Infliximab therapy inhibits inflammation-induced angiogenesis in the mucosa of patients with Crohn's disease.

OBJECTIVES: Inflammation-driven angiogenesis contributes to the pathogenesis of inflammatory bowel disease (IBD). In line with this, the efficacy of inhibitors of angiogenesis has been demonstrated in experimental models of colitis. Currently, the ability of infliximab, an anti-tumor necrosis factor-α (TNF-α) agent that is highly beneficial in patients with IBD, to affect mucosal angiogenesis in patients with Crohn's disease (CD) and ulcerative colitis (UC) is unknown. METHODS: Patients with active CD (n=14) were treated with infliximab for 1 year, and peripheral blood and intestinal mucosa samples were collected before and after treatment. Mucosal angiogenesis was evaluated by CD31 and Ki-67 staining in endoscopic biopsies at baseline (week 0) and at week 54. The release of vascular endothelial growth factor-A (VEGF-A) by cultured mucosal extracts was measured by enzyme-linked immunosorbent assay (ELISA), before and after administration of infliximab, as well as in cultures of human intestinal fibroblasts (HIFs) stimulated with TNF-α in the presence or absence of infliximab. Migration of human intestinal microvascular endothelial cells (HIMECs) was investigated by migration assays. RESULTS: Microvessel density was significantly higher in the mucosa from patients with CD compared with tissue from healthy control individuals. Of the 14 patients, 8 (57%) showed a clinical remission in response to infliximab, which was associated with a significant reduction of microvascular density. Morphometric vessel analysis further confirmed the significant reduction of the area of vascular section after administration of infliximab. Furthermore, the expression levels of the proliferation marker Ki-67 in endothelial cells were significantly reduced after treatment. The mucosal concentration of VEGF-A was also significantly decreased, whereas in vitro exposure of HIF to infliximab virtually abolished TNF-α-induced VEGF-A production. These phenomena did not occur in patients who showed no clinical response to infliximab. CONCLUSIONS: Administration of infliximab downregulates mucosal angiogenesis in patients with CD and restrains production of VEGF-A by mucosal fibroblasts. It is proposed that this ameliorates inflammation-driven angiogenesis in the gut mucosa and contributes to the therapeutic efficacy of blockade of TNF-α.

Diminished expression of S100A2, a putative tumour suppressor, is an independent predictive factor of neck node relapse in laryngeal squamous cell carcinoma.

PURPOSE: In primary squamous cell carcinoma of the larynx (LSCC), Ca(2+) binding S100A2 protein underexpression was already found to be associated with poor tumour differentiation and shorter overall survival. In the present work, the role of S100A2 protein expression in the prediction of regional metastasis-free survival (MFS) was investigated to guide neck management in LSCC. EXPERIMENTAL DESIGN: Specimens of LSCC from 62 consecutive untreated patients were examined for S100A2 content by immunocytochemistry; the patients were followed up for a median of 44 months (range 2-90 months) after initial surgical resection. MFS was calculated from the date of first surgery to that of regional neck node recurrence. RESULTS: S100A2 was detected in 18 of 19 (95%) low-grade tumours and in 22 of 43 (51%) high-grade tumours. The 5-year regional MFS was 81% for patients with S100A2-positive tumours and 55% for patients with S100A2-negative tumours. By multivariate analysis, the S100A2 status appeared to be a significant independent predictive factor for MFS (p = .02). CONCLUSIONS: Our results suggest that the assessment of S100A2 status at diagnosis may identify a subset of LSCC patients highly susceptible to neck node metastases and may thus help define therapy accordingly.

DNA damage and apoptosis induction by the pesticide Mancozeb in rat cells: involvement of the oxidative mechanism.

