Fenofibrate mildly stimulates browning-associated expression in white adipose tissues of young but not old male rats.

The aim of this study was to examine the effects of the hypolipemic drug fenofibrate (FF) and aging on the expression of factors/enzymes involved in brown adipose tissue (BAT) function and browning of white adipose tissue epididymal (eWAT) and subcutaneous (sWAT) depots. Young-adult and old male Wistar rats were fed standard chow (control) or supplemented with 0.1% or 0.5% FF for 30 days. Tissue samples were analysed for gene expression and protein content, and stained with Oil Red O or hematoxylin and eosin. In BAT of young rats, 0.5% FF increased only Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1 (CITED1) protein content and Fgf21 and Gpr109A mRNA expression. The expression of oxidative metabolism related genes (Pgc1α, Cpt1b, Mcad) decreased after 0.5% FF. In BAT of old rats, FF did not affect UCP1 and CITED1 content and had little effect on gene expression. Oil Red O staining of BAT revealed no changes in lipid droplet area upon treatment in either age group. In eWAT of young rats, 0.1FF elevated UCP1 protein content and Ucp1, Pgc-1α, and Mcad expression, whereas 0.5% FF increased PPARα content and Pgc-1α, Cpt1b, Mcad, and Gpr109A levels. In eWAT of old rats, only 0.1FF increased Pgc1α and Mcad expression. In both age groups median cell area of eWAT adipocytes was reduced after 0.5% FF. In sWAT Ucp1 gene expression was very low and UCP1 protein was undetectable. FF upregulated Ucp1, Cited1, Eva1, and Cpt1b expression in sWAT of young rats, with diminished effects in old rats. In both age groups 0.5% FF increased Fgf21 expression in sWAT. Median cell area of sWAT adipocytes decreased only in young rats treated with 0.5% FF. Our results reveal that fenofibrate differentially affects gene expression in BAT, with diminished effects in old compared to young rats. In WAT of young rats FF modestly stimulates the expression of factors/enzymes involved in lipid oxidative metabolism and browning. Aging reduces both these effects. Gpr109A may present a novel gene target upregulated by FF in BAT and eWAT.

The role of occlusion and micro-incontinence in the pathogenesis of penile lichen sclerosus: an observational study of pro-inflammatory cytokines' gene expression.

PURPOSE: To assess the expression of selected cytokines in penile lichen sclerosus (PLS) and associate them with the occurrence of micro-incontinence (MI) in different stages of PLS. METHODS: The skin biopsies from 49 PLS affected, and 13 from nonlesional foreskins (healthy control adult males undergoing circumcision due to phimosis caused by short frenulum) were obtained. All specimens were used for RNA extraction and RT-qPCR. Quantitative assessment of the gene expression of interleukin 1-A (IL-1A), interleukin 1-B (IL-1B), interleukin 1 receptor antagonist (IL-1RN), interleukin 6 (IL-6), transforming growth factor ß1 (TGF-ß1), and interferon-gamma (INF-γ) was performed. To determinate the presence of MI, the patients were asked about voiding patterns, especially leaking tiny drops of urine from the urethral meatus after urination. RESULTS: IL-1A, IL-6, and INF-γ mRNA levels were approximately 150, 16, and 59 times higher in PLS than in control samples, respectively. The highest IL-1A mRNA levels were observed in early PLS (n = 13), INF-γ in moderate PLS (n = 32), while IL-6 in severe PLS (n = 4). MI was noted in 45 PLS patients vs. 0 in control (p < 0.0001). IL-1A and IL-6 vs control ratios were concentration (ca.) 400 and 30 times higher, respectively, in MI PLS samples than in PLS without MI. CONCLUSION: Occlusion and irritating urine effect are associated with the clinical progression of penile LS with increased mRNA expression of IL-1A, INF-γ, and IL-6 pro-inflammatory cytokines in the foreskin.

The wine polyphenol resveratrol modulates autophagy and induces apoptosis in MOLT-4 and HL-60 human leukemia cells.

