Selective gamma-ketoaldehyde scavengers protect Nav1.5 from oxidant-induced inactivation.

The cardiac sodium channel (SCN5A, Na(V)1.5) is a key determinant of electrical impulse conduction in cardiac tissue. Acute myocardial infarction leads to diminished sodium channel availability, both because of decreased channel expression and because of greater inactivation of channels already present. Myocardial infarction leads to significant increases in reactive oxygen species and their downstream effectors including lipoxidation products. The effects of reactive oxygen species on Na(V)1.5 function in whole hearts can be modeled in cultured myocytes, where oxidants shift the availability curve of I(Na) to hyperpolarized potentials, decreasing cardiac sodium current at the normal activation threshold. We recently examined potential mediators of the oxidant-induced inactivation and found that one specific lipoxidation product, the isoketals, recapitulated the effects of oxidant on sodium currents. Isoketals are highly reactive gamma-ketoaldehydes formed by the peroxidation of arachidonic acid that covalently modify the lysine residues of proteins. We now confirm that exposure to oxidants induces lipoxidative modification of Na(V)1.5 and that the selective isoketal scavengers block voltage-dependent changes in sodium current by the oxidant tert-butylhydroperoxide, both in cells heterologously expressing Na(V)1.5 and in a mouse cardiac myocyte cell line (HL-1). Thus, inhibition of this lipoxidative modification pathway is sufficient to protect the sodium channel from oxidant induced inactivation and suggests the potential use of isoketal scavengers as novel therapeutics to prevent arrhythmogenesis during myocardial infarction.

Growth and differentiation of human gastrointestinal adenocarcinoma stem cells in soft agarose.

A soft agarose clonogenic assay is presented which has been optimized for the growth of human gastrointestinal adenocarcinomas. Samples from 15 gastric and colonic solid tumors and from 2 noncancerous stomachs (control cultures) were disaggregated by treatment with collagenase at 37 degrees overnight. Colonies appeared 10 to 15 days after plating, with a cloning efficiency between 0 and 0.82%, which was markedly improved by a fibroblastic feeder layer. The results suggest a correlation between cloning efficiency and the degree of differentiation of the initial tumor. Histochemistry, electron microscopy, and a carcinoembryonic antigen immunofluorescence assay showed that the colonies consisted of cells with the same characteristics as those of the original tumor.. This colony formation assay appears to be potentially useful for assessing the stem cell pool of gastrointestinal tumors. It will be valuable for studying their response to chemotherapeutic agents in vitro. This clonogenic assay may also permit the establishment of cancer cell lines.

Characterization of a newly established human gastric cancer cell line HGT-1 bearing histamine H2-receptors.

A human gastric adenocarcinoma cell line, HGT-1, was established in vitro from the primary tumor of a 60-year-old patient. Histological examination of the tumor revealed a poorly differentiated adenocarcinoma. Primary tumor cells were cloned in soft agarose and gave rise to tumor colonies. The procedures enabling us to form a continuous cell line from the agarose colonies are described. The cultured cells grew as monolayers of closely apposed polygonal cells with a population-doubling time of 19.48 +/- 1.20 (S.E.) hr during exponential growth at passage 59. They had an epithelial morphology. Ultrastructural studies revealed the presence of microvilli and tight junctions. The HGT-1 cell line is tumorigenic in nude mice and has a hyperdiploid karyotype with a modal number of 57 chromosomes. It exhibits numerous marker chromosomes. These human gastric epithelial cells do not secrete mucus or carcinoembryonic antigen. They exhibit functional histamine H2-receptors mediating cellular cyclic adenosine 3':5'-monophosphate production and adenylate cyclase activation. In conclusion, the use of a soft-agarose clonogenic assay permitted us to develop a cancer cell line without the problems of fibroblastic cell contamination. The existence of histamine H2-receptors on gastric HGT-1 cells stresses the importance of this line as a model for studies of regulatory mechanisms involved in gastric secretion.

Novel SCN5A mutation leading either to isolated cardiac conduction defect or Brugada syndrome in a large French family.

