TIMP3 regulates migration, invasion and in vivo tumorigenicity of thyroid tumor cells.

Papillary thyroid carcinoma (PTC) arises from the thyroid follicular epithelium and represents the most frequent thyroid malignancy. PTC is associated with gene rearrangements generating RET/PTC and TRK oncogenes, and to the BRAFV600E activating point mutation. A role of tumor-suppressor genes in the pathogenesis of PTC has not been assessed yet. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene, encoding a metalloproteinases inhibitor and capable of inhibiting growth, angiogenesis, invasion and metastasis of several cancers, was found to be silenced by promoter methylation in a consistent fraction of PTCs, in association with tumor aggressiveness and BRAFV600E mutation, thus suggesting an oncosuppressor role. To explore this possibility, in this study we performed gene expression and functional studies. Analysis of gene expression data produced in our laboratory as well as meta-analysis of publicly available data sets confirmed the downregulation of TIMP3 gene expression in PTC with respect to normal thyroid. The functional consequences of TIMP3 downregulation were investigated in the PTC-derived NIM1 cell line, in which the expression of TIMP3 is silenced. Restoration of TIMP3 expression by exposure to soluble TIMP3 protein or by complementary DNA transfection had no effect on the growth rate of NIM1 cells. Instead, it affected the adhesive, migratory and invasive capabilities of NIM1 cells by modulating several proteins involved in these processes. A striking effect was observed in vivo, as TIMP3 reduced the tumorigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. Our data indicate that the loss of TIMP3 expression exerts a functional role in the pathogenesis of PTC.

IGFBP7: an oncosuppressor gene in thyroid carcinogenesis.

Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted protein involved in several cellular processes, including proliferation, senescence and apoptosis. Loss of IGFBP7 expression is a critical step in the development of human tumors, including melanoma and colon cancer. By microarray gene expression studies, we have detected downregulation of IGFBP7 gene expression in follicular and papillary thyroid tumors in comparison with normal thyroid tissue. Evaluation of publicly available PTC microarray gene expression data sets confirmed, in a consistent fraction of tumors, the downregulation of IGFBP7 transcript levels. The functional consequence of IGFBP7 downregulation was addressed in the PTC-derived NIM1 cell line in which IGFBP7 expression is repressed by promoter hypermethylation. Exposure to soluble IGFBP7 protein or restoration of IGFBP7 expression by complementary DNA transfection reduced growth rate, migration, anchorage-independent growth and tumorigenicity of NIM1 cells. We show that the effects of IGFBP7 are related to apoptosis. Our data suggest that loss of IGFBP7 expression has a functional role in thyroid carcinogenesis, and it may represent a possible basis for therapeutic strategies.

Detection of anti-endomysial and anti-tissue transglutaminase autoantibodies in media following culture of oral biopsies from patients with untreated coeliac disease.

BACKGROUND AND AIMS: Coeliac disease is an autoimmune disorder characterised by high levels of anti-endomysial and anti-tissue transglutaminase autoantibodies in sera and media of cultured intestinal mucosa biopsies from affected patients. In this study, we wished to investigate whether anti-endomysial and anti-tissue transglutaminase antibodies can also be detected in culture media of oral mucosa specimens, and whether the mouth can be used as an area of immunological testing for coeliac disease. METHODS: Small intestine and cheek biopsy samples taken from 16 patients with active coeliac disease and from 11 controls were cultured in vitro for 48 h at 37 degrees C in presence of medium alone. Anti-endomysial and anti-tissue transglutaminase were detected in sera and in supernatants of these cultured biopsy samples by indirect immunofluorescence and enzyme immunoassay (EIA), respectively. RESULTS: Anti-endomysial and anti-tissue transglutaminase were positive in sera of 15/16 coeliac disease patients. Culture media of intestinal mucosa samples from 14/16 coeliac disease patients were anti-endomysial positive, while the same antibodies were positive in supernatants of cultured oral mucosa samples from 15/16 coeliac disease patients. Anti-tissue transglutaminase were positive in both intestinal and oral culture media of 15/16 coeliac disease patients. Neither anti-endomysial nor anti-tissue transglutaminase were found in sera or in culture supernatants of both intestinal and oral biopsy samples from 11 controls. CONCLUSIONS: Our study suggests a new immunological site to detect the pathognomonic autoantibodies of coeliac disease and confirms that the mouth is involved in this illness.