The DNA damaging and proapoptotic effects of Mancozeb, a widely used fungicide of the ethylene-bis-dithiocarbamate (EBDC) group, were studied in RAT-1 fibroblasts cultured in vitro and in peripheral blood mononucleated cells (PBMC) isolated from Wistar rats. After 1 h exposition to Mancozeb (up to 500 ng/ml), cells produced a dose-dependent induction in DNA single strand break (SSB) formation, measured by single cell gel electrophoresis (SCGE). Concomitantly, a concentration-dependent increase in the levels of the oxidative markers of DNA oxidation, the DNA adduct 8-hydroxy-2'-deoxyguanosine (8-OHdG) and of reactive oxygen species (ROS) were observed, suggesting a prooxidant action of Mancozeb. PBMC were less responsive than fibroblasts to the oxidative insult carried out by Mancozeb, as shown by the lower increase in the levels of ROS, 8-OHdG adducts and SSB measured in these cells after exposure to the pesticide. A 4-h treatment with Mancozeb induced also apoptosis in both PBMC and RAT-1 cells, even though leukocytes were less sensitive than fibroblasts to the proapoptotic action. This effect was dose-dependent and was inhibited by the action of the antioxidant alpha-tocopherol. The proapoptotic effect was accompanied by the altered expression of several proteins involved in the regulation of apoptosis, such as the prosurvival protein BCL-2 and the proapoptotic protein c-MYC. Exposition of cells to higher concentrations of Mancozeb or for longer periods (>4 h) caused post-apoptotic, necrotic alterations in cell membrane integrity. The data herein presented demonstrate the oxidative effect of Mancozeb and suggest that its prooxidant action may be involved in the proapoptotic effect exerted by this compound in rat cells. It appears possible that the observed oxidative and genotoxic damage may be involved in the pathogenesis of various pathologies associated with the chronic exposition to Mancozeb, including cancer. On the other hand, the proapoptotic effect of Mancozeb suggests its possible relevance in the pathogenesis of neurodegenerative diseases, often related to the exposition of pesticides.

Telomerase inhibition by stable RNA interference impairs tumor growth and angiogenesis in glioblastoma xenografts.

Telomerase is highly expressed in advanced stages of most cancers where it allows the clonal expansion of transformed cells by counteracting telomere erosion. Telomerase may also contribute to tumor progression through still undefined cell growth-promoting functions. Here, we inhibited telomerase activity in 2 human glioblastoma (GBM) cell lines, TB10 and U87MG, by targeting the catalytic subunit, hTERT, via stable RNA interference (RNAi). Although the reduction in telomerase activity had no effect on GBM cell growth in vitro, the development of tumors in subcutaneously and intracranially grafted nude mice was significantly inhibited by antitelomerase RNAi. The in vivo effect was observed within a relatively small number of population doublings, suggesting that telomerase inhibition may hinder cancer cell growth in vivo prior to a substantial shortening of telomere length. Tumor xenografts that arose from telomerase-inhibited GBM cells also showed a less-malignant phenotype due both to the absence of massive necrosis and to reduced angiogenesis.

IgA nephropathy in an Italian child with familial Mediterranean fever.

Familial Mediterranean fever is an autosomal recessive disorder characterized by transient attacks of fever and polyserositis with substantial risk of developing amyloidotic nephropathy over time. We report an Italian child with familial Mediterranean fever presenting with hematuria during attacks in whom kidney biopsy documented the presence of mesangial IgA deposits and the absence of amyloidosis. Kidney biopsy should be performed in patients showing microscopic or gross hematuria during attacks of familial Mediterranean fever in order to gain additional epidemiological data about specific features of renal involvement and to allow adequate treatment.

Impaired primary hemostasis with normal platelet function in Duchenne muscular dystrophy during highly-invasive spinal surgery.

A defective, normal or enhanced hemostasis has been reported in Duchenne muscular dystrophy (DMD). A retrospective analysis of intra-and postoperative (up to 36 h) estimated blood losses was performed in 156 patients undergoing spinal surgery for: DMD (n = 31), idiopathic scoliosis (IS) (n = 70), poliomyelitis (n = 10), cerebral palsy (CP) (n = 28), spinal muscular atrophy (SMA) (n = 17). Platelet aggregation and bleeding times were also investigated in DMD patients. Immunohistochemistry for dystrophin was performed in platelets, megakaryocytes and blood vessels of normal tissues. DMD patients showed significantly higher intraoperative estimated blood losses (DMD: 3495+/-890 ml; IS: 2269+/-804 ml; poliomyelitis: 2582+/-1252 ml; CP: 2071+/-683 ml; SMA: 2464+/-806 ml; P < 0.05), while postoperative blood losses were similar among different groups. Higher estimated blood losses in DMD were independent of the duration of surgery, body weight, gender, age, vertebral levels or preoperative Cobb angle. DMD children had significantly prolonged bleeding times, but retained normal platelet function. From control samples dystrophin was expressed in vascular smooth muscle cells, but not in platelets. DMD appears to be characterized by immediate bleeding during highly-invasive surgery and increased bleeding time without platelet abnormalities. Considering dystrophin expression in normal vascular smooth muscle cells, these results altogether suggest a selective defect of primary hemostasis in DMD, likely to be due to impaired vessel reactivity.

