LncRNA LUCAT1 as a novel prognostic biomarker for patients with papillary thyroid cancer.

In recent years, long non-coding RNAs have emerged as a novel class of regulators of cancer biological processes. While they are dysregulated in many cancer types, little is known about their expression and functional profiles. This study has been focused on the determination of the role of a specific lncRNA in papillary thyroid cancer. Quantitative reverse transcription PCR was performed to detect the expression levels of 84 lncRNAs in 61 papillary thyroid carcinoma tissues and their adjacent non-tumor tissues. The highest fold-change was obtained for lung cancer associated transcript 1 LUCAT1, and thus, this study determines the expression and biological implication of lncRNA LUCAT1 through different in vitro and ex vivo approaches in this tumor. LUCAT1 was specifically located at the cell nucleus in tumoral regions of patient tissues. Furthermore, LUCAT1 knockdown significantly reduced both cell proliferation and invasion ex vivo and induced cell-cycle arrest and apoptosis. These facts were corroborated by an enhanced expression of P21, P57, P53 and BAX, and a reduced expression of EZH2 and HDAC1. In addition, a significant decrease was observed on DNMT1 and NRF2 genes, helping to clarify the role of LUCAT1 on PTC. Our study reveals the involvement of LUCAT1 in PTC development, through acting in cell-cycle regulation, proliferation, epigenetic modifications through LUCAT1/ CDK1/ EZH2/ P57/ P21/ HDAC1/ DNMT1/ P53/ BAX axis and apoptosis, via extrinsic pathway activating caspases. These findings indicate that LUCAT1 is maybe a potential therapeutic target and molecular biomarker for PTC.

Epigenetics in ENS development and Hirschsprung disease.

Hirschsprung disease (HSCR, OMIM 142623) is a neurocristopathy caused by a failure of the enteric nervous system (ENS) progenitors derived from neural crest cells (NCCs), to migrate, proliferate, differentiate or survive to and within the gastrointestinal tract, resulting in aganglionosis in the distal colon. The formation of the ENS is a complex process, which is regulated by a large range of molecules and signalling pathways involving both the NCCs and the intestinal environment. This tightly regulated process needs correct regulation of the expression of ENS specific genes. Alterations in the expression of these genes can have dramatic consequences. Several mechanisms that control the expression of genes have been described, such as DNA modification (epigenetic mechanisms), regulation of transcription (transcription factor, enhancers, repressors and silencers), post-transcriptional regulation (3'UTR and miRNAs) and regulation of translation. In this review, we focus on the epigenetic DNA modifications that have been described so far in the context of the ENS development. Moreover we describe the changes that are found in relation to the onset of HSCR.

Genetic disorders of pediatric MEN2A patients in the south of Spain.

INTRODUCTION: Medullary thyroid carcinoma (MTC) is observed in nearly 100 % of patients with multiple endocrine neoplasia type 2A (MEN2A). The gene responsible for MEN2A is the RET proto-oncogene and about 95 % of MEN2A patients have germline mutations in five specific cysteine codons (609, 611, 618, 620 and 634). MATERIALS AND METHODS: A retrospective study of children from families with MEN2A in our geographic area was performed. Variables analyzed included demographic data, kinship relations, age at genetic screening, age at prophylactic thyroidectomy, genetic mutation subtype and histological findings. The genetic study consisted in direct molecular analysis by automatic sequencing of RET mutated exon in the studied family. RESULTS: We performed 13 prophylactic total thyroidectomies from 1997 to 2013, 8 females and 5 males. The mean age at genetic diagnosis was 3.8 years (range 2-5.9). All children belonged to four interconnected families living in the same geographic area and presenting C634Y mutation in all the cases. The mean age at prophylactic thyroidectomy was 5.6 years (range 4-8.5). Histopathological findings demonstrated seven cases of C-cells nodular hyperplasia, one lymphocytic thyroiditis, two without evidence of disease, two micro-carcinomas and one multicentric carcinoma. CONCLUSION: The mutation found in the RET proto-oncogene responsible for MEN2A in pediatric patients in the south of Spain is the C635Y. It is considered a high-risk mutation, associated with an earlier malignant transformation and development of MTC.