Resveratrol is a naturally occurring polyphenolic compound present in many plant species and wine. It possesses a wide range of beneficial biological properties including anticancer activity. Resveratrol has been demonstrated to induce both autophagy and apoptosis in several human cancer cell lines. The aim of this study was to investigate whether resveratrol modulates autophagy and apoptosis in MOLT-4 human lymphoblastic leukemia and HL-60 human promyelocytic leukemia cells. Cell viability was evaluated by the neutral red uptake assay. Cell cycle distribution, phosphatidylserine externalization, caspase-3 activation, changes of the mitochondrial membrane potential, intracellular production of reactive oxygen species were evaluated by flow cytometry. LC3-I to LC3-II conversion was examined based on Western blotting and immunofluorescence analyses. The level of p62/SQSTM1 protein and PARP1 cleavage were analyzed by Western blotting. The DNA degradation was assessed by gel electrophoresis. We found that resveratrol is able to modulate autophagy in MOLT-4 and HL-60 cells, as evidenced by the detection of an increased level of LC3-II and p62/SQSTM1 proteins. Moreover, resveratrol induced apoptosis in both cell lines which was associated with phosphatidylserine externalization, disruption of the mitochondrial membrane potential, caspase-3 activation, internucleosomal DNA fragmentation, PARP1 cleavage, chromatin condensation, and fragmentation of cell nuclei. The present study provides evidence that resveratrol can act as an autophagy modulator as well as an apoptosis inducer in MOLT-4 and HL-60 human leukemia cells. Our findings imply that resveratrol can be a promising chemotherapeutic agent in the treatment of leukemia.

The Immunoexpression of YAP1 and LATS1 Proteins in Clear Cell Renal Cell Carcinoma: Impact on Patients' Survival.

The aim of the study was to determine by immunohistochemistry cellular localization and immunoreactivity levels of YAP1 and LATS1 proteins in paired sections of tumor and unchanged renal tissues of 54 clear cell renal cell carcinoma (ccRCC) patients. Associations between clinical-pathological and overall survival (OS; median follow-up was 40.6 months) data of patients and YAP1 and LATS1 immunoreactivity were analyzed by uni- and multivariate Cox regression model and log-rank test. YAP1 immunoreactivity was found in the nuclei of tumor cells in 64.8% of ccRCC patients, whereas only 24.1% of tumors revealed cytoplasmic YAP1 expression. LATS1 immunoexpression was observed only in the cytoplasm of tumor cells in 59.3% of patients. LATS1 immunoreactivity in cancer cells negatively correlated with the size of primary tumor. The overall YAP1 immunoreactivity did not correlate with clinical-pathological data of patients. However, the subgroup of ccRCC patients who presented with cytoplasmic YAP1 immunoexpression had significantly shorter OS (median = 26.8 months) than patients without cytoplasmic YAP1 expression (median undefined). Multivariate Cox analysis revealed that increased cytoplasmic YAP1 (HR = 4.53) and decreased LATS1 immunoreactivity levels (HR = 0.90) were associated with worse prognosis, being independent prognostic factors. These results suggest that YAP1 and LATS1 can be considered as new prognostic factors in ccRCC.

The role of low-calorie diets and intermittent fasting in the treatment of obesity and type-2 diabetes.

Obesity is a condition associated with an increased risk of metabolic disorders, and in particular of type-2 diabetes (T2D). The treatment and prevention of obesity and associated metabolic disorders present great medical challenges. A major therapeutic goal in T2D is to control blood glucose levels, which can be achieved by pharmacological and nonpharmacological measures. The latter include increased physical activity and reduction of body fat mass by limiting dietary caloric content. Low-calorie diets (LCDs) involve a reduction in daily caloric intake by 25% to 30%. LCDs should be individualized depending on the patient's energy requirements, the severity of the obesity, and any accompanying diseases and treatments. Intermittent fasting (IF) involves caloric restriction for one or several days a week, or every day as the prolongation of the overnight fast. The results of recent clinical trials have shown that LCDs and intermittent fasting in patients with obesity (including those with coexisting T2D) can lead to a reduction in body fat mass and metabolic parameter improvements. These beneficial effects arise not only from the loss of body mass, but also from the activation of metabolic pathways specific to fasting conditions. However, the paucity of large-scale randomized controlled trials makes it difficult to prescribe LCDs or IF as reliable, routine methods for successful and stable weight loss.

Differential antitumor effects of vitamin D analogues on colorectal carcinoma in culture.