Anti-endomysial antibody of IgG1 isotype detection strongly increases the prevalence of coeliac disease in patients affected by type I diabetes mellitus.

A strong association between type 1 insulin-dependent diabetes mellitus (IDDM1) and coeliac disease (CD) is well documented, but it is known that prevalence values are underestimated. Serum anti-endomysial antibodies (EMA), considered diagnostic for CD because of their high sensitivity and specificity, belong to the IgA class, but the existence of EMA of IgG1 isotype in the presence or absence of IgA deficiency was reported. In order to re-evaluate the occurrence of CD in IDDM1 patients we performed a screening in IDDM1 patients using EMA of both isotypes. Ninety-four adults affected by IDDM1 (unaffected by CD before enrolling) were enrolled and 83 blood donors as controls. All subjects were on a gluten-containing diet. Histology and biopsy culture were performed. EMA IgA and IgG1 in sera and culture supernatants were detected. Serum EMA were positive in 13 of 94 IDDM1 patients (13.8%). Six of 13 presented IgA-EMA, seven of 13 presented IgG1-EMA. No EMA were found in the control population. Total intestinal atrophy was found in all six patients with serum IgA-EMA and in five of seven with serum IgG1-EMA. Diagnosis of CD was confirmed by histology and organ culture in all 13 patients with serum EMA. The prevalence of CD in the patients affected by IDDM1 was 6.4% for IgA-EMA-positive and 7.4% for IgG1-EMA-positive patients. We confirmed the prevalence of CD in the IDDM1 population obtained with IgA-EMA screening only (6.4%). This prevalence value increases dramatically to 13.8% when IgG1-EMA are also used in the screening. We conclude that IgG1-EMA should also be sought whenever an IDDM1 patient undergoes screening for CD.

Immunologic evidence of no harmful effect of oats in celiac disease.

BACKGROUND: It was recently shown that antiendomysial antibodies (EMAs), which are highly sensitive and specific for celiac disease, are produced by intestinal mucosa. Furthermore, EMAs were detected previously in supernatant fluid from cultured duodenal mucosa specimens collected from untreated celiac disease patients and in culture media of biopsy specimens collected from treated celiac disease patients after an in vitro challenge with gliadin. Moreover, it was recently shown in vivo that oats are not toxic to celiac disease patients, suggesting the safety of oats in a gluten free-diet. OBJECTIVE: The objective was to better define the controversial role of oats in celiac disease to determine whether oats can be safely included in a gluten-free diet. DESIGN: We used an in vitro model to test whether oats induce EMA production in supernatant fluid from cultured duodenal mucosa specimens collected from 13 treated celiac disease patients. The biopsy specimens were cultured with and without peptic-tryptic digest (PT) of gliadin and avenin (from oats) and in medium alone. Samples from 5 of the 13 patients were cultured with the C fraction of PT-avenin. Indirect immunofluorescence was used to detect EMAs. RESULTS: EMAs were detected in specimens from all 13 patients after the challenge with gliadin but not after culture in medium alone. By contrast, no EMAs were detected in any of the specimens cultured with PT-avenin and its C fraction. CONCLUSIONS: Because the in vitro challenge with PT-avenin and its C fraction did not induce EMA production in treated celiac disease patients, it appears that oats have no harmful effect on celiac disease. Therefore, oats can be safely included in a gluten-free diet.

Identification of a new coeliac disease subgroup: antiendomysial and anti-transglutaminase antibodies of IgG class in the absence of selective IgA deficiency.