Metástasis cutáneas de origen visceral / Cutaneous Metastases of Internal Tumors

Las metástasis cutáneas son relativamente raras en la práctica clínica. Su diagnóstico requiere un alto índice de sospecha, pues los hallazgos clínicos pueden ser sutiles. Las metástasis cutáneas ponen de manifiesto la presencia de un tumor maligno diseminado y pueden permitir el diagnóstico de neoplasias internas no conocidas, o indicar la diseminación o recurrencia de otras ya diagnosticadas. Su reconocimiento temprano puede llevar a un diagnóstico preciso y rápido, con el consiguiente tratamiento oportuno, aunque en la mayoría de los casos son indicativas de un pronóstico infausto. Algunos tumores tienen predilección por metastatizar en áreas específicas. El reconocimiento de esos patrones es esencial para dirigir la búsqueda del tumor subyacente (AU)

Cutaneous metastases are relatively rare in clinical practice and their diagnosis requires a high index of suspicion because clinical findings can be subtle. These metastases reveal the presence of disseminated malignant disease and can lead to the diagnosis of unsuspected internal tumors or the spread or recurrence of an already diagnosed tumor. Early recognition of cutaneous metastases can facilitate prompt and accurate diagnosis resulting in early treatment; however, they are generally indicative of a poor prognosis. Some tumors have a predilection to metastasize to specific areas. Recognition of these patterns provides essential information that can guide the search for the underlying tumor (AU)

Mejora de la profilaxis antibiótica en cirugía colo-rectal mediante el análisis de las infecciones incisionales: estudio observacional / Improving antibiotic prophylaxis in colorectal surgery through the analysis of incisional infections: observational study

INTRODUCCIÓN. Las infecciones del sitio quirúrgico son muy frecuentes tras cirugía colo-rectal. El objetivo de este estudio es analizar cuantitativa y cualitativamente dichas infecciones en nuestro servicio. MATERIAL Y MÉTODOS. Se realizó un estudio observacional en 23 enfermos sometidos a resecciones colo-rectales programadas a los que se administró la profilaxis antibiótica habitual de nuestro servicio, gentamicina y metronidazol (G+M). Se analizó la presencia de infecciones incisionales superficiales o profundas y los gérmenes causantes. Ante los resultados, y guiados por los cultivos, se decidió cambiar la profilaxis por amoxicilina y ácido clavulánico (AMC), y continuar el estudio en 38 enfermos. RESULTADOS. Los enfermos que recibieron como profilaxis G+M tuvieron un índice de infecciones incisionales del 48%. En el 90% de esas infecciones había Escherichia coli, y en un 80% enterococos o estreptococos. En el grupo de AMC hubo un índice de infecciones incisionales del 19%, siendo la diferencia con el grupo de G+M estadísticamente significativa (p=0,021). En los cultivos de sus heridas no había enterococos ni estreptococos. DISCUSIÓN. El índice de infecciones incisionales del grupo G+M es superior al comunicado en cirugía colo-rectal programada. El predominio de Escherichia coli en estas infecciones es habitual, no así la elevada presencia de cocos positivos, especialmente enterococos. Estos resultados exigen un cambio en nuestra profilaxis antibiótica, para cubrir estreptococos y enterococos, además de bacilos negativos y anaerobios. AMC parece la opción más lógica. Nuestros resultados corroboran esta hipótesis (AU)

BACKGROUND. Surgical site infections are very common after colorectal surgery. The objective of this study is to analyze quantitatively and qualitatively such infections in our service. METHOD. An observational study was performed in 23 patients undergoing elective colorectal resections who received the usual antibiotic prophylaxis of our service, gentamicin and metronidazole (G+M). Superficial and deep incisional infections, as well as microbes that cause them, were analyzed. Given the results, and guided by the cultures, it was decided to change the prophylaxis to amoxicillin and clavulanic acid (AMC), and continue the study in 38 patients. RESULTS. Patients who were given G+M for prophylaxis had incisional infection rate of 48%. Escherichia coli was present in 90% of these infections, enterococci or streptococci were present in 80% of these infections. In the AMC group there was an incisional infection rate of 19%. The observed difference with the G+M group is statistically significant (p = 0.021). Enterococci and streptococci were not isolated in their incisions. CONCLUSION. The rate of incisional infections in the G+M group is higher than the usually reported in elective colorectal surgery. The predominance of Escherichia coli is usual in these infections, but not the high presence of positive cocci, especially enterococci. These results call for a change in our antibiotic prophylaxis to cover streptococci and enterococci, as well as gram-negative bacilli and anaerobes. AMC seems the most logical choice. Our results support this hypothesis (AU)

Cutaneous metastases of internal tumors.

