The transcriptional regulators, the immune system and the the circadian clock.

The immune system function oscillates with a 24-hour period driving circadian rhythmicity of immune responses. A circadian timing system comprising central and peripheral oscillators entrains body rhythmicity of physiology and behavior to environmental cues by means of humoral signals and autonomic neural outputs. In every single cell an oscillator goes ticking through a molecular clock operated by transcriptional/translational feedback loops driven by the rhythmic expression of circadian genes. This clock gene machinery steers daily oscillations in the regulation of immune cell activity, driving the periodicity in immune system function. The transcriptional networks that regulate temporal variation in gene expression in immunocompetent cells and tissues respond to diverse physiological clues, addressing well-timed adjustments of transcription and translation processes. Nuclear receptors comprise a unique class of transcriptional regulators that are capable of gauging hormones, metabolites, endobiotics and xenobiotics, linking ligand sensing to transcriptional responses in various cell types through switching between coactivator and corepressor recruitment. The expression of coregulators is highly responsive to physiological signals, and plays an important role in the control of rhythmic patterns of gene expression, optimizing the switch between nycthemeral patterns, and synchronizing circadian rhythmicity with changing physiological demands across the light-dark cycle. The nuclear receptors and transcription factors expressed in the immune components contribute to the cross-talk between the circadian timing system, the clock gene machinery and the immune system, influencing transcriptional activities and directing cell-type specific gene expression programs linked to innate and adaptive immune responses.

REV-ERBα and the clock gene machinery in mouse peripheral tissues: a possible role as a synchronizing hinge.

Rhythmic oscillations of cellular biological processes are driven by translational-transcriptional feedback loops that realize molecular clocks ticking in every single cell, driven by neural and humoral outputs from the suprachiasmatic nuclei of the hypothalamus that are entrained by environmental photon inputs. The nuclear receptor REV-ERBα has the capability to reset the molecular oscillators of peripheral tissues. The aim of our study was to evaluate the clock gene machinery function in light/dark cycles (LD) and in constant darkness (DD) exploiting in particular the REV-ERBα pattern of expression by using data from two independent experimental settings to reduce procedure related influences. In the LD study C57BL/6 male mice housed on a 12L:12D cycle were sacrificed at 4 h intervals. Liver, kidney, spleen, thymus and testis were harvested and blood was collected. Expression levels of PER1, PER2, CRY1, CRY2, BMAL1, REV-ERBα, CLOCK were evaluated by qRT-PCR. In the DD study Balb/c male mice in the third DD cycle as a continuation of the dark phase of the last LD cycle were sacrificed at 4 h intervals. Lung, heart, liver, stomach, kidney, spleen, and testis were harvested and mRNA expression of PER1, PER2, CRY1, CRY2, BMAL1, REV-ERBα, CLOCK, was evaluated by qRT-PCR. A statistically significant difference was found for the size of the semi-interquartile range of acrophases of clock gene expression in different organs evaluated in LD and DD conditions (4:38∓1:12h versus 1:16∓0:10h, p=0.026). A statistically significant difference was found for the acrophases of clock gene expression in different organs evaluated in LD (p=0.01) and in DD (p<0.0001). In LD study only REV-ERBα showed concomitant expression in the different peripheral tissues with the phase peaking around 07:03∓0.8h. In the DD study all the core clock genes showed concomitant phases in different peripheral mouse tissues and REV-ERB alpha expression peaked around 07:09∓0.9h. In conclusion, REV-ERBα is the only clock gene that maintains its timing of oscillation in the LD study and in the DD study and its phase of expression remains concomitant in the different mouse peripheral tissues in the presence of LD alternance, or in constant darkness. Oscillation in REV-ERBα ligands (heme, carbon monoxide) may affect not only the phase and amplitude of circadian rhythms, but also physiological outputs of the circadian system and REV-ERBalpha may participate in the entrainment of central and peripheral clocks, functioning as a synchronizing hinge of the clock gene machinery.

Clock gene expression in mouse kidney and testis: analysis of periodical and dynamical patterns.