OBJECTIVE: The aim of the present study was to increase the sensitivity of the antiendomysial antibody (EMA) test by evaluating also EMAs of IgG1 isotype. DESIGN AND SUBJECTS: Over the last 2 years, serum EMAs IgA and IgG1 were determined in 1399 patients, referred to our gastrointestinal unit due to clinical suspicion of malabsorption. Serum anti-tissue transglutaminase (tTG) antibodies IgA and IgG, as well as total IgA levels, were also investigated. Furthermore, EMAs IgA and IgG1 were evaluated in biopsy culture supernatants. Biopsy specimens were also admitted to histological and immunohistochemical evaluation. Twenty-six patients with gastroenterological disease other than coeliac disease (CD) were used as a disease control group. Ninety-nine blood donors were used as a healthy control group. RESULTS: Diagnosis of CD was based on histological findings in the 110/1399 patients showing EMA IgA positivity, and in a further 56/1399 patients presenting both EMA IgA and IgG1 positivity in sera as well as in culture supernatants. Of the remaining 1233 EMA IgA-negative patients, 60 showed only EMA IgG1 positivity both in sera and in culture supernatants. It is noteworthy that anti-tissue transglutaminase antibodies IgG (anti-tTG) were positive in all 60 EMA IgG1-positive patients as well. By contrast, a selective IgA deficiency was found in only 11 out of the 60 EMA IgG1-positive patients. Villous height/crypt depth ratio was < 3:1 in 38 of the 60 EMA IgG1-positive patients (63.3%), whilst overexpression of ICAM-1 and CD25 was observed in all these patients. CONCLUSIONS: In this study, we observed a group of CD patients who were EMA IgG1-positive even in the absence of EMA IgA positivity and IgA deficiency. The diagnosis was based on the finding of the gluten-dependent clinical and histological features typical of CD. Data emerging from the present investigation thus suggest that the prevalence of CD should be reassessed and that the determination of EMA IgG1 could offer a new tool in the diagnostic armamentarium of CD.

Celiac disease diagnosis in misdiagnosed children.

Antiendomysial antibodies (EMA) are today considered the most sensitive and specific serological marker of celiac disease (CD). The aim of the present study was to assess the occurrence of EMA of IgG isotype in EMA IgA negative children with clinical suspicion of malabsorption and their relationship with CD. Serum EMA IgG1 determination was performed on 30 EMA IgA negative children with clinical suspicion of CD. Total serum IgA levels were further investigated. Sixty children with gastroenterological diseases other than CD were used as control disease patients and 63 healthy children were evaluated as the control group. Eighteen out of 30 children in the study showed EMA IgG1 positivity in sera and a villous height/crypt depth ratio <3:1 as index of intestinal atrophy. It is noticeable that a selective IgA deficiency was present in only 9 of 18 EMA IgG1 positive children. In addition, clinical symptoms, EMA IgG1, and mucosal atrophy disappeared after 8-10 mo on a gluten-free diet. Neither EMA IgA nor EMA IgG1 were detected in the children in the control groups. The other 12 children in study group showed no histologic abnormalities and were EMA IgG1 negative. In this study, we reveal a group of EMA IgG1 CD children without IgA deficiency. The diagnosis was based on the presence of gluten-dependent typical serological and histologic features of CD. Our data suggest that EMA IgG1 determination could be a new tool in the diagnostic workup of CD, useful in avoiding possible misdiagnosis.

31-43 amino acid sequence of the alpha-gliadin induces anti-endomysial antibody production during in vitro challenge.

