa cells, western blot analysis revealed the gal-1-mediated suppression of the cytochrome P450-dependent cholesterol side chain cleavage enzyme (P450(SCC)) that catalyzes the conversion of cholesterol to pregnenolone. In the presence of 30 mM lactose, the gal-1-reduced P450(SCC) expression was prevented. Strongly reduced mRNA levels were recorded for P450(SCC) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) when FSH-stimulated granulosa cells were cultured in the presence of gal-1. We conclude that gal-1 exerts its inhibitory effect on steroidogenic activity of granulosa cells by interfering the hormone-receptor interaction resulting in decreased responses to FSH stimulation.

A comprehensive survey for polymorphisms in the bovine IFN-gamma gene reveals a highly polymorphic intronic DNA sequence allowing improved genotyping of Bovinae.

This study aimed to identify interferon-gamma (IFN-gamma) gene variants in cattle for diagnostic purposes. Therefore, the entire bovine IFN-gamma gene (BoIFNG) and 2605 bp of its promoter DNA were sequenced. The BoIFNG DNA sequence conforms to the published part of Bo-IFN-gamma cDNA. Primer extension experiments show the presence of a 5' extension of exon 1 by 42 nucleotides (nt). One SINE element (Bov-A2) is located in the 5'-region, and two SINE elements (Bov-tA, Bov-B) are contained in the 3'-region of BoIFNG. The variants were detected by comparative sequence analysis of PCR amplicons from different bovine species. Four polymorphic mononucleotide repeats are situated in the promoter and in intron 1. Four distinct series of single nucleotide polymorphisms (SNP) were found in functionally important regions of BoIFNG. The region between the two intron 1 microsatellites contains the highest density of SNPs in Bos taurus breeds. One G-T transversion in the coding region of exon 1 causes a Gly(14) to Val(14) exchange in the BoIFNG signal peptide of different bovine species. A G-A transition in exon 2 encodes a Ser(19) to Asn(19) change in the mature protein of the Tibetan yak. Genotyping of randomly sampled Holstein Friesian cows at selected SNPs and of both intron 1 microsatellites revealed two dominant BoIFNG microhaplotypes. The detected SNPs improve the recently reported genotyping system of cattle.

Effect of GnRH and its antagonist (Antarelix) on LH release from cultured bovine anterior pituitary cells.

In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6 h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10(-5) or 10(-4) M Antarelix followed by 10(-6) M GnRH coincubation for a further 6 h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10(-6) M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10(-4) and 10(-5) M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRH-stimulated LH secretion with a maximal effect of 10(-4) M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.