Repeated exposure to pyrrolidine-dithiocarbamate induces peripheral nerve alterations in rats.

Pyrrolidine-dithiocarbamate (PDTC), a synthetic compound widely used in cell biological investigations, recently attracted considerable interest as a putative anticancer agent. However, different dithiocarbamates have previously shown to cause neurological symptoms and morphological alterations in peripheral nerves. The purpose of the present study was to determine whether a 15-day oral administration with low doses of PDTC may produce adverse effects in peripheral nerves of rats. Female Wistar rats were assigned to receive PDTC [0.1, 0.5 or 1.0mmol/(kg body weight/day)] by gavage for 15 days. Reduced conduction velocity was observed by electrophysiological analysis in tibial nerves of treated animals, accompanied by a marked decrease in Shwann cell S100-protein expression determined by immunohistochemistry. Electron microscopy evaluation revealed marked myelin degeneration in the fibers of treated animals. In particular, both morphological and electrophysiological data suggested an impairment of large, fast conducting fibers, whereas the smallest and slowest ones remained intact. However, the activity of plasma and liver alkaline-phosphatase, an enzymic marker of hepatic dithiocarbamate toxicity, was not altered by the treatment. The total contents of the redox-active metal copper increased in tibial nerves of treated rats and was accompanied by raised levels of lipid peroxidation products. This finding suggests a role for oxidative stress in the development of PDTC-induced pathological and functional alterations of tibial nerves. The observation that a 15-day treatment with low doses of PDTC causes functional and morphological derangement of peripheral nerves advices against the possible use of this compound as a chemopreventive agent against cancer.

Parathyroid hormone-related peptide (hPTHrP) and parathyroid hormone-related peptide receptor type 1 (PTHR1) expression in human thymus.

Parathyroid hormone-related peptide (hPTHrP) is expressed in human tissues and regulates cellular proliferation, differentiation, and apoptosis by an autocrine/paracrine loop. In rodent thymus, both parathormone and parathyroid hormone-related peptide (PTHrP) are expressed by thymic epithelial cells (TECs). The present study demonstrated by RT-PCR and immunohistochemistry that hPTHrP and parathyroid hormone-related peptide receptor type 1 (PTHR1) were expressed in human thymus at both RNA and protein levels. hPTHrP was expressed mainly in the thymic medulla by epithelial (cytokeratin-positive), mature dendritic (CD40+/86+) and plasmacytoid interleukin (IL)-3Ralpha1 cells. This protein was also present in some cells forming Hassall's bodies and a few subcapsular and cortical TECs. PTHR1 was expressed by scattered subcapsular and cortical TECs and by rare TECs in the medulla. Thymocytes did not express either hPTHrP or PTHR1. Primary cultures of human TECs revealed the presence of both hPTHrP and PTHR1 mRNAs, confirming the capacity of TECs to synthesize both peptides. Moreover, synthetic (1-39) hPTHrP peptide administered on cultured TECs induced the expression of IL-6 mRNA, suggesting that hPTHrP can regulate thymic functions by inducing in TECs the expression of IL-6, which is involved in the development and maturation of thymocytes.

Dilated intercellular spaces of esophageal epithelium in nonerosive reflux disease patients with physiological esophageal acid exposure.