Colorectal cancer (CRC) is an emerging global problem with the rapid increase in its incidence being associated with an unhealthy lifestyle. Epidemiological studies have shown that decreased levels of vitamin D3 significantly increases the risk of CRC. Furthermore, negative effects of vitamin D3 deficiency can be compensated by appropriate supplementation. Vitamin D3 was shown to inhibit growth and induce differentiation of cancer cells, however, excessive vitamin D3 intake leads to hypercalcemia. Thus, development of efficient vitamin D3 analogues with limited impact on calcium homeostasis is an important scientific and clinically relevant task. The aims of the present study were to compare the antiproliferative potential of classic vitamin D3 metabolites (1α,25(OH)2D3 and 25(OH)D3) with selected low calcemic analogues (calcipotriol and 20(OH)D3) on CRC cell lines and to investigate the expression of vitamin D-related genes in CRC cell lines and clinical samples. Vitamin D3 analogues exerted anti-proliferative effects on all CRC cell lines tested. Calcipotriol proved to be as potent as 1α,25(OH)2D3 and had more efficacy than 20-hydroxyvitamin D3. In addition, the analogs tested effectively inhibited the formation of colonies in Matrigel. The expression of genes involved in 1α,25(OH)2D3 signaling and metabolism varied in cell lines analysed, which explains in part their different sensitivities to the various analogues. In CRC biopsies, there was decreased VDR expression in tumor samples in comparison to the surgical margin and healthy colon samples (p<0.01). The present study indicates that vitamin D3 analogues which have low calcemic activity, such as calcipotriol or 20(OH)D3, are very promising candidates for CRC therapy. Moreover, expression profiling of vitamin D-related genes is likely to be a powerful tool in the planning of anticancer therapy. Decreased levels of VDR and increased CYP24A1 expression in clinical samples underline the importance of deregulation of vitamin D pathways in the development of CRC.

TL1A as a Potential Local Inducer of IL17A Expression in Colon Mucosa of Inflammatory Bowel Disease Patients.

Interaction between TL1A and death receptor 3 (DR3) co-stimulates T cells, induces production of several pro-inflammatory cytokines and has been linked to pathogenesis of inflammatory bowel disease (IBD). This study aimed to establish a link between expression of TL1A and selected TL1A-induced pro-inflammatory cytokines involved in IBD pathogenesis (IL-4, IL-13, IL-17A and IFN-γ) and to investigate a connection between serum concentration of TL1A in patients with IBD and activation of peripheral blood T cells. Elevated levels of IL-4 (2.91-fold) and IL-13 (4.05-fold) mRNA were detected in the inflamed colon mucosa of patients with ulcerative colitis (UC), IFN-γ mRNA was upregulated (3.23-fold) in the inflamed colon mucosa of patients with Crohn's disease (CD), whereas upregulation of IL-17A and TL1A mRNA was present in the inflamed colon mucosa of patients with both CD and UC (IL-17A: 4.48-fold and 2.74-fold, TL1A: 3.19-fold and 3.22-fold, respectively) vs. control subjects. We did not detect any changes in DR3 mRNA expression in the investigated groups of patients. TL1A mRNA level in colon mucosa of patients with IBD correlated only with the level of IL-17A mRNA but no other investigated cytokines. In colon mucosa, expression of TL1A and DR3 was localized to enterocytes and lamina propria mononuclear cells. We did not find any correlation between serum concentrations of TL1A and IL-17A or changes of CD4(+) or CD8(+) lymphocytes phenotype in patients with IBD. Therefore, our data indicate that TL1A may contribute to pathogenesis of IBD via local but not systemic induction of IL-17A but not IL-4, IL-13 or IFN-γ.

3-Fluoromethcathinone, a structural analog of mephedrone, inhibits growth and induces cell cycle arrest in HT22 mouse hippocampal cells.

Recently, the abuse of recreational drugs has become an important problem in many countries. Among these psychoactive substances are synthetic cathinones, a group of compounds derived from the alkaloid cathinone, that have gained widespread popularity. Many cathinones have demonstrated neurotoxic effects. The aim of this study was to examine the effects of 3-fluoromethcathinone, a structural analog of mephedrone, on HT22 mouse hippocampal cells. Cell viability was assessed using the sulforhodamine B assay. Flow cytometry was used to study the cell cycle distribution. We found that 3-fluoromethcathinone inhibits growth of HT22 cells. Our results also revealed that it induces G0/G1-phase cell cycle arrest. To our knowledge, this is the first study to demonstrate the cytotoxic action of 3-fluoromethcathinone. Our findings suggest that abuse of this cathinone derivative may not be without risk.

Short-term calorie restriction and refeeding differently affect lipogenic enzymes in major white adipose tissue depots of young and old rats.