BACKGROUND: The SCN5A gene encoding the human cardiac sodium channel alpha subunit plays a key role in cardiac electrophysiology. Mutations in SCN5A lead to a large spectrum of phenotypes, including long-QT syndrome, Brugada syndrome, and isolated progressive cardiac conduction defect (Lenègre disease). METHODS AND RESULTS: In the present study, we report the identification of a novel single SCN5A missense mutation causing either Brugada syndrome or an isolated cardiac conduction defect in the same family. A G-to-T mutation at position 4372 was identified by direct sequencing and was predicted to change a glycine for an arginine (G1406R) between the DIII-S5 and DIII-S6 domain of the sodium channel protein. Among 45 family members, 13 were carrying the G1406R SCN5A mutation. Four individuals from 2 family collateral branches showed typical Brugada phenotypes, including ST-segment elevation in the right precordial leads and right bundle branch block. One symptomatic patient with the Brugada phenotype required implantation of a cardioverter-defibrillator. Seven individuals from 3 other family collateral branches had isolated cardiac conduction defects but no Brugada phenotype. Three flecainide test were negative. One patient with an isolated cardiac conduction defect had an episode of syncope and required pacemaker implantation. An expression study of the G1406R-mutated SCN5A showed no detectable Na(+) current but normal protein trafficking. CONCLUSIONS: We conclude that the same mutation in the SCN5A gene can lead either to Brugada syndrome or to an isolated cardiac conduction defect. Our findings suggest that modifier gene(s) may influence the phenotypic consequences of a SCN5A mutation.

Non-invasive testing of acquired long QT syndrome: evidence for multiple arrhythmogenic substrates.

BACKGROUND: Although well-defined clinically and electrocardiographically, Acquired Long QT Syndrome (LQTS) remains elusive from a pathophysiologic point of view. An increasingly accepted hypothesis is that it represents an attenuated form of Congenital Long QT Syndrome. To test this hypothesis further, we investigated patients with Acquired LQTS, using various investigations that are known to give information in patients with Congenital LQTS. METHODS: All the investigations were performed in patients with a history of Acquired Long QT Syndrome, defined by marked transient QT lengthening (QT>600 ms) and/or torsades de pointes. Measurement of the QT interval dispersion, the interlead difference for the QT interval on a 12-lead ECG, was performed in 18 patients and compared with 18 controls, matched for age and sex. To assess sympathetic myocardial innervation, I-123 Meta-iodobenzylguanidine (I-123-MIBG) scintigraphy was performed in 12 patients, together with Thallium scintigraphy, to rule out abnormal myocardial perfusion. Time-frequency analysis of a high-resolution ECG using a wavelet technique, was made for nine patients and compared with 38 healthy controls. Finally, genetic studies were performed prospectively in 16 consecutive patients, to look for HERG, KCNE1, KCNE2 and KCNQ1 mutations. The functional profile of a mutated HERG protein was performed using the patch-clamp technique. RESULTS: Compared with the control group, a significant increase in QT dispersion was observed in the patients with a history of Acquired LQTS (55+/-15 vs. 33+/-9 ms, P<0.001). In another group of patients with Acquired LQTS, 123 I-MIBG tomoscintigraphy demonstrated a decrease in the sympathetic myocardial innervation. Time--frequency analysis using wavelet transform, demonstrated an abnormal frequency content within the QRS complexes, in the patients with Acquired LQTS, similar to that found in Congenital LQTS patients. Molecular screening in 16 consecutive patients, identified one patient with a missense mutation on HERG, one of the LQTS genes. Expression of the mutated HERG protein led to altered K(+) channel function. CONCLUSION: Our results suggest that Acquired and Congenital Long QT Syndromes have some common features. They allow the mechanism of the clinical heterogeneity, found in both syndromes, to be understood. Further multi-facet approaches are needed to decipher the complex interplay between the main determinants of these arrhythmogenic diseases.

Fibrosing alveolitis and hepatitis B surface antigen-associated chronic active hepatitis in a patient with immunoglobulin A deficiency.

Fibrosing alveolitis is described in a 22 year old woman with immunoglobulin A (IgA) deficiency and hepatitis B surface antigen (HBsAg)-associated chronic active hepatitis. At lung biopsy HBsAg was detected by indirect immunofluorescence in the alveolar space but not in the septal fibrosis. We discuss the possible relationships between IgA deficiency on the one hand, and HBsAg-associated lung and liver diseases on the other hand.