Molecular clocks drive circadian rhythmicity of cellular functions in peripheral tissues and organs, kidney included, whereas in the testis this clockwork seems constitutively active. We have evaluated the periodicity and the dynamics of expression of the clock genes BMAL1, CLOCK, PER1, PER2, CRY1, CRY2 and REV ERBalpha over 24 h in the kidney and testis using a mouse model. The periodicity was explored by single cosinor, and dynamics were explored by calculation of fractional variations of gene expression related to time intervals. Kidney and testis were harvested at 4-h intervals over a 24-h period from eight-week-old C57BL/6 male mice housed individually on a 12 h light (L)-dark (D) cycle (lights on at 08:00 h; lights off at 20:00 h) and mRNA was extracted and analyzed by Quantitative Real-time Reverse Transcription PCR. A statistically significant difference was evidenced between kidney and testis for the original values of expression level of BMAL1, PER1, PER2 CRY1, CRY2 and REV ERBα. A statistically significant difference was evidenced between kidney and testis for the fractional variation of BMAL1, PER2, CRY1, CRY2 and REV ERBα. A significant 24-h rhythmic component was found for BMAL1, CLOCK, PER1, PER2, CRY1, CRY2 and REV ERBα in the kidney, whereas no core clock gene showed circadian rhythmicity in the testis. Fractional variations provided significant circadian rhythms for BMAL1, PER2, CRY, CRY2 and REV ERBα in the kidney, whereas in the testis the fractional variation calculations showed no circadian rhythmicity, but quantitative comparison showed statistically significant differences in only 16.7 percent of the time points studied. In conclusion, in the kidney the clock gene machinery shows circadian oscillation of mRNA levels and time-related variations in the rate of change of clock gene expression. In the testis the clock genes do not show circadian rhythmicity of expression and the dynamics of variation are not characterized by a periodical pattern, but are quantitatively similar to those observed in the kidney. These data suggest that in the testis the clock gene machinery shows a tissue-specific pattern of function and clock genes may play a different role in the testis with regard to other peripheral tissues, maybe in relation to the presence of developmental and differentiation phenomena.

Hypothalamus-hypophysis-thyroid axis function in healthy aging.

There is an increased frequency of dysthyroidism in elderly people. We investigated whether there are differences among healthy young middle-aged and elderly people in the 24 hour secretory profiles of TRH, TSH and free thyroxine. The study was carried out on fifteen healthy young, middle-aged subjects (range 36-55 years, mean age±s.e. 44.1±1.7) and fifteen healthy elderly subjects (range 67-79 years, mean age±s.e. 68.5±1.2). TRH, TSH and free thyroxine serum levels were measured in blood samples collected every four hours for 24 hours. The area under the curve (AUC), the mean of 06:00h-10:00h-14:00h and the mean of 18:00h-22:00h-02:00h hormone serum levels and the presence of circadian rhythmicity were evaluated. A normal circadian rhythmicity was recognizable for TRH and TSH in young, middle-aged subjects and for TSH in elderly subjects. Elderly subjects presented lower TSH levels, whereas there was no statistically significant difference in TRH and free thyroxine serum levels between young, middle-aged and elderly subjects. Aging is associated with an altered TSH secretion.

Statistical assessment of functional categories of genes deregulated in pathological conditions by using microarray data.

MOTIVATION: A major challenge in current biomedical research is the identification of cellular processes deregulated in a given pathology through the analysis of gene expression profiles. To this end, predefined lists of genes, coding specific functions, are compared with a list of genes ordered according to their values of differential expression measured by suitable univariate statistics. RESULTS: We propose a statistically well-founded method for measuring the relevance of predefined lists of genes and for assessing their statistical significance starting from their raw expression levels as recorded on the microarray. We use prediction accuracy as a measure of relevance of the list. The rationale is that a functional category, coded through a list of genes, is perturbed in a given pathology if it is possible to correctly predict the occurrence of the disease in new subjects on the basis of the expression levels of the genes belonging to the list only. The accuracy is estimated with multiple random validation strategy and its statistical significance is assessed against a couple of null hypothesis, by using two independent permutation tests. The utility of the proposed methodology is illustrated by analyzing the relevance of Gene Ontology terms belonging to biological process category in colon and prostate cancer, by using three different microarray data sets and by comparing it with current approaches. AVAILABILITY: Source code for the algorithms is available from author upon request. SUPPLEMENTARY INFORMATION: Colon cancer data set and a complete description of experimental results are available at: ftp://bioftp:76bioftpxxx@marx.ba.issia.cnr.it/supp-info.htm.

