Immortalization of neuro-endocrine cells from adrenal tumors arising in SV40 T-transgenic mice.

Pheochromocytomas are adrenal medullary tumors which arise from the transformation of neural crest-derived cells. In the course of studies of mice transgenic for an SV40 T-gene ectopically expressed in the adrenal medulla, we observed the occurrence of large, mainly bilateral tumors in a high proportion of transgenic animals. From these tumors we established immortalized cell lines which grow in vitro at 32 degrees C (the permissive temperature for the tsA58 T-protein encoded by the transgene), but not at 38 C. These cells demonstrate characteristics of both neuronal (160 kd neurofilament) and endocrine (chromogranins) cells. The expression of Mash-1 and ret supports their initial characterization as early bipotential neuro-endocrine progenitors.

Octreotide 24-h prophylaxis in patients at high risk for post-ERCP pancreatitis: results of a multicenter, randomized, controlled trial.

BACKGROUND: Pharmacological prophylaxis of post-ERCP pancreatitis is costly and not useful in most non-selected patients, in whom the incidence of pancreatitis is 5% or less. However, it could be useful and probably cost-effective, in patients at high risk for this complication, where the post-procedure pancreatitis rate is 10% and more. AIM: To assess the efficacy of octreotide in reducing the incidence and severity of post-ERCP pancreatitis and procedure-related hospital stay, in subjects with known patient-related risk factors. METHODS: A total of 120 patients were randomly allocated to receive octreotide or not, in a multicentre, randomized, controlled trial. The drug was given subcutaneously, 200 microg t.d.s., starting 24 h before the ERCP procedure, in patients with either sphincter of Oddi dysfunction, or a history of relapsing pancreatitis or post-ERCP pancreatitis, or who were aged under 35 years, or who had a small common bile duct diameter (< 8 mm). RESULTS: A total of 114 patients (58 in the octreotide group and 56 in the control group) completed the trial. Post-procedure pancreatitis occurred in seven octreotide-treated patients (12.0%) and eight controls (14.3%). The two groups showed no significant differences in the incidence or severity of pancreatitis. Twenty-four hours after the procedure, severe hyperamylasemia (more than five times the upper normal limit) without pancreatic-like pain was recorded in three octreotide-treated patients (5.2%) and six controls (10.7%), the difference being not significant. CONCLUSION: Twenty-four-hour prophylaxis with octreotide proved ineffective in preventing post-ERCP pancreatitis and in avoiding 24-h severe hyperamylasemia in high-risk patients.

Feasibility study of colorectal cancer screening by immunochemical faecal occult blood testing: results in a northern Italian community.

AIM: Screening by means of faecal occult blood testing (FOBT) has proved to be effective in reducing colorectal cancer incidence and mortality. We performed a pilot screening for colorectal cancer by latex immunological FOBT in two municipalities of the region Valle d'Aosta, Italy, focusing on problems and obtaining indications for the feasibility and extension of the screening programme on a regional basis. METHODS: A total of 2961 subjects aged 50-74 years were invited by mail to perform a one-day immunochemical FOBT without any dietary restrictions and with a positive threshold put at 100 ng/ml. Patients with positive tests were then invited to undergo colonoscopy and double-contrast barium enema if colonoscopy was incomplete. RESULTS: A total of 1631 subjects performed the screening test with an overall compliance of 55.1%. Seventy-two subjects had a positive FOBT. Detection rates for cancer and adenomas were 1.8 per thousand and 16.6 per thousand, respectively. Positive predictive values (PPVs) for cancer and adenomas were 4.5% and 40.3%, respectively. CONCLUSIONS: Screening had an adequate attendance rate and the majority of the indicators were satisfactory. The use of a one-day quantitative latex FOBT with no dietary restrictions, automation of the analytical procedure, and a positive threshold of 100 ng/ml has shown that a programme based on this test is feasible in both organizational and attendance terms. On the basis of this experience, the extension of the screening on a regional basis is suggested.

Evaluation of a commercial serological kit for detection of salivary immunoglobulin G to Helicobacter pylori: a multicentre study.