Superficial squamous cell carcinoma of the esophagus. A report of 76 cases and review of the literature.

Superficial squamous carcinoma of the esophagus, defined as carcinoma limited to mucosa or submucosa regardless of lymph node status, is being increasingly recognized in the Western hemisphere. Seventy-six cases of this entity are herein presented. Five macroscopic types were recognized: normal flat (eight cases), coarse (21 cases), verrucous (25 cases), polypoid (17 cases), and ulcerating infiltrating (five cases). Histological typing included 65 conventional squamous cell carcinomas, six squamous carcinomas with spindle cell features, and five adenoid cystic carcinomas. Four cases were strictly intraepithelial, 10 cases were confined to the mucosa, nine cases encroached onto the muscularis mucosae, and 53 extended into the submucosa. Cases with intraepithelial and infiltrating carcinomas confined to the mucosa showed no lymph node involvement. Thirty percent of cases extending into the submucosa developed lymph node metastases. Thirty-eight patients survived surgical resection from 1 to 96 months; 34 of these 38 were free of neoplastic disease. Fourteen patients had an associated bronchial or oropharyngolaryngeal carcinoma either simultaneously or asynchronously. We conclude that patients with superficial squamous carcinoma of the esophagus can benefit from early diagnosis and prompt surgery.

Pathological features and mucin histochemistry of primary gastric stump carcinoma associated with gastritis cystica polyposa. A study of six cases.

Six unusual cases of primary gastric stump carcinoma associated with gastritis cystica polyposa and arising in old gastrojejunostomy stomas are presented. The clinical data, the gross and microscopic pathological features, and the histochemical mucin profile of these two lesions are described in detail. A review is undertaken of the most relevant and previously published reports concerning, separately, either primary gastric stump carcinoma or gastritis cystica polyposa. The findings available in these reports are compared with those observed in our six cases. Some aspects of gastritis cystica polyposa are not unlike those seen in the solitary ulcer syndrome of the rectum and Ménétrier's disease. The histological type and mucin profile of primary gastric stump carcinoma parallel those recorded in gastric cancer arising in the unoperated stomach. However, the role of intestinal metaplasia and its histochemical typing appear somewhat different in primary gastric stump carcinoma.

Surgical pathology of adenocarcinoma arising in Barrett's esophagus. Analysis of 67 cases.

Numerous reviews of adenocarcinoma arising in Barrett's esophagus have been reported, but detailed pathologic findings or survival analysis have rarely been provided. This retrospective study analyzed 67 patients (mean age, 64 years; male-to-female ratio, 10:1) with an adenocarcinoma arising in Barrett's esophagus treated by surgical resection. Prevalence of smokers was 63%, alcohol users, 45%, and patients with hiatal hernia, 73%. Five patients had another synchronous cancer, and seven patients, previous esophageal surgery. Forty percent of the tumors were well differentiated, 31% moderately differentiated, 15% poorly differentiated, 7% mucinous, and 6% composed of signet-ring cells. Depth of invasion in the esophageal wall was limited to mucosa in 13% of cases and submucosa in 18%. Invasive adenocarcinomas extended to the muscular layer in 12% of cases, to adventitia in 33%, and to periesophageal tissue in 24%. Vascular and perineural neoplastic invasion was present in 67 and 38% of cases. Regional lymph node involvement and distant metastases were found in 51 and 9% of cases. Overall, 1-, 2-, and 5-year survival rates were 63, 41, and 32%, respectively. Five-year survival rate was significantly better for patients with superficial cancer limited to mucosa or submucosa (82 vs. 12%) or without regional lymph node involvement (59 vs. 10%). Tumor differentiation, vascular and perineural invasion, extranodal spread, distant metastases, and resection margins status also had a significant prognostic value on univariate analysis. In a multivariate Cox regression analysis for overall survival, depth of invasion in the esophageal wall and regional lymph node involvement were independent prognostic factors. Careful pathologic staging is of value in determining the prognosis of patients with adenocarcinoma arising in Barrett's esophagus.