BACKGROUND: Wheat gliadin is the culprit antigen of coeliac disease (CD). Two short sequences of NH2-terminal portion of gliadin seem to be responsible for CD. Antiendomysial antibodies (EMA), highly sensitive and specific for CD, are detectable in the culture media from treated CD patients, after in vitro challenge with peptic-tryptic (PT) digest of gliadin. In this study we detected EMA production after in vitro challenge with 31-43 peptide. We used 56-68 peptide, lacking toxic sequences, as a negative control. METHODS: Duodenal samples from 11 treated CD patients and 9 control patients were cultured with 31-43 and 56-68 peptides and PT gliadin. Indirect immunofluorescence analysis was used for EMA detection. RESULTS: EMA were detected in culture media of 10 of 11 specimens challenged with PT-gliadin and in the media of all specimens challenged with 31-43 peptide. No EMA were detectable in any treated patients cultured with 56-68 peptide or with medium alone. No EMA were observed in cultures of control specimens. DISCUSSION: The ability of the 31-43 sequence of the alpha-gliadin to induce EMA production suggests its involvement in the pathogenesis of CD. Furthermore, it may be a more useful antigenic substance than PT gliadin for both in vitro and in vivo studies of CD.

Does pretreatment with omeprazole decrease the chance of eradication of Helicobacter pylori in peptic ulcer patients?

OBJECTIVE: It has been reported that pretreatment with omeprazole could decrease the efficacy of Helicobacter pylori eradication. Our aim was to compare the efficacy, safety, and tolerability of the eradicating regimen, omeprazole/amoxicillin/metronidazole. The two antibiotics were scheduled either during the first or during the last 2 wk of omeprazole administration. METHODS: In this prospective controlled study conducted in a single center, 78 symptomatic peptic ulcer patients were treated for 4 wk with omeprazole 40 mg o.m.; the patients were randomly assigned to receive amoxicillin 1 g t.i.d. postprandially and metronidazole 250 mg t.i.d. postprandially, either during the first 2 wk (group A, n = 40) or the last 2 wk of therapy with omeprazole (group B, n = 38). H. pylori status was assessed by culture, histology, urease test, and IgG antibodies. Each patient's course was followed for 1 yr. RESULTS: H. pylori infection was cured in 97.4% of group A (95% CI: 0.84-0.99) and in 89% of group B (95% CI: 0.73-0.96, p = 0.28). Healing was achieved in 80% of the patients in group A (95% CI: 0.63-0.90) and in 75.7% of patients in group B (95% CI: 0.60-0.90, p = 0.60) At 12-month follow-up, 72 patients were evaluated: 37/38 (97%) of patients in group A and 33/33 (100%) in group B were confirmed as cured of the infection (NS). Peptic ulcer healing rate reached 100% in the two groups. Furthermore, between the two groups, there were no significant differences in symptom relief or improvement. Both regimens were well tolerated, and no patient had to be withdrawn from therapy because of an adverse event. Minor side-effects appeared to be similar in the two groups (40% vs. 38%). CONCLUSIONS: This randomized study clearly indicates that omeprazole pretreatment does not significantly reduce the efficacy of eradicating therapy for H. pylori in peptic ulcer patients.

Pharmacological characterization of [Leu-13-psi-CH2NH-Leu14]-bombesin as a specific bombesin receptor antagonist on isolated smooth muscle cells.

Isolated smooth muscle cells from guinea pig stomach were used to study the pharmacological characteristics of a newly synthetized bombesin analog, [Leu13-psi-CH2NH-Leu14]-bombesin (psi 13,14-BN) to function as antagonist of bombesin-induced contractile response. The antagonism caused by this new analog was compared to that obtained with the substance P analog [D-Arg1,D-Pro2,D-Trp7,9,Leu11]Substance P [( APTTL]SP), which has been used until now to characterize bombesin receptors on smooth muscle cells. psi 13,14-BN resulted to be more potent than [APTTL]SP as antagonist of bombesin action on smooth muscle. Comparing the IC50, psi 13,14-BN (IC50 70 nM) was 8 times more potent than [APTTL]SP (IC50 600 nM). In contrast to [APTTL] SP, the action of psi 13,14-BN was shown to be specific toward bombesin receptors in that it does not interfere with receptors for other agents (i.e., cholecystokinin, acetylcholine or substance P). The antagonism induced by both compounds was competitive inasmuch as the slope of the regression lines obtained by Schild plot analysis were not significantly different from the unity. The apparent affinity for the bombesin receptor was 0.8 nM for psi 13,14-BN and 7.8 nM for [APTTL]SP. These results indicate that psi 13,14-BN acts on isolated gastric smooth muscle cells as a competitive bombesin receptor antagonist, with a higher affinity and specificity than the substance P analog used previously.