OBJECTIVES: It has been demonstrated that dilation of intercellular spaces of esophageal epithelium is a marker of tissue injury in GERD patients with a pathological esophageal acid exposure time. To evaluate the relationship among ultrastructural changes, acid esophageal exposure, and GERD symptoms, intercellular space diameters have been assessed in nonerosive reflux disease (NERD) patients with/without abnormal acid exposure time. METHODS: Following a pharmacological wash-out, 20 NERD patients underwent upper endoscopy, esophageal manometry, and 24-h pH monitoring. Biopsies were taken at 5 cm above the lower esophageal sphincter and intercellular space diameters were measured on transmission electron microscopy photomicrographs. Seven asymptomatic controls underwent the same protocol. RESULTS: Acid exposure time was in the normal range in all controls and in 11 patients (NERD pH-negative); it was abnormal in 9 patients (NERD pH-positive). Mean intercellular space diameter in NERD pH-negative and in NERD pH-positive patients was three times greater than in controls (1.45 and 1.49 microm vs 0.45, p < 0.001). Mean values of maximum intercellular spaces in all NERD patients were greater, two-fold or more, than those in controls (p < 0.001). No difference in mean and maximal space diameters was observed between NERD pH-positive and pH-negative patients. CONCLUSIONS: Dilation of intercellular spaces is a feature of NERD patients, irrespective of esophageal acid exposure, and can be considered an objective, structural marker of GERD symptoms. Impaired esophageal mucosal resistance, even to small amounts of acid refluxate, plays a key role in the pathophysiology of NERD.

RGDS peptide inhibits activation of lymphocytes and adhesion of activated lymphocytes to human umbilical vein endothelial cells in vitro.

The Arg-Gly-Asp (RGD) motif is known to mediate cell adhesion to several extracellular matrix components as well as cell-cell interactions. In the present study, we investigated whether the RGDS peptide interferes with cell-cell recognition-based events such as allogeneic activation of PBMC and PBMC adhesion to human umbilical vein endothelial cells (HUVEC). We show here for the first time, to our knowledge, that RGDS significantly inhibits adhesion of activated PBMC to HUVEC; in addition, RGDS inhibits PBMC allogenenic activation in human mixed lymphocyte reaction assays. Caspases played a pivotal role in both events, because preventing their activation abolished or strongly reduced the observed inhibitory effect. The RGDS antirecognition effect was strongly increased by pretreatment of HUVEC with RGDS, which affected mostly T lymphocyte adhesion to HUVEC. These results indicate that PBMC allogeneic activation, as well as reciprocal recognition between activated PBMC and endothelial cells, are RGDS-dependent events that occur through a dual effect involving anti-adhesive and caspase-dependent mechanisms. These data suggest a potential role of RGDS in cell-mediated immunity, inflammation and organ transplantation.

Docosahexaenoic acid enhances the susceptibility of human colorectal cancer cells to 5-fluorouracil.

PURPOSE: Powerful growth-inhibitory action has been shown for n-3 polyunsaturated fatty acids against colon cancer cells. We have previously described their ability to inhibit proliferation of colon epithelial cells in patients at high risk of colon cancer. In the work reported here we investigated the ability of docosahexaenoic acid (DHA) to potentiate the antineoplastic activity of 5-fluorouracil (5-FU) in p53-wildtype (LS-174 and Colo 320) and p53-mutant (HT-29 and Colo 205) human colon cancer cells. METHODS: When in combination with DHA, 5-FU was used at concentrations ranging from 0.1 to 1.0 microM, much lower than those currently found in plasma patients after infusion of this drug. Similarly, the DHA concentrations (< or =10 microM) used in combination with 5-FU were lower than those widely used in vitro and known to cause peroxidative effects in vivo. RESULTS: Whereas the cells showed different sensitivity to the growth-inhibitory action of 5-FU, DHA reduced cell growth independently of p53 cellular status. DHA synergized with 5-FU in reducing colon cancer cell growth. The potentiating effect of DHA was attributable to the enhancement of the proapoptotic effect of 5-FU. DHA markedly increased the inhibitory effect of 5-FU on the expression of the antiapoptotic proteins BCL-2 and BCL-XL, and induced overexpression of c-MYC which has recently been shown to drive apoptosis and, when overexpressed, to sensitize cancer cells to the action of proapoptotic agents, including 5-FU. CONCLUSION: Our results indicate that DHA strongly increases the antineoplastic effects of low concentrations of 5-FU. Overall, the results suggest that combinations of low doses of the two compounds could represent a chemotherapeutic approach with low toxicity.

beta-Carotene downregulates the steady-state and heregulin-alpha-induced COX-2 pathways in colon cancer cells.

