Three novel human sporadic melanoma cell lines: signaling pathways controlled by MC1R, BRAF and ß-catenins.

We studied the behaviour of three novel human sporadic melanoma cell lines (hmel1, hmel9, hmel11) extracted from tumors with different degrees of malignancy, concerning the cell signalling pathways controlled by MC1R, BRAF, NRAS and ß-catenins. The novel cell lines were compared to metastatic cell lines (HBL, LND1), wild type (wt) for MC1R and BRAF genes, that have been extensively characterised and were used as control. All the novel cell lines have silent or no MC1R mutations even though MC1R signalling is severely impaired. Conversely, they harbour BRAF mutations at the V600 residue. These mutations determine a constitutive ERK phosphorylation in all the three cell lines. Our new melanoma cell lines were BRAF mutated in hetero- and homozygosis, even with a wild type MC1R, and unresponsive to NDP-MSH treatment. Quantity and subcellular localization of ß-catenin were analyzed in both novel and control cell lines. In HBL and LND1 there were high levels of beta-catenin distributed in the cytoplasm/nucleus, while in the novel melanoma cell lines ß-catenins were less abundant and seemed to be located at the plasma membrane/cytoplasm and absent in the nucleus. We sequenced beta-catenin cDNA for all the melanoma cell lines, and found mutations in HBL, LND1 and hmel1, while hmel9 and hmel11 were wt. We found that beta-catenin levels were not influenced by the RAS/RAF/MAPK pathway because inhibition with PD98059 (a MEK inhibitor) did not produce any effect on beta-catenin stability and/or localization.

Molecular characterization of novel melanoma cell lines.

We isolated two novel cell lines from different types of sporadic human malignant melanoma: the hmel1 line was obtained from a melanoma skin metastasis and the hmel9 cell line from a primary superficial spreading melanoma. The karyotype and pigmentation parameters were assessed in these cell lines. Cytogenetic analysis in early stages of culture revealed that both cell lines had chromosome instability and simultaneous growth of heteroploid subpopulations. The molecular analysis of some genes involved in melanoma showed that both cell lines harbor BRAF mutations. The unpigmented hmel1 and the pigmented hmel9 lines were found to express the tyrosinase gene. The tyrosine hydroxylase activity was detectable only in hmel9 cells and practically absent in the hmel1 cell line. This activity was found to be correlated with the relative tyrosinase protein amount in both melanoma cell lines. The biological behaviour in the two melanoma cell lines, derived from two different types of melanoma lesions displaying distinct clinical and histopathological features, confirms the heterogeneous characteristics of sporadic melanoma. Similarities and/or differences between cell lines extracted from different melanoma cases could be useful in the future for diagnostic, prognostic and therapeutic purposes.

Normal values in ambulatory oesophageal pH monitoring at two levels in Spain.

AIM: Upper oesophageal pH monitoring may play a significant role in the study of extra-oesophageal GERD, but limited normal data are available to date. Our aim was to develop a large series of normal values of proximal oesophageal acidification. METHODS: 155 healthy volunteers (74 male) participated in a multi-centre national study including oesophageal manometry and 24 hours oesophageal pH monitoring using two electrodes individually located 5 cm above the LOS and 3 cm below the UOS. RESULTS: 130 participants with normal manometry completed all the study. Twelve of them were excluded for inadequate pH tests. Twenty-seven subjects had abnormal conventional pH. The remaining 91 subjects (37 M; 18-72 yrs age range) formed the reference group for normality. At the level of the upper oesophagus, the 95th percentile of the total number of reflux events was 30, after eliminating the meal periods 22, and after eliminating also the pseudo-reflux events 18. Duration of the longest episodes was 5, 4 and 4 min, respectively (3.5 min in upright and 0.5 min in supine). The upper limit for the percentage of acid exposure time was 1.35, 1.05 and 0.95%, respectively. No reflux events were recorded in the upper oesophagus in 8 cases. CONCLUSION: This is the largest series of normal values of proximal oesophageal reflux that confirm the existence of acid reflux at that level in healthy subjects, in small quantity and unrelated to age or gender. Our data support the convenience of excluding pseudo-reflux events and meal periods from analysis.

[Recurrent adenomatous polyp of the bladder with prostatic type epithelium]. / Pólipo adenomatoso vesical recidivante con epitelio de tipo prostático.

We present a fresh case of adenomatous polyp with prostatic type epithelium located in the vesical neck, with urethral orientation, in a 59-year-old male. The interest lies in that it is a relatively uncommon lesion, which generally starts off with hematuria, thus causing vesical carcinoma to be suspected, and which in our case displayed recurring capacity despite confirmation of its benign characteristics.

[Renal dysplasia and vesico-ureteral reflux]. / Displasia renal y reflujo vesicoureteral.

We studied two groups of patients in order to evaluate the frequency of association between renal dysplasia and vesicoureteral reflux. The first was composed of 19 patients with dysplastic kidneys, 15 of whom were studied by means of micturition cystourethrography. The second group was made up of 86 patients with 124 kidneys with reflux. In 16 of these we were able to obtain a histopathological diagnosis of the renal lesions (12 nephrectomies, 4 biopsies). In three cases the renal dysplasia was associated with a vesicoureteral reflux, which represents an incidence of reflux in 20% of the patients with renal dysplasia, and an incidence of dysplasia in 19% of the kidneys with reflux examined histopathologically. We contrast these results with the literature, discussing the pathogenic hypotheses that enable us to explain this association as well as the clinical consequences thereof.

Morphometric and immunohistochemical characterization of bladder carcinoma in situ and its preneoplastic lesions.