On the statistical assessment of classifiers using DNA microarray data.

BACKGROUND: In this paper we present a method for the statistical assessment of cancer predictors which make use of gene expression profiles. The methodology is applied to a new data set of microarray gene expression data collected in Casa Sollievo della Sofferenza Hospital, Foggia--Italy. The data set is made up of normal (22) and tumor (25) specimens extracted from 25 patients affected by colon cancer. We propose to give answers to some questions which are relevant for the automatic diagnosis of cancer such as: Is the size of the available data set sufficient to build accurate classifiers? What is the statistical significance of the associated error rates? In what ways can accuracy be considered dependant on the adopted classification scheme? How many genes are correlated with the pathology and how many are sufficient for an accurate colon cancer classification? The method we propose answers these questions whilst avoiding the potential pitfalls hidden in the analysis and interpretation of microarray data. RESULTS: We estimate the generalization error, evaluated through the Leave-K-Out Cross Validation error, for three different classification schemes by varying the number of training examples and the number of the genes used. The statistical significance of the error rate is measured by using a permutation test. We provide a statistical analysis in terms of the frequencies of the genes involved in the classification. Using the whole set of genes, we found that the Weighted Voting Algorithm (WVA) classifier learns the distinction between normal and tumor specimens with 25 training examples, providing e = 21% (p = 0.045) as an error rate. This remains constant even when the number of examples increases. Moreover, Regularized Least Squares (RLS) and Support Vector Machines (SVM) classifiers can learn with only 15 training examples, with an error rate of e = 19% (p = 0.035) and e = 18% (p = 0.037) respectively. Moreover, the error rate decreases as the training set size increases, reaching its best performances with 35 training examples. In this case, RLS and SVM have error rates of e = 14% (p = 0.027) and e = 11% (p = 0.019). Concerning the number of genes, we found about 6000 genes (p < 0.05) correlated with the pathology, resulting from the signal-to-noise statistic. Moreover the performances of RLS and SVM classifiers do not change when 74% of genes is used. They progressively reduce up to e = 16% (p < 0.05) when only 2 genes are employed. The biological relevance of a set of genes determined by our statistical analysis and the major roles they play in colorectal tumorigenesis is discussed. CONCLUSIONS: The method proposed provides statistically significant answers to precise questions relevant for the diagnosis and prognosis of cancer. We found that, with as few as 15 examples, it is possible to train statistically significant classifiers for colon cancer diagnosis. As for the definition of the number of genes sufficient for a reliable classification of colon cancer, our results suggest that it depends on the accuracy required.

Proteomic screening identifies calreticulin as a miR-27a direct target repressing MHC class I cell surface exposure in colorectal cancer.

Impairment of the immune response and aberrant expression of microRNAs are emerging hallmarks of tumour initiation/progression, in addition to driver gene mutations and epigenetic modifications. We performed a preliminary survey of independent adenoma and colorectal cancer (CRC) miRnoma data sets and, among the most dysregulated miRNAs, we selected miR-27a and disclosed that it is already upregulated in adenoma and further increases during the evolution to adenocarcinoma. To identify novel genes and pathways regulated by this miRNA, we employed a differential 2DE-DIGE proteome analysis. We showed that miR-27a modulates a group of proteins involved in MHC class I cell surface exposure and, mechanistically, demonstrated that calreticulin is a miR-27a direct target responsible for most downstream effects in epistasis experiments. In vitro miR-27a affected cell proliferation and angiogenesis; mouse xenografts of human CRC cell lines expressing different miR-27a levels confirmed the protein variations and recapitulated the cell growth and apoptosis effects. In vivo miR-27a inversely correlated with MHC class I molecules and calreticulin expression, CD8(+) T cells infiltration and cytotoxic activity (LAMP-1 exposure and perforin release). Tumours with high miR-27a, low calreticulin and CD8(+) T cells' infiltration were associated with distant metastasis and poor prognosis. Our data demonstrate that miR-27a acts as an oncomiRNA, represses MHC class I expression through calreticulin downregulation and affects tumour progression. These results may pave the way for better diagnosis, patient stratification and novel therapeutic approaches.