OBJECTIVE: To evaluate the usefulness of a commercial serological enzyme immunoassay (EIA) for detecting salivary immunoglobulin (Ig) G to Helicobacter pylori. DESIGN: Prospective, multicentre study. SETTING: The study was conducted at 11 gastroenterology hospital centres throughout Italy. PARTICIPANTS AND INTERVENTIONS: Two hundred and thirteen dyspeptic patients underwent gastroscopy with antral biopsies. At each centre, two of the following three tests for H. pylori diagnosis were performed: urease quick test, histology, and 13C-urea breath test. Samples of unstimulated saliva and venous blood were collected from each patient. Salivary and serum H. pylori IgG were determined with the EIA Helori-test IgG (Eurospital, Trieste, Italy). MAIN OUTCOME MEASURES: Sensitivity, specificity, positive and negative predictive values, and accuracy of the salivary and serum EIAs. RESULTS: By using a receiver operating characteristic curve, salivary EIA yielded 81% (95% confidence interval 73-87%) sensitivity, 73% (62-83%) specificity, 84% (76-90%) positive predictive value, 69% (58-79%) negative predictive value, and 78% (72-84%) accuracy. At the cut-off suggested by the manufacturer, serum EIA had 90% (84-95%) sensitivity, 78% (67-86%) specificity, 88% (81-93%) positive predictive value, 82% (71-90%) negative predictive value, and 86% (81-91%) accuracy. CONCLUSION: In this large multicentre study, detection of salivary H. pylori IgG with a commercial serological EIA was a fairly accurate diagnostic method. Data confirm that saliva testing does not compare favourably with serology in the assessment of H. pylori status.

[Ileal adenocarcinoma in Crohn's disease. A report of 2 cases]. / Adenocarcinoma ileale nel morbo di Crohn. Descrizione di 2 casi.

The incidence of intestinal adenocarcinoma in Crohn's ileal disease is reported in the literature to be higher than in the normal population. The authors report two cases of adenocarcinoma observed during the course of Crohn's ileitis and, by comparing these findings with earlier published data, highlight their characteristics. Two patients, a 53-year-old woman and a 62-year-old man, are reported as suffering from Crohn's ileitis. The woman underwent resection of the right colon with ileo-transversostomy due to stenosis twenty years earlier, whereas diagnosis in the male patient dates back to an appendicectomy 11 years earlier. Surgery, which was performed in both due to subocclusion which failed to respond to steroid therapy, showed the typical alterations of Crohn's disease. An adenocarcinoma was also found in both patients in the stenotic zone with highly dysplasic foci. The man presented a monoclonal gammopathy of an IgA-type with light lambda chains and Bence-Jones' proteinuria. Both patients are living and enjoy apparently good health respectively 2 and 6 years after surgery. The authors emphasize that the association between Crohn's ileal disease and intestinal adenocarcinoma cannot be considered casual. In the literature the relative risk is reported to be between 6 and 320 times higher, with approximately 1 out of 350 patients suffering from both diseases contemporaneously. In the series reported earlier, 2 cases of carcinoma were diagnosed in 38 Crohn's patients studied over a period of 12 years out of a total population of 114000 inhabitants, with a frequency of carcinoma of 5.13% compared to 0.3% reported in the literature. The incidence of Crohn's disease was 2.84/100000 per year.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased prevalence of migraine in patients with uninvestigated dyspepsia referred for open-access upper gastrointestinal endoscopy.