Neutralization of the anionic sites of cultured rat mesangial cells by poly-L-lysine.

Changes in eicosanoid synthesis are a feature of the functional response of rat glomerular epithelial cells to neutralization of the cell surface polyanion. In order to ascertain if this property is common to other glomerular cell types, we have investigated the effect of neutralization of the negatively charged sites of cultured rat mesangial cells by poly-L-lysine on prostaglandin E2 (PGE2) production. Addition of poly-L-lysine (10 micrograms/ml) to the cells stimulated PGE2 synthesis after an initial latency of approximately two minutes, reaching maximum levels at 60 minutes. Poly-L-lysine also progressively increased cytosolic free calcium ([Ca2+]i) in fura-2 loaded monolayers with a similar initial lag time. Poly-L-lysine-evoked PGE2 synthesis and [Ca2+]i increases were dose dependent and prevented by addition of the polyanions, heparin and albumin. Additionally, heparin was also capable of reversing the effect of poly-L-lysine on [Ca2+]i. Removal of extracellular Ca2+ prevented the increase of [Ca2+]i and PGE2 synthesis. Increased PGE2 synthesis following neutralization of mesangial cell anionic sites may play a role in the hemodynamic dysfunction and cellular derangements of glomerular inflammation.

Somatostatin infused during acute pancreatitis retains its biological activity.

Somatostatin (SST) is used in the treatment of acute pancreatitis (AP) to inhibit pancreatic exocrine secretion, which represents one of the goals of medical treatment in this disease. Its therapeutic efficacy, however, is poor. One hypothesis, which has not yet been investigated, is that i.v. SST might be broken down by blood proteolytic enzymes. In order to evaluate the structural integrity and biological activity of infused SST, somatostatin-like immunoreactivity (SLI) and levels of pancreatic enzymes were monitored in the blood stream during the infusion of SST-14 (3,5 micrograms/kg/h for 48 h) in eight patients with severe acute pancreatitis. SLI was measured by both radioimmunoassay (RIA) and high-pressure liquid chromatography (HPLC). The results indicate that SLI levels increase promptly after the beginning of infusion, with a slower increase between 6 and 36 h, and a rapid increase again at 48 h. HPLC analysis shows a single peak of SLI with the same retention time as standard SST-14. Total amylase, lipase, and trypsinogen significantly decreased compared with pretreatment values (48, 63.1, and 77.4%, respectively) after 24 h of SST infusion, while a decrease in elastase 1 (62.6%) was observed later at 48 h. These results indicate that in severe AP, somatostatin recovered in plasma retains its biological activity: it inhibits pancreatic circulating enzymes, an action not influenced by breakdown of the peptide, as demonstrated by HPLC of the SLI measured in plasma.

Brief footshock analgesia: long-lasting enhancement induced by cathinone, an amphetamine-like agent.

In view of the analgesic effects produced by cathinone (CATH), an amphetamine-like agent, and of the interaction of amphetamines with stressful environmental stimuli, the present study evaluated in rats the influence of CATH on the nonopioid analgesia induced by a brief electric footshock (FSA; 3 min of continuous 2.5 mA current). The influence of this combination on body temperature was also evaluated. CATH (5 mg/kg, i.p.) alone induced a brief and slight increase in latency during the hot plate test (HPT), but enhanced and prolonged the analgesic effect induced by FS. In addition, the presentation of the environment (shock box with unelectrified grid) where other rats received FS, caused CATH to induce a slow-rising analgesic effect for 180 min. A hyperthermic response paralleling the analgesic effect was observed in shocked and nonshocked rats receiving CATH. After 24 h, rats that had received both CATH and FS on the previous day showed prolonged latencies on the HPT before and after a 1-min presentation of unelectrified grid. These animals also showed an increased analgesic response to the subsequent application of a 15-second FS. At the same time no differences in body temperature were observed between treatment groups. These results suggest that CATH can interact with environmental stimuli to induce an analgesic effect, the time-course of which depends upon the intensity of the stimulus applied.