Experimental studies have shown that beta-carotene inhibited the growth of colon cancer cells, and human trials have demonstrated that the carotenoid reduces colon cell proliferation of adenomatous polyps; however, molecular mechanisms underlying this chemopreventive activity remain unclear. Because COX-2 has been implicated as a causative factor in colon carcinogenesis, the present study was designed to investigate the relation between the growth-inhibitory effect of the carotenoid and COX-2 expression in colon cancer cells. We evaluated the effects of beta-carotene on the growth of human colon adenocarcinoma cells overexpressing (LS-174, HT-29, WiDr) or not expressing (HCT116) COX-2. We also studied COX-2 expression induced by heregulin-alpha, apoptosis induction, reactive oxygen species (ROS) production, and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. beta-Carotene (0.5-2.0 micromol/L) decreased COX-2 expression (P < 0.05) and prostaglandin E(2) (PGE(2)) production (P < 0.05) in colon cancer cells. This effect was not observed in cells treated with retinoic acid or retinol. The downregulation of COX-2 by the carotenoid occurred in both untreated and heregulin-treated cells. It was accompanied by an increased ability of cells to undergo apoptosis and by a decrease in intracellular ROS production and in the activation of ERK1/2. Moreover, cells not expressing COX-2 were insensitive to the growth-inhibitory and proapoptotic effects of the carotenoid. Here, we report that the suppression of COX-2 by beta-carotene may represent a molecular mechanism by which this compound acts as an antitumor agent in colon carcinogenesis.

Abrogation of hepatocyte apoptosis and early appearance of liver dysplasia in ethanol-fed p53-deficient mice.

Ethanol consumption represents a major risk factor for cancer development, and a significant fraction of hepatocarcinomas arises in alcoholic liver cirrhosis. Increasing evidence indicates that ethanol acts as a tumor promoter on genetically initiated cells, by increasing the intracellular concentration of reactive oxygen species and promoting tissue necrosis/regeneration and cell proliferation. The tumor suppressor p53 restrains the expansion of carcinogen-initiated cells by inducing cell cycle arrest and apoptosis; accordingly, p53-deficient mice develop spontaneous and chemically induced neoplasms at a much higher frequency than normal mice. In normal mice exposed to a subacute (3 weeks) ethanol intoxication, a significant increase in the number of apoptotic hepatocytes was observed in concomitance with the up-regulation of the mitochondrial superoxide scavenger MnSOD, a reliable indicator of oxidative stress. Cell death occurred in the absence of liver inflammation and necrosis. Ethanol-induced hepatocyte apoptosis was completely abrogated in the p53 null background, suggesting that the tumor suppressor is necessary for hepatocyte death by ethanol. Accordingly, p53 -/- MEF were, unlike wild type cells, completely insensitive up to 0.5M ethanol in the culture medium. Strikingly, marked and widespread signs of dysplasia, with nuclear pleomorphisms and initial loss of normal architecture, heralding malignant transformation, were scored in all the mutant mice exposed to ethanol, but not in the control-fed littermates nor in ethanol-fed normal mice. These observations suggest that p53-dependent apoptosis restrains the tumorigenic effect of ethanol on liver cells, in agreement with the frequent loss of p53 function in HCC, and reveal an unexpected carcinogenic potential of alcohol which appears to be independent from the induction of cirrhosis and hepatocyte regeneration.

Modulation of the expression and activity of cyclooxygenases in normal and accelerated erythropoiesis.

OBJECTIVE: The present study was aimed at characterizing the expression and activity of cyclooxygenase (COX) isoenzymes in erythropoiesis. METHODS: The expression and activity of cyclooxygenase (COX) and prostaglandin (PG) synthases were investigated in: 1) erythroblasts developed in culture from human CD34(+) hematopoietic progenitors, 2) erythroblasts in bone marrow specimens, and 3) peripheral erythrocytes isolated from healthy donors and from patients with a high regeneration rate of erythrocytes. RESULTS: While COX-1 protein was observed at each stage of erythroblast development, COX-2 protein was induced at later stages through a p38/MAPK-dependent pathway. Both COX isoforms were also observed in mature erythroblasts of the bone marrow. Erythroblasts developed in culture synthesized significantly more PGE(2) than TXB(2) and indomethacin delayed erythroid maturation. COX-1 and COX-2 were also observed in erythrocytes by immunostainings, although COX expression was confined to a fraction of circulating erythrocytes. Peripheral erythrocytes synthesized low but detectable amounts of PGE(2) and TXB(2). Similarly to erythroblast progenitors, PGE(2) was the prevalent prostanoid released by erythrocytes. This biosynthetic capacity was significantly increased in erythrocytes from patients with accelerated erythropoiesis as compared to controls. CONCLUSIONS: Both COX isoforms are present and enzymatically active during human erythropoiesis, although with different kinetics, and COX-derived prostanoids may play a role in erythroid maturation. Furthermore, peripheral erythrocytes retain in part the capacity of expressing COX and synthesizing prostanoids, which may contribute to the hemostatic/thrombotic response to vascular injury in different diseases, including congenital hemolytic disorders.