The metabolic effects of short-term calorie restriction (SCR) and subsequent refeeding were compared in different white adipose tissue (WAT) depots of young (5-month old) and old (24-month) male Wistar rats. The animals were subjected to a 40% calorie restricted diet (i.e. 40% lower food supply than of control rats) for 30 days, and then re-fed for 0, 2, or 4 days. WAT samples from perirenal (pWAT), epididymal (eWAT), and subcutaneous (sWAT) depots were analysed for the enzymatic activities of ATP-citrate lyase (ACL), fatty acid synthase (FAS), and glucose-6-phosphate dehydrogenase (G6PD). The total WAT mass almost doubled in old rats, however, aging did not alter the relative proportions of the major regional fat depots. Serum leptin concentration was prominently higher in old rats, in which SCR resulted in less suppression of leptin level than in young animals, whereas refeeding increased leptin concentration in young, but not old, rats. In young rats refeeding elevated leptin gene expression only in pWAT, while in old rats the expression was induced first in eWAT, and later in pWAT. A prominent age-related decrease of ACL and FAS activities, but not of G6PD activity, was found in all the studied WAT depots. In young control rats, ACL activity was highest in pWAT, FAS activity was similar in all WAT depots, and G6PD activity was lowest in eWAT. In old rats, the enzymatic activities were lower in eWAT than in the other depots. The patterns of response to SCR and refeeding varied by age and WAT location. SCR stimulated ACL activity in pWAT but not in other depots of young rats, while FAS activity in pWAT and sWAT did not change in young and decreased in the old animals. Among the studied depots, pWAT was most responsive to refeeding in both age groups. In conclusion, SCR in old rats, as compared to the young, may be accompanied by reduced 'rebound effect' upon returning to unrestricted diet.

The presence of a supporting person during delivery affects cord blood haematopoietic stem cells.

Several randomised trials have shown that support during labour improves the outcomes of both mother and the newborn. There is a lack of information concerning the influence of the supporting person on cord blood haematopoietic stem cells (CB HSCs), thus, these cells have been determined to be a suitable graft source for haematopoietic transplantations. This study was aimed to examine the relation between the presence of the accompanying person during labour and some features of CB HSCs. Interestingly, we found that supported deliveries were characterised by lower CB volume and lower counts of HSCs and mononuclear cells in CB. We concluded that the presence of a supporting person during labour seems to affect the yield of HSCs.

Influence of receptor activator of nuclear factor kappa B ligand, osteoprotegerin and interleukin-33 on bone metabolism in patients with long-standing ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic disease with periods of remission and recurrences. Dysfunction of the local immune response leads to chronic inflammation within the large intestine which triggers morphological changes in the intestinal wall as well as induces the synthesis of numerous factors that have an adverse impact on the bone metabolism. The aim of the study was to determine the expression of RANKL, OPG and IL-33 in mucosal biopsies of UC patients with long disease duration as well as serum level of these cytokines in the context of bone density and bone metabolism. MATERIALS AND METHODS: The UC group consisted of 56 patients with average disease duration of 16y. The control group comprised 37 healthy individuals. Local expression of cytokines was assessed in the biopsies of colonic mucosa by the real-time PCR and immunohistochemistry (IHC), and their serum concentration was measured by ELISA. RESULTS: The increased bone resorption observed in patients with UC was reflected by low bone density and high serum level of C-terminal telopeptide (CTX). Mucosal RANKL expression and serum concentration were similar in UC group and healthy subjects, however, UC patients had higher local expression of OPG and serum OPG concentration. Increased IL-33 gene expression was observed only in UC at the mRNA level. We propose that bone resorption in UC patients despite OPG up-regulation could be caused by IL-33-induced mucosal synthesis of a potent proinflammatory cytokine, such as TNF-α, known as a possible inducer of osteoclastogenesis in the way independent of RANKL.

Pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 human leukemia cells.