Two novel mutations and a new STK11/LKB1 gene isoform in Peutz-Jeghers patients.

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominantly inherited disorder with variable expression and incomplete penetrance characterized by mucocutaneous pigmentation, predisposition to hamartomatous intestinal polyposis, and various other neoplasms. It occurs in approximately 1 in 8,300 to 29,000 live births. In nearly 50% of patients PJS is caused by germ line mutations in the STK11/LKB1 serine/threonine kinase gene, the only kinase gene currently known to act as a tumor suppressor. We have performed a mutation search in the STK11/LKB1 gene in 8 sporadic cases and 3 PJS families using a combination of different screening techniques. We have identified four mutations, two of which I177N and the IVS2+1A->G, were previously unreported. We have also evaluated the presence of cDNA alterations by means of RT-PCR analysis and direct cDNA sequencing and have found two aberrant transcripts in a single PJS case despite the lack of any apparent genomic alteration. Finally, we report the presence of a novel STK11/LKB1 cDNA isoform observed in all the normal subjects studied as well as in the majority of the PJS patients.

Genetic analysis in Italian families with inflammatory bowel disease supports linkage to the IBD1 locus--a GISC study.

Epidemiological studies suggest that inherited factors influence susceptibility to inflammatory bowel disease (IBD), and some candidate loci have been described. In order to verify whether the same loci are responsible for predisposition to IBD in our population, we carried out a linkage study in a series of 58 Italian families with Crohn's disease (CD) and ulcerative colitis (UC). HLA-DQ alleles, motilin gene, and 34 microsatellites flanking the previously described loci on chromosomes 3, 6, 7, 12 and 16 were analysed by non-parametric linkage analysis in 16 and 23 families with CD and UC, respectively, and in 19 families where CD and UC coexisted. Non parametric analysis using GENEHUNTER yielded maximum NPL scores for marker D16S408 in all IBD families combined (2.71, P = 0.003), for marker D16S419 in CD (1.97, P = 0.026) and for marker D16S514 in UC families (2.44, P = 0.007). These markers map in the previously described IBD1 region. No significant linkage was found for markers of chromosomes 3, 6, 7 and 12. The present study performed in a Southern European population provides additional support for the conclusion with the IBD1 locus has a clear role in the genetic susceptibility to IBD.

Appropriateness of urea breath test: a prospective observational study based on Maastricht 2000 guidelines.

BACKGROUND: The urea breath test is routinely used for diagnosing or confirming the eradication of Helicobacter pylori. AIM: To evaluate the appropriateness of urea breath test referrals. METHODS: The age, sex, symptoms, endoscopic findings, use of non-steroidal anti-inflammatory drugs, family history of gastric cancer or H. pylori infection and concomitant diseases of patients referred for urea breath testing in a 1-year period were recorded. The appropriateness of urea breath test referrals was judged according to Maastricht guidelines. RESULTS: One thousand, three hundred and twenty subjects (47 +/- 16 years) were referred in 2001: 578 (43.8%) for the diagnosis and 742 (56.2%) for confirmation of the eradication of H. pylori. The urea breath test was considered to be appropriate in 836 (63.3%) patients, inappropriate in 192 (14.5%) and appropriate but avoidable in 292 (22.1%). The appropriateness ratios of urea breath test referrals were 4.6 and 9.0 (P < 0.0001) for general practitioners and gastroenterologists, respectively. Of the patients (n=230) with un investigated dyspepsia, who underwent urea breath testing according to a 'test and treat' strategy, 98 (42.6%) presented at least one risk factor for organic disease. CONCLUSIONS: In Italy, nearly 36% of urea breath test referrals are inappropriate or could be avoided if all dyspeptic patients with risk factors were referred for endoscopy or all dyspeptic patients undergoing endoscopy were tested for H. pylori infection with biopsy methods. Both general practitioners and, to a lesser extent, gastroenterologists require educational programmes to deal effectively with H. pylori.

Anti-Saccharomyces cerevisiae mannan antibodies in inflammatory bowel disease: comparison of different assays and correlation with clinical features.