n-3 PUFAs reduce VEGF expression in human colon cancer cells modulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-1alpha induction pathway.

n-3 Polyunsaturated fatty acids (PUFAs) inhibit the development of microvessels in mammary tumors growing in mice. Human colorectal tumors produce vascular endothelial growth factor (VEGF) whose expression is up-regulated in tumor cells by both cyclooxygenase-2 (COX-2) and PGE(2) and directly correlated to neoangiogenesis and clinical outcome. The goal of this study was to examine the capability of n-3 PUFAs to regulate VEGF expression in HT-29 human colorectal cells in vitro and in vivo. Constitutive VEGF expression was augmented in cultured HT-29 cells by serum starvation and the effects of eicosapentaenoic (EPA) or docosahexaenoic acid (DHA) on VEGF, COX-2, phosphorylated extracellular signal-regulated kinase (ERK)-1 and -2 and hypoxia-inducible-factor 1-alpha (HIF-1alpha) expression and PGE(2) levels were assessed. Tumor growth, VEGF, COX and PGE(2) analysis were carried out in tumors derived from HT-29 cells transplanted in nude mice fed with either EPA or DHA. Both EPA and DHA reduced VEGF and COX-2 expression and PGE(2) levels in HT-29 cells cultured in vitro. Moreover, they inhibited ERK-1 and -2 phosphorylation and HIF-1alpha protein over-expression, critical steps in the PGE(2)-induced signaling pathway leading to the augmented expression of VEGF in colon cancer cells. EPA always showed higher efficacy than DHA in vitro. Both fatty acids decreased the growth of the tumors obtained by inoculating HT-29 cells in nude mice, microvessel formation and the levels of VEGF, COX-2 and PGE(2) in tumors. The data provide evidence that these n-3 PUFAs are able to inhibit VEGF expression in colon cancer cells and suggest that one possible mechanism involved may be the negative regulation of the COX-2/PGE(2) pathway. Their potential clinical application as anti-angiogenic compounds in colon cancer therapy is proposed.

Role of p16/INK4a in gastrointestinal stromal tumor progression.

Because the p16 locus is involved consistently in chromosomal losses found in malignant gastrointestinal stromal tumors (GISTs), we studied p16 in a series of 21 GISTs with complete follow-up using immunohistochemical analysis, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and methylation-specific PCR (MSP). A fraction of cells of more than 20% with low or absent p16 immunostaining was detected in 12 GISTs, including all showing malignancy. RT-PCR revealed decreased p16 transcription in all except 2 p16 protein-deficient GISTs. By MSP, 7 cases showed p16 promoter methylation (all hypoexpressing p16; 6 malignant). A fraction of p16-deficient cells of more than 20% was associated with clinical malignancy (P = .003; log-rank test). The percentage of cells underexpressing p16, size, cellularity, mitotic count, and coagulative necrosis were associated with malignancy by Cox proportional hazards univariate analysis; only the former factor was selected by multivariate analysis (P = .039). Thus, p16 down-regulation, partly due to p16 promoter methylation, is implied in GIST progression. Furthermore, p16 immunohistochemical assessment seems a promising method for GIST prognostication.

Lactobacillus acidophilus protects tight junctions from aspirin damage in HT-29 cells.