BACKGROUND AND STUDY AIMS: The association between gastrointestinal symptoms and headache is frequently unrecognized. The aim of the present study was to determine the prevalence of migraine in dyspeptic outpatients referred for upper gastrointestinal endoscopy. PATIENTS AND METHODS: Patients aged 18 - 55 years undergoing upper gastrointestinal endoscopy for dyspeptic symptoms in three endoscopic units were recruited consecutively. All of the patients were given a validated questionnaire on headache symptoms in order to determine the prevalence of migraine. Patients were divided into four groups (ulcer-like dyspepsia, reflux-like dyspepsia, dysmotility-like dyspepsia, only nausea and/or vomiting). Age-matched blood donors were given the same questionnaires and served as controls. RESULTS: A total of 378 patients (mean age 40 +/- 11, 52 % men) and 310 controls (mean age 39 +/- 11, 56 % men) were enrolled. No differences were observed between the two groups in the prevalence of migraine (15 % vs. 11 %; P = 0.12). A higher prevalence of migraine was found among women in both groups (P < 0.006). In patients with reflux-like and ulcer-like dyspepsia, the prevalence of migraine did not differ from that in the control individuals (8 % and 7 %, respectively), whereas a higher prevalence of migraine was noted in patients with dysmotility-like dyspepsia (23 %; P < 0.02 vs. controls, those with ulcer-like dyspepsia and those with reflux-like dyspepsia) and in patients with nausea and/or vomiting alone (53 %; P < 0.002 vs. all other groups). The multivariate analysis confirmed that the symptom pattern and sex were the only variables independently associated with migraine. CONCLUSIONS: A diagnosis of migraine should be considered in young patients referred for upper gastrointestinal endoscopy due to nausea and/or vomiting or for dysmotility-like dyspepsia.

Transcriptional and posttranscriptional regulation of the expression of the erythropoietin receptor gene in human erythropoietin-responsive cell lines.

With erythroid differentiation, committed progenitor cells acquire the ability to respond to erythropoietin (Epo). Epo interacts with target cells through the Epo receptor (Epo-R), whose expression is tightly regulated in a lineage-specific fashion. Epo-R expression is presumed to be progressively activated or repressed as cells progress along the erythroid or the myeloid pathway, respectively. Little is known of the mechanisms that underlie the erythroid-specific expression of the Epo-R gene. GATA-1, the major known transcription factor involved in Epo-R gene regulation, is not erythroid-specific. We have studied the regulation of the expression of the Epo-R gene in two related human Epo-responsive cell lines, UT-7 and UT-7 Epo. These lines express Epo-R at high levels because of amplification of the endogenous gene, which is apparently not rearranged. Treatment for 6 to 24 hours with the tumor promoter, phorbol myristate acetate (PMA), or 24 hours of growth factor starvation (Epo or granulocyte/macrophage colony-stimulating factor [GM-CSF]) decreased or increased the levels of Epo-R mRNA, respectively. In the case of growth factor starvation, the increase (approximately equal to threefold) in the level of Epo-R mRNA correlated directly with an increase in the rate of Epo-R gene transcription as measured by run-off assay. Both increases were observed as early as 3 hours after the growth factor was withdrawn and were reversible; levels of mRNA and transcription rates returned to baseline 3 hours after the cells were reexposed to growth factors. The changes in Epo-R expression after growth factor starvation were coordinated with changes in the level of expression of GATA-1 that were detected both at the mRNA and at the gene transcription level under these conditions (suggesting that GATA-1 was responsible for this upregulation). During PMA treatment, after a transient increase in Epo-R mRNA at 1 hour, a progressive decline in the level of Epo-R mRNA was observed; the level of Epo-R mRNA decreased by 50%, and fell below the level of detection by 6 and 24 hours, respectively. This decrement was explained in part by a fourfold reduction in the rate of gene transcription as well as a reduction (measured as levels of Epo-R mRNA in the presence of actinomycin D) in mRNA stability. The changes in transcription rate occurred in the absence of changes in the level of GATA-1 binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)

An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1.

The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the cell-type specific expression of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40 promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.

Life, activation and death of intrahepatic lymphocytes in chronic hepatitis C.