Pterostilbene, a naturally occurring structural analog of resveratrol, has been reported to exert antiproliferative and proapoptotic effects in various cancer types. Recently, it has been demonstrated to induce both autophagy and apoptosis in human bladder and breast cancer cell lines. The aim of this study was to evaluate the effects of pterostilbene on HL60 human leukemia cells. Cell morphology was examined using confocal and electron microscopy. Cell viability was determined by MTT, neutral red uptake and trypan blue exclusion assays. LC3 processing was studied based on Western blotting and immunofluorescence analyses. Flow cytometry was used to study cell cycle distribution, phosphatidylserine externalization, caspase activation, disruption of mitochondrial membrane potential and intracellular production of reactive oxygen species. DNA degradation was examined by gel electrophoresis. We found that treatment of HL60 cells with pterostilbene at the IC90 concentration resulted in the G0/G1 cell cycle arrest. Pterostilbene induced conversion of cytosolic LC3-I to membrane-bound LC3-II and accumulation of large LC3-positive vacuolar structures. Pterostilbene also led to phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase activation and disruption of mitochondrial membrane potential. Moreover, it did not induce oxidative stress. Our results suggest that pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 cells.

The effect of lateral electrical surface stimulation (LESS) on motor end-plates in an animal model of experimental scoliosis.

The treatment of idiopathic scoliosis is challenging because of its diverse etiology, age of onset, and long duration of intensive treatment. We examined the effect of lateral electrical surface stimulation (LESS) in an animal model of experimental scoliosis (ES) assessing the number of motor end-plates (MEPs) as a study end-point. The control group (n=5) was adapted to the experimental apparatus without stimulation, whereas ES was induced in rabbits by one-sided LESS of the longissimus dorsi muscle (LDM) for a duration of 2 months. The ES group (n=5) were subjected to a short-term corrective electrostimulation applied at the contralateral side of the spine compared to the previous LESS stimulation for 2 h daily for 3 (n=5) or 6 months (n=5). Another group of ES rabbits was subjected to a long-term corrective electrostimulation applied for 9 h daily for 3 (n=5) or 6 months (n=5). LESS applied for 2 months (ES), significantly increased the number of MEPs in LDM. The short-term corrective electrostimulation for 3 months resulted in an increased number of MEPs. However, a decrease was observed in the animals treated for 6 months. The long-term corrective electrostimulation for 3 months did not change the density of MEPs in the LDM, but for 6 months the number of MEPs in the LMD significantly decreased by ES and control groups. Thus, the results of the present study clearly show that the short-term LESS is able to influence both the number of MEPs and the effectiveness of muscle correctional adaptation in a more efficient and harmless manner than the long-term procedure.

Structural and biochemical characteristics of various white adipose tissue depots.

It is now widely accepted that white adipose tissue (WAT) is not merely a fuel storage organ, but also a key component of metabolic homoeostatic mechanisms. Apart from its major role in lipid and glucose metabolism, adipose tissue is also involved in a wide array of other biological processes. The hormones and adipokines, as well as other biologically active agents released from fat cells, affect many physiological and pathological processes. WAT is neither uniform nor inflexible because it undergoes constant remodelling, adapting the size and number of adipocytes to changes in nutrients' availability and hormonal milieu. Fat depots from different areas of the body display distinct structural and functional properties and have disparate roles in pathology. The two major types of WAT are visceral fat, localized within the abdominal cavity and mediastinum, and subcutaneous fat in the hypodermis. Visceral obesity correlates with increased risk of insulin resistance and cardiovascular diseases, while increase of subcutaneous fat is associated with favourable plasma lipid profiles. Visceral adipocytes show higher lipogenic and lipolytic activities and produce more pro-inflammatory cytokines, while subcutaneous adipocytes are the main source of leptin and adiponectin. Moreover, adipose tissue associated with skeletal muscles (intramyocellular and intermuscular fat) and with the epicardium is believed to provide fuels for skeletal and cardiac muscle contraction. However, increased mass of either epicardial or intermuscular adipose tissue correlates with cardiovascular risk, while the presence of the intramyocellular fat is a risk factor for the development of insulin resistance. This review summarizes results of mainly human studies related to the differential characteristics of various WAT depots.

Sensitivity of natural killer cells to activation in the process of ageing is related to the oxidative and inflammatory status of the elderly.