BACKGROUND: Anti-Saccharomyces cerevisiae mannan antibodies have been proposed as a new serological marker associated with Crohn's disease. However, their clinical value is still unclear; furthermore, a standardization of anti-S. cerevisiae mannan measurements is lacking. AIM: In this study, we aimed to assess the correlation between anti-S. cerevisiae mannan detection and specific clinical features in Crohn's disease and ulcerative colitis. Moreover, we tested the concordance of four different anti-S. cerevisiae mannan assays. MATERIALS AND METHODS: Serum samples from 196 patients with Crohn's disease, 197 patients with ulcerative colitis and 100 unrelated healthy controls were tested for anti-S. cerevisiae mannan with a standard enzyme-linked immunosorbent assay method (Lille) by one of the authors (VP). Subsequently, 60 randomly selected serum samples (27 Crohn's disease, 28 ulcerative colitis and five healthy controls) were tested for anti-S. cerevisiae mannan with three different commercial kits. RESULTS: With the Lille assay, anti-S. cerevisiae mannan were detected in 100 of 196 patients with Crohn's disease (51%; P < 0.0001 vs. controls), 32 of 197 patients with ulcerative colitis (16%; P < 0.02 vs. controls), and six of 100 controls (6%). No correlation between presence of anti-S. cerevisiae mannan and specific clinical features was found in both ulcerative colitis and Crohn's disease patients. The percentages of anti-S. cerevisiae mannan detected with four different assays ranged from 28 (Bouty) up to 43% (Inova), but these differences did not reach statistical significance. The concordance rate of anti-S. cerevisiae mannan detection in the four assays was very low (11 concordant results of 60 samples, 18.3%) (k = 0.15). No improvement of the concordance rate was obtained by modifying the suggested cut-off values (k = 0.20). CONCLUSION: In this study, we confirm that anti-S. cerevisiae mannan are significantly more frequent in Crohn's disease patients compared with ulcerative colitis patients (P < 0.0001) and controls. However, no correlation with clinical features was found in both ulcerative colitis and Crohn's disease. The low prevalence of anti-S. cerevisiae mannan, at least in our population, and the low concordance rate between different assays, makes the clinical role of this marker questionable.

Effect of somatostatin on human gallbladder motility: an in vitro study.

In vivo studies have demonstrated that somatostatin induces human gallbladder relaxation. To determine whether this polypeptide acts directly on the gallbladder muscle, its effect on strips of human gallbladder was studied in vitro. Strips of gallbladder were set up isometrically in an organ bath containing oxygenated Krebs' solution. Dose-response curves to cholecystokinin-octapeptide and carbachol were first established. The ability of somatostatin to cause relaxation under basal conditions and during 50% maximal stimulation by cholecystokinin-octapeptide (7.2 x 10(-8) M) and carbachol (3.5 x 10(-6) M) was assessed in 32 strips at 4.3 x 10(-6) M concentration which mimics the plasma concentrations found in patients with somatostatinoma and in 12 additional strips at 4.3 x 10(-8) M concentration. Somatostatin action on the intrinsic innervation by using electrical field stimulation (EFS) (200 mA 5 msec in duration, 30 Hz; 400 mA, 1 msec in duration, 10 Hz) was also evaluated in 39 strips. Somatostatin had no effect on the basal or carbachol-generated tensions. On the contrary, somatostatin (4.3 x 10(-6) M) reduced cholecystokinin-octapeptide-generated tensions by 8% (P < 0.001) and reduced EFS-generated tensions at 30 Hz by 7.7% (P < 0.01) and those at 10 Hz by 41.2% (P < 0.01). All responses to cholecystokinin-octapeptide and carbachol were abolished by dibutyryl-guanosine 3', 5'-cyclic monophosphate (5 x 10(-3) M) and atropine (10(-5) M), respectively (P < 0.0002 and P < 0.0002). All responses to electrical field stimulation were reduced or abolished by tetrodotoxin (2 x 10(-6) M) (P < 0.001 and P < 0.0001, respectively). Our findings show that somatostatin exerts its inhibitory action on the response to cholecystokinin-octapeptide and on the intrinsic innervation of the gallbladder smooth muscle. The probable neurotransmitter is the acetylcholine.

Gallbladder motility in vitro in men with gallstones following Billroth II gastric resection.