Recurrence of Fabry's disease in a renal allograft eleven years after successful renal transplantation.

A case of Fabry's disease in a renal transplant recipient with a follow-up period of 11 years is reported. The patient suffered from renal, skin, peripheral nerve lesions, and asymptomatic cardiomegaly. Fabry's disease symptoms disappeared after transplantation. Improvement of renal function was rapidly observed, and it remained satisfactory during the whole posttransplantation period. The patient died of a severe, uncontrolled infection and of biliary peritonitis. Autopsy showed a polyvisceral accumulation of sphingolipids deposits. The engrafted kidney was histologically free of disease. Ultrastructurally, it revealed numerous sphingolipid inclusions in the endothelial cells of capillaries. The explanation of this complication could be attributed to: (1) high circulating levels of plasma substrates locally overwhelming the enzymatic capability of the graft endothelial cells; and (2) the endothelial cells originated from the recipient but not from the donor, an occurrence that has been described after transplantation. Rejection and the newly formed deposits in the endothelial cells may lead to the loss of the engrafted organ. As a consequence of the increasing possibility of organ transplantation, this complication should be detected by studying the blood vessels ultrastructurally in order to evaluate the condition of the transplant.

Gastrointestinal Richter's syndrome.

The development of a diffuse large cell lymphoma of the stomach in a patient who had chronic lymphocytic leukemia is reported. Richter's syndrome localized to the gut has not been described previously. Morphologic and immunologic studies suggest that the diffuse large cell lymphoma arose from the same clonal proliferation, IgG lambda, as the initially detected serum monoclonal protein associated with the chronic lymphocytic leukemia.

Expression of cadherins in benign, borderline, and malignant ovarian epithelial tumors: a clinicopathologic study of 60 cases.

We have analyzed the expression of E- and N-cadherins in benign, borderline, and maligant ovarian tumors, and we have correlated the pattern of cadherin expression with the standard clinicopathological parameters. An immunohistochemical technique has been applied to formalin-fixed, paraffin-embedded samples of 20 benign cystic tumors, 20 borderline tumors, and 20 cancers. Expression of E- and N-cadherins immunostaining were compared with the histological type, degree of histological differentiation, International Federation of Gynecology and Obstetrics (FIGO) stage, presence of ascites, occurrence of recurrence, and survival. E-cadherin was homogeneosuly expressed in benign tumors but was heterogeneously expressed or undetectable in most borderline and malignant tumors. In contrast, N-cadherin was detected in most benign and borderline tumors but was absent or heterogeneous in most carcinomas. The difference of expression of E-cadherin and N-cadherin between the three groups of ovarian tumors was statistically significant (respectively, P = .03 and P < .001). In ovarian carcinoma, patients with negative E-cadherin staining present a significantly shorter survival. No correlation was found between cadherin expression and clinicopathological parameters in borderline tumors. Our results suggest that alterations in E-cadherin and N-cadherin expressions are differentially involved in ovarian carcinogenesis and may have diagnostic and prognostic values.

Hepatocytic PAS-positive diastase-resistance inclusions in the absence of alpha-1-antitrypsin deficiency--high prevalence in alcoholic cirrhosis.

The presence of PAS-positive, diastase-resistant inclusions in the cytoplasm of the hepatocytes is characteristic of alpha-1-antitrypsin deficiency. The purpose of this investigation was to determine whether the presence of these inclusions is a specific feature, permitting the recognition of alpha-1-antitrypsin deficiency in patients with liver disease. We examined the liver specimens from 20 patients suffering from alcoholic cirrhosis with the Pi M phenotype, i.e., in whom alpha-1-antitrypsin deficiency was excluded. In seven of these patients, PAS-positive, diastase-resistant inclusions were seen in the hepatocytes; in two patients, these inclusions contained a material antigenically similar to alpha-1-antitrypsin. These inclusions might represent deposits of glycoproteins poorly excreted by the diseased hepatocytes. It is concluded that, in patients with liver disease, the presence of PAS-positive, diastase-resistant inclusions--even containing alpha-1-antitrypsin--in the cytoplasm of the hepatocytes does not permit the hepatic lesions to be ascribed to alpha-1-antitrypsin deficiency.