The healthy liver of adult humans has little or no lymphocyte component and the histological finding of intrahepatic lymphocytes (IHL) is evidence of liver pathology. In a liver injured by chronic hepatitis C, the most common chronic liver disease, most IHL are activated/pro-inflammatory cells, which are particularly enriched for effectors of innate immunity (natural killer (NK), natural T, and other NK-like T cells). IHL do not undergo clonal expansion in the liver but migrate from extrahepatic sites to the chronically infected liver, where they display effector function and subsequently die, suggesting that maintenance of the IHL pool depends on continuous lymphocyte migration. The cytotoxic and inflammatory functions of these IHL have three potential outcomes: 1) they could be helpful in clearing the virus (a rare case in hepatitis C virus (HCV) infection); 2) they could be useless and have no effect on the infection; or 3) they could be harmful, whereby overaggressive lymphocyte responses destroy the liver in a continuous and unsuccessful attempt to clear the virus. Unfortunately, we do not know as of yet which of these possibilities is the case and, therefore, a more complete picture of the intrahepatic immune response will be relevant to the development of new therapeutic strategies against HCV. Additionally and from a more general perspective, due to the availability of biopsied material and the high prevalence (approximately 3%) of HCV infection worldwide, studying the chronically inflamed liver of hepatitis C patients is an ideal model to investigate the poorly understood processes of lymphocyte trafficking, activation and death to non-lymphoid sites of chronic inflammation in man.

Progressive inactivation of the expression of an erythroid transcriptional factor in GM- and G-CSF-dependent myeloid cell lines.

The transcriptional binding protein NFE-1 (also called GF-1 and Ery-f1) is thought to play a necessary, but not sufficient, role in the regulation of differentiation-related gene expression in a subset of hematopoietic lineages (erythroid, megakaryocytic, and basophil-mast cell). In order to clarify the mechanism which underlies the lineage-specificity of the NFE-1 expression, as well as the relationship between the expression of this factor and growth factor responsiveness, we have evaluated the capacity of erythropoietin (Epo)-, granulomonocytic (GM)-colony stimulating factor (CSF)-, and granulocyte (G)-CSF-dependent subclones derived from the interleukin 3 (IL-3)-dependent cell line 32D, to express 1) NFE-1 mRNA, 2) NFE-1-related nuclear proteins, and 3) chloramphenicol acetyl transferase (CAT) activity when transfected with a CAT gene under the control of NFE-1 cognate sequences. NFE-1 mRNA was found to be expressed not only in cells with mast cell (IL-3-dependent 32D) and erythroid (Epo-dependent 32D Epo1) phenotypes, but also in cells with predominantly granulocyte/macrophage properties, such as the GM-CSF- (early myelomonocytic) and G-CSF- (myelocytic) dependent subclones of 32D. However, a gradient of expression, correlating with the lineage, the stage of differentiation, and the growth factor responsiveness of the cell lines, was found among the different subclones: Epo greater than or equal to IL-3 greater than GM-CSF greater than G-CSF. Binding experiments demonstrated NFE-1 activity in all cell lines except the G-CSF-dependent line. Function of the NFE-1 protein was assessed by the expression of the CAT gene linked to the SV40 promoter and a mutant (-175 T----C) HPFH gamma-globin promoter. High level CAT expression was seen only in the Epo1 cells although low level expression was also seen in the parent 32D. These results demonstrate that the specificity of the expression of NFE-1 for the erythroid--megakaryocytic--mast cell lineages is obtained by progressive inactivation of its expression in alternative lineages.

Response to erythropoietin in erythroid subclones of the factor-dependent cell line 32D is determined by translocation of the erythropoietin receptor to the cell surface.

Regulation of the expression of the erythropoietin (Epo) receptor (EpoR) gene is under the control of transcriptional regulatory factor GATA-1. GATA-1 is expressed widely among the nonerythroid, factor-dependent subclones of the interleukin 3-dependent mouse cell line 32D. Consequently, to determine whether GATA-1 and EpoR gene expression are linked even in nonerythroid cells, we have studied the correlation of GATA-1 expression with expression and function of EpoR in these cell lines. EpoR mRNA (by RNase protection analysis) and EpoR protein (by specific antibody immunoprecipitation of metabolically labeled EpoR protein) were detectable not only in 32D and 32D Epo (an Epo-dependent subclone) but also in 32D GM, a subclone dependent for growth on granulocyte/macrophage colony-stimulating factor. EpoR mRNA also was detectable by PCR in 32D G, a subclone dependent for growth on granulocyte colony-stimulating factor. However, only 32D Epo cells bound 125I-labeled Epo and expressed EpoR protein on the cell surface, as determined by immunoprecipitation of surface-labeled proteins. These results indicate that, in these factor-dependent cell lines, the major regulatory step determining the erythroid-specific response to Epo is the efficiency of EpoR protein translocation to the cell surface. Mechanisms that could affect lineage-specific translocation are the presence of a chaperone protein, erythroid-specific editing of EpoR mRNA, or altered processing of the EpoR protein to the cell surface. In this model, lineage-restricted responses to growth factors such as Epo are determined not by expression of the genes for growth factor receptors but, rather, by appropriate processing of the receptor protein.