Gastric surgery induces an increased incidence of gallstones. To investigate the changes in gallbladder kinetics after gastric resection, 20 male patients were studied: ten patients undergoing cholecystectomy for gallstones developed after Billroth II gastric resection and ten patients undergoing cholecystectomy for cholelithiasis without previous abdominal surgery. Longitudinal strips from the gallbladder wall were suspended in an organ bath and the isometric tension recorded. Dose-response curves to cholecystokinin-octapeptide and carbachol were obtained. Half the maximal response to cholecysto-kinin-octapeptide was 0.50 +/- 0.11 x 10(-7) M in the first group and 1.36 +/- 0.37 x 10(-7) M in the second group (P < 0.05). The ED50 to carbachol was 24.33 +/- 2.69 x 10(-7) M in the gastrectomy group and 40.39 +/- 5.01 x 10(-7) M in the control group (P < 0.01). There was no significant difference in the maximal contractile response either to cholecystokinin-octa-peptide or carbachol in the two groups. Our study shows an increased gallbladder sensitivity to cholecystokinin-octapeptide and carbachol in patients with gallstones developed after Billroth II gastric resection.

Colonic smooth muscle responses in patients with diverticular disease of the colon: effect of the NK2 receptor antagonist SR48968.

BACKGROUND: Little is known about the pathophysiology of diverticular disease. AIM: To compare passive and active stress and the response to carbachol of colonic smooth muscle specimens from patients with diverticular disease and patients with colon cancer. The effect of the NK2 receptor antagonist, SR48968, on electrically evoked contractions of circular muscle was also investigated. PATIENTS: Sigmoid colon segments were obtained from 16 patients (51-83 years) undergoing elective sigmoid resection for diverticular disease and 39 patients (50-88 years) undergoing left hemicolectomy for non-obstructive sigmoid colon cancer. METHODS: Isometric tension was measured on circular or longitudinal taenial muscle. Strips were stretched gradually to Lo (length allowing the development of optimal active tension with carbachol) and were also exposed to increasing carbachol concentrations. The effects of atropine, tetrodotoxin and SR48968 on electrically evoked (supramaximal strength, 0.3 ms, 0.1-10 Hz) contractions of circular strips from 8 patients with diverticular disease and 19 patients with colon cancer were also studied. RESULTS: Both passive and active stress in circular muscle strips obtained from patients with diverticular disease was higher than in patients with colon cancer (P < 0.05). Electrically evoked contractions were significantly reduced by atropine in all preparations and were virtually suppressed by combined SR48968 and atropine. Tetrodotoxin suppressed electrically evoked contractions only in patients with colon cancer, whereas a tetrodotoxin-resistant component was identified in patients with diverticular disease. CONCLUSIONS: The changes in both passive and active stress in specimens from patients with diverticular disease may reflect circular smooth muscle dysfunction. Acetylcholine and tachykinins are the main excitatory neurotransmitters mediating electrically evoked contractions in human sigmoid colon circular muscle.

Effects of in vivo treatment with interleukins 1beta and 6 on rat mesenteric vascular bed reactivity.

1. Inflammatory bowel disease (IBD) is a condition that involves proinflammatory cytokines such as interleukins 1beta and 6 (ILs). In this disease, it has been shown that an abnormal microcirculatory system is implicated. 2. Therefore, the effects of in vivo treatment for three days with interleukins 1beta and 6 were investigated on rat isolated mesenteric vascular bed (MVB). 3. A significant concentration-dependent increase in vascular response to noradrenaline (NA) was found, with a significant difference in Emax between control (93.01 +/- 16.78 mmHg) and treated preparations (137.91 +/- 5.20 mmHg). Endothelin-1(ET-1) induced a significantly greater increase of perfusion pressure in treated rats in comparison with control rats at the highest concentration used (0.1 microm). 4. The concentration-dependent decrease of perfusion pressure induced by acetylcholine (ACh) in MVB precontracted with NA was significantly reduced in specimens from treated rats in comparison with control rats, with a significant difference in Emax between control and treated preparations. 5. Perivascular nerve stimulation (PNS) evoked contractions with no difference between treatments. Similarly, no difference in relaxant effect was found after PNS in specimens precontracted with NA, in the presence of guanethidine. 6. These findings indicate that the precocious inflammation acts only at postsynaptic level, facilitating vascular contraction. These data seem to support the hypothesis that vascular dysfunction caused by overproduction of ILs may contribute, among other immunological factors, to vasculitis in IBD that leads to intestinal ischaemia through vasoconstriction.