Possible involvement of amine oxidase inhibition in the sympathetic activation induced by khat (Catha edulis) chewing in humans.

The possibility that phenylalkylamines (cathinone and cathine) of khat, like amphetamines, are amine oxidase inhibitors with clinical significance is evaluated. Results show that khat chewing induces a significant increase in blood pressure, body temperature and urinary catecholamines in khat naive subjects. On the other hand, in habitual consumers a significant enhancement only of blood pressure is observed. Urinary concentrations of vanilmandelic acid show an inhibitory trend in both groups of subjects. This has been related to a decrease in catecholamine deamination. In fact, oxidative deamination of benzylamine catalyzed by beef plasma amine oxidase is non-competitively inhibited in vitro by cathinone (Ki = 0.05 mM) and cathine (Ki = 10 mM) as well as by amphetamine (Ki = 7.9 mM).

Prolonged analgesia induced by cathinone. The role of stress and opioid and nonopioid mechanisms.

Cathinone, the active principle of Catha edulis (khat), shows long-lasting analgesic effects when the tail-flick test is used in rats. The involvement of monoamines, endogenous opioids and stress in this analgesic effect was tested. Both early (30 min) and late (24 h) analgesic effects of cathinone were prevented by reserpine or p-chlorophenylalanine, which deplete catecholamines or serotonin, respectively, and by nomifensine, which prevents neuronal uptake of biogenic amines and amphetamines. The same inhibitory effect was obtained with a high dose (4 mg/kg) of naloxone. However, rats made tolerant to morphine retained both early and late analgesic response to cathinone. The increase in plasma ACTH induced by the tail-flick test at 30 min and 24 h was significantly enhanced by cathinone, in a naloxone-reversible way. However, the analgesic responses shown at these times were not prevented by either dexamethasone or adrenalectomy. We conclude that the prolonged analgesia induced by cathinone is primarily due to an amphetamine-like activation of monoaminergic pathways, but requires the integrity of non-mu-opioid mechanisms. The involvement of the adrenohypophyseal axis in this cathinone effect is less probable.

Correlations of DNA, RNA and protein levels in duodenal mucosa with antiinflammatory potency and disposition to gut damage of non-steroidal agents. Comparative behaviour of glucametacine, indometacin, phenylbutazone and ibuprofen.

As far as molar ratio is concerned, glucametacine was half as potent as indometacin, twice as active as phenylbutazone and four times more effective than ibuprofen in preventing cotton granuloma. Both indometacin and phenylbutazone induced dose related gastrointestinal ulcerations and increase of 51Cr tagged erythrocytes in feces. The former drug displayed gut toxicity at anti-inflammatory doses, the latter at doses approximately four times larger. Glucametacine was still devoid of damaging effects at a dose ten times larger than the minimal one capable of inhibiting granuloma growth. Ibuprofen, too, failed to induce ulcers at all doses examined; however, it displayed a trend toward gut bleeding when doses that increased blood corticosterone were attained. Studies on duodenal mucosa showed that in rats on cotton granuloma, DNA, proteins and DNA:RNA ratio increase as compared to unimplanted rats. Glucametacine and phenylbutazone reversed the increase of DNA and proteins, respectively. Indometacin decreased all forementioned constituents of duodenal mucosa while inducing haemorrhages and ulcers on gut. Furthermore, in naive rats, unlike glucametacine and phenylbutazone, indometacin induced a decrease in protein content of duodenal mucosa. Differences in disposition of gut toxicity among glucametacine and other anti-inflammatory drugs are discussed.