We performed a morphometric and immunohistochemical study of 26 bladder carcinoma in situ (Cis) specimens, compared with normal urothelium and urothelial preneoplastic lesions. The following morphometric parameters were evaluated: nuclear area, nuclear perimeter and maximum nuclear diameter. For the immunohistochemical study we used five lectins, antibodies against four epithelial differentiation antigens, and antibodies against blood group isoantigens. A progressive increase in nuclear size from normal urothelium to dysplastic urothelium and Cis was detected. Nuclear size values in Cis and in high-grade, high-stage bladder carcinomas were similar. The most relevant immunohistochemical results were obtained with CEA, CK, UEA-1 and ABH isoantigens, which show significant immunoreactivity changes in preneoplastic urothelium and Cis when compared with normal urothelium. We conclude that bladder Cis behaves morphometrically and immunohistochemically as an invasive bladder carcinoma, and we emphasize the usefulness of these techniques for detecting flat dysplastic and neoplastic lesions in random bladder biopsies.

Presence of a protective allele for achalasia on the central region of the major histocompatibility complex.

Idiopathic achalasia is a motility disorder of the esophagus whose etiology is unknown. An association between HLA genes and susceptibility to achalasia which suggests a possible immunogenetic mechanism has been reported recently. This study was designed to examine the HLA class II association in a large group of achalasia patients further and to investigate the distribution of TNFa and TNFb microsatellites in these patients. The study population, all Spanish, white and unrelated, consisted of 115 consecutive patients and 339 healthy controls. All of the patients had been diagnosed with primary achalasia of the esophagus with manometric, radiographic and endoscopic studies. All studies were performed on DNA samples after locus-specific amplification with the polymerase chain reaction: HLA-DRB1, DQA1 and DQB1 were typed by dot-blot hybridization and the size of the TNFa and TNFb microsatellites was measured using a semiautomatic method. The broad allele HLA-DQ1 was seen to be weakly associated with achalasia. The TNFa11 allele and the DRB1*1501-DQA1*0102-DQB1*0602 haplotype were reduced in achalasia patients but the stratified analyses showed that this was true only when both were present in the same individual. These results confirm the association between achalasia and HLA-DQ1 allele and suggest that TNFa11 is a marker for a protective allele for the disease, present on the B7-DRB1*1501 (7.1) ancestral haplotype in our population.

Behavior of epithelial differentiation antigens (carcinoembryonic antigen, epithelial membrane antigen, keratin and cytokeratin) in transitional cell carcinomas of the bladder.

Results of an immunohistochemical study in normal urothelium and transitional cell carcinomas of the bladder are presented. Paraffin-embedded material was confronted with immunoantisera against carcinoembryonic antigen (CEA), keratin (K), cytokeratin (CK) and epithelial membrane antigen (EMA). Immunohistochemical findings confirm the changes in reactivity of dysplastic urothelium and carcinoma in situ for CEA, CK and EMA, in comparison with normal urothelium. Statistically significant differences were also found, depending upon tumor stage, in staining of transitional cell carcinomas for K and CK. Expression of CK correlated with the tumor differentiation grade: normal urothelium and well-differentiated carcinomas showed a specific pattern of immunostaining for the basal cells, this pattern being lost in poorly differentiated carcinomas.

DNA-ploidy, morphometric-stereological and P-glycoprotein study of superficial bladder carcinomas.

We carried out a DNA-ploidy, morphometric-stereologic and P-glycoprotein study on 40 newly diagnosed superficial bladder cancer patients (G1-G2), correlating the results with histological grade and clinical outcome. Variations in the number of patients who present recurrences, progression or remain tumor-free during the whole follow-up period (at least 5 years) were not significant when related to nuclear size, proliferative diploid index, presence of aneuploidy and expression of P-glycoprotein. It is striking how the majority of disease-free subjects showed a proliferative diploid index higher than 10%. Moreover, 3 of them presented an aneuploid cell population. In our study, only histological grade showed a significant discriminatory level in terms of progression versus no progression in patients with superficial bladder cancer.

Anisakis simplex: una causa de pseudobstrucción intestinal / Anisakis simplex: a cause of intestinal pseudo-obstruction

OBJETIVOS: la ingesta de la larva viva del parásito Anisakis simplex puede provocar la aparición de síntomas gastrointestinales. Siendo España el segundo país consumidor de pescado por habitante, es destacable el bajo número de casos de Anisakiasis gastrointestinal recogidos en la bibliografía, más aún cuando se consume frecuentemente crudo o poco cocinado, criterios que posibilitan dicha parasitación. Pretendemos demostrar que se observarían una mayor incidencia de casos si se realizasen técnicas complementarias cuando exista sospecha clínica. DISEÑO EXPERIMENTAL: se han estudiado seis pacientes diagnosticados de pseudobstrucción intestinal e ingesta de pescado previamente. Se realizaron pruebas cutáneas en prick con extractos de inhalantes, pescados y Anisakis simplex, determinación de IgE total, IgE específica frente a Anisakis mediante CAP radioinmunoensayo e inmunoblot y provocación oral controlada con larva de Anisakis previamente congelada. RESULTADOS: presentaron cifras elevadas de IgE total e IgE específica frente a Anísakís y Prick-test positivo con extracto del parásito. No se objetivó un patrón homogéneo en el inmunoblotting, pero todos reconocían antígenos de bajo peso molecular (14-18 Kd) y de mediano peso molecular (30-50). Los pacientes, sometidos a provocación, no refirieron sintomatología gastrointestinal o sistémica. CONCLUSIONES: presentamos seis pacientes en los que los hallazgos clínicos y de laboratorio orientan a considerar la parasitación por la larva de Anisakis simplex como factor etiológico del cuadro abdominal. Sería conveniente protocolizar el estudio complementario cuando exista sospecha clínica de anisakiasis. La ingesta de larva muerta no produce afectación clínica como así lo manifiesta la tolerancia al test de provocación (AU)