Mucin histochemistry of the columnar epithelium of the oesophagus (Barrett's oesophagus): a prospective biopsy study.

Columnar epithelium-lined oesophagus (CELO) is an acquired disorder associated with a high incidence of cancer. CELO consists of three histological types of epithelium: gastric-fundic, junctional, and specialised columnar, the last resembling intestinal metaplasia of the stomach. In a previous study of CELO an incompletely differentiated variant of intestinal metaplasia secreting sulphomucins (type II B) was found. This was shown to be associated with well differentiated adenocarcinoma, as in the stomach. The purpose of this paper has been to define by histochemistry the mucin profile of CELO in 17 patients and to compare it with the mucin profile of the gastroesophageal junction in 27 patients without CELO. In CELO a specialised columnar epithelium was always found and type II B intestinal metaplasia (with sulphomucins) showed the highest incidence (53%). In normal subjects, this type of intestinal metaplasia was found in only three of 27 cases. Type II B intestinal metaplasia has often been considered as a precancerous lesion or as an equivalent of dysplasia; consequently, its high incidence in our study on CELO raises the question of whether this lesion should be considered a high risk condition for adenocarcinoma of the lower oesophagus.

Expression of c-erbB-2 oncogene product in Barrett's adenocarcinoma: pathological and prognostic correlations.

AIMS: To establish the prevalence of c-erbB-2 protein expression in a surgical series of Barrett's adenocarcinomas; and to correlate this expression with clinicopathological data and prognosis. METHODS: Sixty six surgical specimens of Barrett's adenocarcinomas were included in this retrospective study. Blocks of the tumour and of non-dysplastic Barrett's mucosa were stained with a polyclonal antibody specific for the intracytoplasmic domain of the c-erbB-2 protein. RESULTS: Seven of 66 tumours showed membrane staining for the c-erbB-2 protein. The non-dysplastic Barrett's mucosa was negative in all cases. There was no difference between c-erbB-2 positive and negative tumours with regard to mean age, sex ratio, percentage of alcohol misusers, percentage of smokers, tumour differentiation, depth of invasion, lymph node response, and proliferative activity, assessed by the percentage of tumour cells positive with the MIB-1 antibody directed against the Ki-67 antigen. All c-erb B2 positive tumours were of Lauren's intestinal type compared with negative c-erbB-2 tumours. Patients with c-erbB-2 positive tumours had a significantly poorer prognosis than patients with negative tumours. CONCLUSIONS: The prevalence of Barrett's adenocarcinomas expressing c-erbB-2 found in this study (11%) was similar to that observed in published series of gastric adenocarcinomas. c-erbB-2 protein expression could be an important prognostic indicator in Barrett's adenocarcinoma.

Lymphoid stromal reaction in gastrointestinal lymphomas: immunohistochemical study of 14 cases.

The lymphoid stromal reaction, particularly the T lymphoid reaction, was studied immunohistochemically on cryostat sections in 14 cases of primary gastrointestinal B lymphomas, and compared with the type and distribution of lymphoid cells in three cases of gastric lymphoid hyperplasia. A pronounced T lymphoid reaction, mainly of the T helper phenotype, occurred in both lesions. Most of these T cells bore HLA-DR antigens, but only a few of them had the receptor for interleukin 2. The T lymphoid reaction was observed inside the lymphomas in seven of a total of 14 cases, and around the lymphomas in four of the six cases clinically classified as stage I. Perivascular mucosal and submucosal nodules, entirely composed of T cells, seemed characteristic of gastric lymphoid hyperplasias. A T lymphoid reaction in lymphoid hyperplasias suggests an amplification of the cell mediated immune response; in lymphomas it could represent a host reaction against the lymphomatous infiltrate, therefore favouring a better prognosis.

Helicobacter pylori infection in patients with Barrett's oesophagus: a prospective immunohistochemical study.