The role of the innate immunity during human ageing is not well understood. The aim of the study was to estimate reactivity of NK (natural killer) cells in the very old (mean age 91 years) and old subjects (mean age 78 years) compared to young individuals (mean age 26 years) in respect to the indices of the oxidative stress (telomere length of NK cells, serum content of -H groups), serum total antioxidant status and serum concentrations of interleukin 6 and tumor necrosis factor-α (TNF-α). The activation state of NK cells, reflected by telomerase activity and intracellular interferon γ (IFNγ) content, was also measured. We found that length of telomeres in NK cells and serum concentration of -SH groups decreased both in the old and the oldest subjects as compared to young individuals. The oldest seniors, on the contrary to the old ones, revealed similar level of serum antioxidant status as the young subjects. The serum level of IL-6, not detectable in the young subjects, did not differ in the oldest and old seniors. TNF-α serum concentrations progressively increased with age. After stimulation, NK cells of both old groups showed higher intracellular levels of IFNγ than young subjects. IL-2-activated NK cells of the oldest seniors showed the highest increase of telomerase activity as compared to the other age groups. Serum level of IL-6 correlated positively with activation markers of NK cells. Moreover, in seniors but not in young subjects, the number of active, IFNγ-expressing NK cells, correlated positively with the serum content of the -SH groups. These findings indicate that sensitivity of NK cells to activation is maintained during ageing and this phenomenon may be related to the oxidative and inflammatory status of the elderly.

Overexpression of the fragile histidine triad (FHIT) gene in inflammatory bowel disease.

OBJECTIVE: FHIT gene encodes human diadenosine triphosphate hydrolase involved in the regulation of cell cycle and nucleotide metabolism and is a candidate tumor suppressor gene. AIM: To investigate expression of FHIT gene at the mRNA and protein levels in sporadic inflammatory bowel disease (IBD). MATERIALS AND METHODS: FHIT mRNA was quantified by the validated real-time PCR (QPCR) and FHIT protein was detected by immunohistochemistry (IHC) in mucosal biopsies of 139 ulcerative colitis (UC), 19 Crohn's disease (CD) and 37 control patients. RESULTS: Significant FHIT gene overexpression was found in 78% of active UC but not in CD. IHC showed comparable results to QPCR. CONCLUSION: The local up-regulation of FHIT gene and protein expression in active UC may represent an adequate response against inflammatory challenge of epithelial cell homeostasis and protect against DNA damage and cell cycle disturbances.

Fragile histidine triad (FHIT) gene is overexpressed in colorectal cancer.

OBJECTIVE: FHIT gene, mapped at FRA3B site, encodes human diadenosine triphosphate hydrolase involved in the regulation of cell cycle and nucleotide metabolism. Decreased FHIT gene expression was previously observed in various types of human cancer, however, quantification of FHIT mRNA was seldom performed. AIM: To investigate loss of heterozygosity (LOH) at FRA3B, expression of FHIT gene at the mRNA and protein levels in sporadic colorectal carcinoma (CRC) and benign colon adenoma. MATERIALS AND METHODS: FHIT mRNA was quantified by the validated realtime PCR (QPCR) in tumor samples of 84 CRC patients and mucosal biopsies of 15 adenomas, in comparison to 37 control patients, whereas subgroup of 57 CRC, 10 adenoma and 10 control cases were selected for immunohistochemical (IHC) detection of the native FHIT protein and LOH determination at FRA3B. RESULTS: Higher level of FHIT mRNA was found in 86% of CRC (P<0.001) and 60% of adenomas (P=0.016). IHC showed comparable results to QPCR (P=0.003), revealing the strongest presence of FHIT protein in Dukes' C/D stages (P<0.001) and N1/N2 lymph nodes metastasis in CRC (P=0.04). FHIT gene expression and Dukes' and G staging were positively correlated in CRC as analyzed by QPCR and IHC. Deletion analysis of the fragile FRA3B site revealed the highest LOH frequency at D3S1234 in 32.5% of CRC informative cases, however, LOH did not correspond to QPCR, IHC or clinical-pathological variables. CONCLUSION: Our data suggest that reduction or absence of the FHIT gene expression is not a prerequisite for colorectal cancer development and progression.

Distribution and neurochemical characterization of sensory dorsal root ganglia neurons supplying porcine urinary bladder.