BACKGROUND/AIMS: Non-steroidal anti-inflammatory drugs cause enterocyte damage inducing an increase of intestinal permeability. Tight junctions are the key structures in the permeability of the intestinal mucosa. ZO-1 is a tight junction associated protein considered a good marker of their integrity. It has been suggested that probiotics could play a protective role in the intestinal barrier function. We determined, in vitro, whether the heat-killed Lactobacillus acidophilus strain LB (LaLB) with its spent culture supernatant protects tight junctions of HT-29 cells from aspirin (ASA) damage. METHODS: HT-29 cells were treated with ASA alone or ASA and LaLB with its spent culture supernatant together. Morphological alterations of tight junctions were evaluated by immunofluorescence using an anti-ZO-1 antibody. Moreover, a semiquantitative assay for ZO-1 was performed by Western blot. RESULTS: Immunofluorescence analysis showed a fragmented and granulous ZO-1 staining, after ASA treatment. Using both ASA and LaLB with its spent culture supernatant together, we found a fine continuous linear web at cell-cell contacts similarly to control. Western blot revealed that ASA inhibited ZO-1 expression and LaLB with its spent culture supernatant counteracted this effect. CONCLUSIONS: This pilot study shows, for the first time, the protective effect of LaLB with its spent culture supernatant on tight junctions from ASA damage. These results suggest that probiotics could play a role in the prevention of ASA-induced alterations of intestinal permeability.

beta-Carotene exacerbates DNA oxidative damage and modifies p53-related pathways of cell proliferation and apoptosis in cultured cells exposed to tobacco smoke condensate.

Human intervention trials have suggested that supplemental beta-carotene resulted in more cancer in smokers, whereas it was protective in non-smokers. However, the mechanisms underlying these effects are still unknown. The aim of this study was to evaluate the effects of an association of cigarette smoke condensate (tar) and beta-carotene on DNA oxidative damage and molecular pathways involved in cell cycle progression and apoptosis in cultured cells. In RAT-1 fibroblasts, tar caused increased levels of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and this effect was enhanced by the concomitant presence of beta-carotene (0.5-4.0 microM) in a dose- and time-dependent manner. In contrast, beta-carotene alone did not significantly modify it. Fibroblasts treated with tar alone decreased their cell growth with respect to control cells through an arrest of cell cycle progression in the G0/G1 phase and an induction of apoptosis. These effects were accompanied by an increased expression of p53, p21 and Bax and by a decreased expression of cyclin D1. In contrast, fibroblasts treated with tar and beta-carotene, after an initial arrest of cell growth at 12 h, re-entered in cell cycle and were unable to undergo apoptosis at 36 h. Concomitantly, their p53 expression, after an increase at 12 h, progressively returned at basal levels at 36 h by a mechanism independent of Mdm2. Such a decrease was followed by a decrease in p21 and Bax expression and by an increase in cyclin D1 expression. Moreover, the presence of the carotenoid remarkably enhanced cyclooxygenase-2 expression induced by tar. During tar treatment, a depletion of beta-carotene was observed in fibroblasts. The effects of tar and beta-carotene on 8-OHdG levels, cell growth and apoptosis were also observed in Mv1Lu lung, MCF-7 mammary, Hep-2 larynx and LS-174 colon cancer cells. This study supports the evidence for potential detrimental effects of an association between beta-carotene and cigarette smoke condensate.

Role of PTEN in gastrointestinal stromal tumor progression.

CONTEXT: Gastrointestinal stromal tumors (GISTs) are Kit/CD117-expressing mesenchymal neoplasms of uncertain malignant potential. The lack of a reliable method of prognostication hampers the selection of patients eligible for STI571 therapy. 10q22-q23 is a region involved in chromosomal losses found in a fraction of malignant primary and metastatic GISTs harboring PTEN (phosphatase and tensin homologue deleted on chromosome 10), a tumor suppressor gene often altered in human neoplasms. OBJECTIVE: To investigate the role of PTEN in GISTs, an issue that to our knowledge has not been addressed previously. DESIGN: PTEN status was determined in a series of 21 GISTs, with follow-up ranging between 6 and 198 months, using immunohistochemistry correlated with clinical data. RESULTS: A greater than 25% fraction of cells with low or absent PTEN immunostaining was detected in 9 GISTs, including all those showing malignancy. By the log-rank test, a fraction of PTEN-deficient cells greater than 25% was associated with malignancy (P <.001). Percentage of cells underexpressing PTEN, size, cellularity, MIB-1 immunoreactivity, and coagulative necrosis proved to be associated with malignancy by Cox proportional hazards univariate analysis; low or absent expression of PTEN was the only factor selected by multivariate analysis (P =.03). CONCLUSIONS: PTEN downregulation is implied in GIST progression. The immunohistochemical assessment of PTEN status appears to be a promising method of GIST prognostication.