The prevalence of Helicobacter pylori infection in patients with Barrett's oesophagus was studied prospectively. A sensitive immunohistochemical staining of H pylori was performed in oesophageal and gastric biopsies of 73 patients from a surveillance group with this condition. H pylori was detected in 11 cases of Barrett's mucosa (15%) and in 26 gastric mucosa specimens (35.6%). All cases positive in Barrett's mucosa were also positive in the stomach. In Barrett's oesophagus, H pylori was never found on specialised epithelium. The percentage of Barrett's mucosa showing inflammatory changes was similar in specimens with and without H pylori, both for chronic (81% v 79%) and acute (9% v 10%) infiltrates. These results indicate that H pylori infection does not play an aetiological role in Barrett's oesophagus and that colonisation of the metaplastic mucosa by this bacteria is related with the presence of gastric type mucosa in the oesophagus and of H pylori infection in the stomach.

Ticlopidine induced colitis: a histopathological study including apoptosis.

AIMS: To describe ticlopidine related microscopic colitis and to assess the occurrence of apoptosis in the colon epithelium. METHODS: A series of colorectal biopsy samples from nine patients with ticlopidine related chronic diarrhoea were analysed. Biopsies were also taken from five of these patients between two and four months after ticlopidine withdrawal. The number of apoptotic cells in the crypts/mm2 (apoptotic index) was calculated using in situ labelling by terminal deoxyribonucleotidyl transferase (TdT) mediated dUTP-biotin nick end labelling (TUNEL). All specimens were matched to normal colorectal specimens from a control group of comparable age and sex distribution. RESULTS: Histological examination of the colon biopsy specimens taken from all nine patients with ticlopidine related chronic diarrhoea showed characteristic features of microscopic colitis. The histology returned to normal when ticlopidine was withdrawn. Apoptotic cells were rarely found in controls, and the mean apoptotic index was 0.53. The apoptotic index was significantly higher (16.53) in ticlopidine related colitis, but decreased dramatically to control value when ticlopidine was withdrawn. CONCLUSION: Microscopic colitis can be induced by ticlopidine and is accompanied by an increase in epithelial apoptosis. Hence, increased apoptosis might be related to drug injury or might be part of microscopic colitis.

Overexpression of p53 protein in Barrett's syndrome with malignant transformation.

AIMS: To study the overexpression of p53 protein in Barrett's oesophagus with adenocarcinoma, and to correlate this expression with the pathological features of Barrett's syndrome. METHODS: Immunohistochemical staining was performed on frozen sections with a monoclonal antibody directed against wild type and mutated p53 protein (Pab 1801). Eleven cases of Barrett's adenocarcinoma were studied, seven of which had extensive sampling of benign Barrett's mucosa. RESULTS: Eight of 11 adenocarcinomas overexpressed the p53 protein. Both early and advanced tumours were positive. In Barrett's mucosa around the p53 positive tumours, high grade dysplasia was positive; low grade dysplasia and non-dysplastic mucosa were negative. CONCLUSIONS: P53 gene mutation with ensuing p53 protein overexpression is a common feature of Barrett's adenocarcinoma, both at early and advanced stages. This mutation appears as a relatively late event during the neoplastic transformation of Barrett's oesophagus.

Improved detection of human papillomavirus infection in genital intraepithelial neoplasia in human immunodeficiency virus positive (HIV +) women by polymerase chain reaction-in situ hybridization.

The prevalence of genital human papillomavirus (HPV) infection was evaluated in 30 consecutive human immunodeficiency virus (HIV) + women by polymerase chain reaction (PCR)-in situ hybridization (ISH) on paraffin-embedded tissue sections and compared with that found with standard ISH. Biopsies were removed from normal or neoplastic areas in the cervix, vagina, and vulva, and ISH was performed with biotinylated or fluorescein isothiocyanate genomic DNA probes. One probe was used for HPV screening and others for HPV typing (types 6, 11, 16, 18, 31, and 33). Sequences were amplified by the "hot-start" PCR method and followed by standard ISH. Among the 30 HIV + women, 90% scored HPV + in one or several locations by PCR-ISH, whereas only 67% were positive by ISH. Oncogenic HPV types were found in 63% by PCR-ISH and in only 43% by ISH. The same HPV types detected by standard ISH were also recognized by PCR-ISH, but with the latter the signal was amplified. Moreover, some HPV types were found with PCR-ISH but not by ISH. We conclude that PCR-ISH is a valuable and sensitive method for specific detection of HPV.