UNLABELLED: Pig has been used recently as an animal model for studying diseases of human urinary tract, however, the sensory innervations of urinary bladder in this species has not been yet described. OBJECTIVE: The present study was aimed at neurochemical characterization of sensory neurons of dorsal root ganglia (DRGs) supplying porcine urinary bladder. METHODS: Retrograde tracer Fast Blue (FB) was injected into the right half of the urinary bladder wall of six juvenile female pigs. Three weeks later ipsi- and contralateral DRGs of interest were harvested from all animals and a neurochemical characterization of retrogradely-labeled neurons was performed using routine single-immunofluorescence labeling technique on 10 microm-thick cryostat sections. RESULTS: 85% of spinal sensory neurons supplying porcine urinary bladder was located in ipsilateral sacral S3-S4 ganglia and in first coccygeal ganglion (Cq1),whereas rest of FB-positive (FB+) nerve cells were found in lumbar L3-L6 DRGs. FB+ neurons belonged mostly to the medium-sized (54%) and small-sized afferent perikarya (45%). Bladder sensory neurons contained substance P (SP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating peptide (PACAP), galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM) and/or calbindin-28k (CB), and these neurons constituted 45%, 36%, 26%, 6%, 6%, 4% and 3% of all retrogradely traced DRGs perikarya, respectively. Distinct differences in the number of traced cells and their neuropeptide content were observed between the lumbar and sacral/coccygeal division of bladder-projecting DRG neurons. Thus, FB+ neurons expressing CGRP, GAL, nNOS or SOM were more numerous in lumbar DRGs (44%, 9%, 9% and 6%, respectively), than in sacral/coccygeal ganglia (23%, 2%, 1.5% and 0.3%, respectively). On the other hand, more FB+ cells expressed PACAP in sacral (31%) than in lumbar DRGs (23%). However, fractions of SP-IR or CB-IR bladder sensory neurons were similar in lumbar and sacral/coccygeal DRGs. CONCLUSIONS: This novel description of both spatial and neurochemical organization pattern of porcine urinary bladder sensory innervation constitutes a basis for further functional studies aimed at unraveling neurogenic mechanisms of urinary bladder diseases.

Decreased Toll-like receptor-5 (TLR-5) expression in the mucosa of ulcerative colitis patients.

OBJECTIVE: Although there is a convincing evidence supporting an important role for microorganisms in the pathogenesis of Inflammatory Bowel Disease (IBD) which comprises ulcerative colitis (UC) and Crohn's disease (CD), the specific mechanisms involved remain unclear. Toll-like receptors (TLR) recognize various molecules of microbiota including flagellin, the principal protein of motile comensal and pathogenic bacteria implicated in the pathogenesis of IBD. AIM: To investigate the expression of the TLR-5 receptors at the mRNA and protein levels in the mucosa of UC patients. MATERIALS AND METHODS: TLR-5 mRNA was quantified by the validated real-time PCR (QPCR) in mucosal biopsies of 99 UC patients and 34 control patients and TLR-5 protein was detected by immunohistochemistry (IHC) in 57 UC and 10 control patients. RESULTS: Significantly decreased TLR-5 gene expression at mRNA and protein level was found in the mucosa of patients with moderate and severe disease activity as compared to patients with low UC activity and control. TLR-5 immunoreactivity was found in the mucosa of UC patients and normal controls in the cytoplasm of enterocytes and at their basolateral domain. However, the intensity of the IHC reaction in specimens from UC patients was substantially lower than in control samples. CONCLUSION: The decreased expression of TLR-5 gene and protein in the mucosa of UC patients suggests that down-regulation of TLR-5 is probably caused by the increased number of ligand molecules in the proximity of epithelial cells in the inflamed tissue.

Central regulation of food intake in ageing.

Energy homeostasis and fuel metabolism undergo significant modifications in the course of ageing. During the second half of life many humans increase their body mass and develop glucose intolerance that may lead to obesity and type 2 diabetes. However, many old people suffer from being underweight, and this "anorexia of elderly" may seriously compromise their health under certain circumstances. Experimental studies into the causes of ageing-related impairments of food intake regulation were performed mainly on rat, and to some extent, on non-human primates. It was found that the expression of NPY, the most potent orexigenic peptide, and of NPY receptors, is highly suppressed in the hypothalamus of old rats. Moreover, the increase of NPY mRNA after fasting was severely blunted in old as compared to young rats. Similar reductions, although of lower magnitude, were reported for other hypothalamic orexigenic compounds such as, AgRP and orexins. Interestingly, ageing does not significantly alter hypothalamic mRNA levels of important anorexigens such as CART and aMSH. The presented findings suggest that, at least in rodents, ageing is associated with the general down-regulation of hypothalamic peptides that stimulate food intake and unchanged expression of anorexigenic peptides. This situation may be responsible for the decreased appetite drive in senescent animals and loss of weight at the end-of-life period. If similar changes of the central control of food intake underly "anorexia of ageing" observed in some elderly, it is possible that therapeutic intervention at this regulatory level may be possible in the future.