Immortalization of multipotent growth-factor dependent hemopoietic progenitors from mice transgenic for GATA-1 driven SV40 tsA58 gene.

The transcription factor GATA-1 is required for the normal development of erythroid cells. GATA-1 is also expressed in other hemopoietic cells, suggesting that it might be initially activated in a multipotent progenitor. To immortalize GATA-1-expressing progenitors, we generated mice transgenic for a thermosensitive SV40 T gene, driven by the GATA-1 promoter-enhancer. Immortalized marrow cells grow in culture at 32 degrees C but not at 38 degrees C, and are dependent on erythropoietin (Epo) or interleukin 3 (IL-3). Epo dependent cells express hemoglobin, high levels of GATA-1, GATA-2 and NF-E2 p45 mRNAs, and are positive for stem cell antigen 2 (Sca-2) and the early myeloid marker ER-MP12. IL-3 dependent cells can be derived from Epo dependent lines, and are hemoglobin-, Sca-2- and ER-MP12-negative, have low GATA-1 and NF-E2 p45 mRNA levels, and express myeloid markers Mac-1, F4/80 and Gr-1. Brief treatment of Epo dependent cells with myeloid growth factors (plus Epo) leads to the induction of Mac-1, F4/80 and Gr-1, concomitant with the disappearance from most cells of Sca-2, ER-MP12 and GATA-1 driven T antigen nuclear expression. Thus, the immortalized Epo dependent cells have the property of a progenitor capable of differentiation towards either the erythroid or myeloid lineages. These cells initiate transcription of a proportion of GATA-1 RNA molecules at an upstream promoter, previously known to be expressed only in testis cells.

Portal hypertensive gastropathy: reproducibility of a classification, prevalence of elementary lesions, sensitivity and specificity in the diagnosis of cirrhosis of the liver. A NIEC multicentre study. New Italian Endoscopic Club.

OBJECTIVE: To classify elementary endoscopic lesions of portal hypertensive gastropathy, assess their reproducibility, prevalences, sensitivity and specificity in the diagnosis of cirrhosis of the liver. METHODS: 1) A classification of portal hypertensive gastropathy elementary lesions was defined. 2) Thirty-two endoscopists evaluated videotapes of endoscopic examinations of patients with liver cirrhosis to assess beyond-chance agreement (kappa). 3) Fifteen centres enrolled consecutive patients with or without cirrhosis of the liver and recorded portal hypertensive gastropathy pattern according to its location. RESULTS: 1) Four elementary lesions (Mosaic-Like Pattern, Red Point Lesions, Cherry Red Spots, Black-Brown Spots) were identified, and graded. 2) A fair to good beyond-chance agreement was obtained for all 4 lesions. 3) portal hypertensive gastropathy prevalence was higher in patients with cirrhosis of the liver (0.63, sensitivity) than in controls (0.17). Mosaic-like pattern was the most prevalent sign (0.54). Specificity of portal hypertensive gastropathy was 0.83. Portal hypertensive gastropathy was tentatively classified as mild or severe when mosaic-like pattern alone or red marks of any kind were present, respectively; this classification led to a further improvement in reproducibility. CONCLUSIONS: Our results suggest that a sufficient degree of agreement can be achieved in recording portal hypertensive gastropathy. Therefore, the New Italian Endoscopic Club classification should be used to evaluate the natural history of this condition.