Functional alterations of mesenteric vascular bed, vas deferens and intestinal tracts in a rat hindlimb unloading model of microgravity.

1. Prolonged bed rest or exposure to microgravity may cause several alterations in autonomic nervous system response (ANSR). 2. Hindlimb unloading (HU) rats were used as an animal model of simulated microgravity to investigate ANSR changes. The experiments were carried out to investigate the effects of simulated microgravity on the autonomic nervous response of the perfused mesenteric vascular bed (MVB), vas deferens and the colon and duodenum from 2-week HU rats. 3. In MVB preparations of HU rats, the frequency-dependent increases in perfusion pressure with perivascular nerve stimulation (PNS; 8-40 Hz) were inhibited, whereas the noradrenaline (NA) concentration-dependent (1-100 microM) perfusion pressure increases were potentiated. The latter most probably reflected up-regulation of alpha-adrenergic receptor function. Relaxant responses of NA-precontracted MVB to PNS (4-30 Hz) or isoprenaline were not different between control and HU preparations, while vasodilation induced by the endothelial agonist ACh was reduced. 4. Transmural stimulation (2-40 Hz) induced frequency-dependent twitches of the vas deferens which were reduced in vas deferens of HU rats, while the sensitivity to NA-induced contraction was significantly increased. 5. In the gastroenteric system of HU rat, direct contractile responses to carbachol or tachykinin as well as relaxant or contractile responses to nervous stimulation appeared unchanged both in the proximal colon rings and in duodenal longitudinal strips. 6. In conclusion, HU treatment affects peripheral tissues in which the main contractile mediators are the adrenergic ones such as resistance vessels and vas deferens, probably by reducing the release of neuromediator. This study validates NA signalling impairment as a widespread process in microgravity, which may most dramatically result in the clinical phenotype of orthostatic intolerance.

Tauroursodeoxycholic acid reduces damaging effects of taurodeoxycholic acid on fundus gastric mucosa.

We investigated the effects of tauroursodeoxycholic acid (TUDCA) to assess whether this acid may also have "protective" effects similar to those found with ursodeoxycholic acid (UDCA). We used a well-known amphibian model of gastric mucosa, and studied the effects of taurodeoxycholic acid (TDCA) on electrical transepithelial parameters, acid secretion and histology in absence or in presence of TUDCA. Mucosal exposure to TDCA, after stimulation with histamine, caused a reduction in transepithelial potential difference (V(t)) and transepithelial resistance (R(t)) and a decrease in acid secretion while mucosal exposure to TUDCA did not cause a significant change in the electrical parameters. Moreover, TDCA primarily affected the neck cells, while TUDCA affected only oxyntic cells, causing a similar degree of injury to that observed in controls. Mucosal exposure to TUDCA plus TDCA caused a reduction in short circuit current (I(sc)) and R(t), whereas acid secretion did not change. These results suggest that: (1) TUDCA reduces the damaging effects of TDCA on fundus gastric mucosa; (2) TUDCA may play an important role in the treatment of gastritis associated with bile reflux.

Clock gene expression levels and relationship with clinical and pathological features in colorectal cancer patients.

The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1? expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p=.002), PER1 (p=.002), PER2 (p=.011), PER3 (p=.003), and CRY2 (p=.012) were lower, whereas the expression level of TIM (p=.044) was higher. No significant difference was observed in the expression levels of CLOCK (p=.778), CRY1 (p=.600), CSNK1 (p=.903), and TIPIN (p=.136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p=.026), and female sex (p=.005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p?=?.015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p= .013) and associated with TNM stages III-IV (p=.005) and microsatellite instability (p=.015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p=.010), PER3 (p= .010), and CSNKIE (p=.024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions.

WITHDRAWN: Neuroendocrine axes function in healthy aging: Evaluation of predictive and manipulable blood serum indexes.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.biomag.2010.09.002. The duplicate article has therefore been withdrawn.