Cutaneous metastases are relatively rare in clinical practice and their diagnosis requires a high index of suspicion because clinical findings can be subtle. These metastases reveal the presence of disseminated malignant disease and can lead to the diagnosis of unsuspected internal tumors or the spread or recurrence of an already diagnosed tumor. Early recognition of cutaneous metastases can facilitate prompt and accurate diagnosis resulting in early treatment; however, they are generally indicative of a poor prognosis. Some tumors have a predilection to metastasize to specific areas. Recognition of these patterns provides essential information that can guide the search for the underlying tumor.

Cyclin D1 interacts and collaborates with Ral GTPases enhancing cell detachment and motility.

Alterations in the levels of adhesion and motility of cells are critical events in the development of metastasis. Cyclin D1 (CycD1) is one of the most frequently amplified oncogenes in many types of cancers and it is also associated with the development of metastasis. Despite this, we still do not know which are all the relevant pathways by which CycD1 induces oncogenic processes. CycD1 functions can be either dependent or independent of the cyclin-dependent kinase Cdk4, and they affect several cellular aspects such as proliferation, cell attachment and migration. In this work, we reveal a novel function of CycD1 that fosters our understanding of the oncogenic potential of CycD1. We show that CycD1 binds to the small GTPases Ral A and B, which are involved, through exocyst regulation, in the progression of metastatic cancers, inducing anchorage-independent growth and cell survival of transformed cells. We show that CycD1 binds active Ral complexes and the exocyst protein Sec6, and co-localizes with Ral GTPases in trans-Golgi and exocyst-rich regions. We have also observed that CycD1-Cdk4 phosphorylates the Ral GEF Rgl2 'in vitro' and that CycD1-Cdk4 activity stimulates accumulation of the Ral GTP active forms. In accordance with this, our data suggest that CycD1-Cdk4 enhances cell detachment and motility in collaboration with Ral GTPases. This new function may help explain the contribution of CycD1 to tumor spreading.

Human umbilical cord stromal stem cell express CD10 and exert contractile properties.

BACKGROUND: It has been demonstrated that human umbilical cord stromal stem cells (UCSSCs) are bio-equivalent to bone marrow mesenchymal stem cells. However, little is known about their tissue origin or in vivo functions, and data on their expansion properties are limited due to early senescence in the culture methods described to date. METHODS: UC sections and cultured UCSSCs were analyzed with a panel of 12 antibodies. UCSSCs were grown in low-FCS containing medium at 5% or 21% oxygen and were assayed for their clonogenic properties, karyotype stability, expression of specific cellular markers, and multi-lineage potential. UCSSC contractile properties were evaluated by using collagen gel contraction assays under cytokine stimulus. RESULTS: Immunohistochemistry studies showed that the UCSSCs were derived from the Wharton's jelly and not from the vascular smooth muscle sheath of the blood vessels. UCSSC growth properties were increased in a 5% oxygen atmosphere in comparison to normoxic culture conditions. In both culture conditions, UCSSCs were CD14-, CD34-, and CD45-negative while expressing high levels of CD73, CD90 and CD105 and maintaining their differentiation potentialities. UCSSCs expressed alpha smooth muscle actin and behaved as functional myofibroblasts when cellular contraction was challenged with appropriate stimuli. CONCLUSIONS: UCSCs are mesenchymal stem cells that reside in the perivascular area of Wharton's jelly and are phenotypically and functionally related to myofibroblasts.

Contribution of RET, NTRK3 and EDN3 to the expression of Hirschsprung disease in a multiplex family.

BACKGROUND: Hirschsprung disease (HSCR) is a developmental disorder caused by a defect in the neural crest neuroblast migration process. It is considered to be a paradigm of complex disorders, with many loci contributing to manifestation of the disease. Although HSCR commonly appears as a sporadic trait, approximately 20% of HSCR cases are familial, with complex patterns of inheritance. METHOD: A multiplex HSCR family with an additive model of inheritance, in which the contribution of three genes (RET, NTRK3, EDN3) leads to the HSCR phenotype is reported. RESULTS AND DISCUSSION: The findings suggest that both RET and NTRK3 mutations acting together are necessary and sufficient for the appearance of the disease, and that the EDN3 mutation is acting as a phenotype-modifier factor in the context of this family, as two different HSCR phenotypes are seen among the affected members: a short segment form, and a total colonic aganglionosis. The results therefore support the complex additive model of inheritance previously proposed for Hirschsprung disease.

A novel point variant in NTRK3, R645C, suggests a role of this gene in the pathogenesis of Hirschsprung disease.

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells in the myenteric and submucosal plexuses due to a defect in the migration process of neural crest neuroblasts. Manifestation of the disease has been linked to the dysfunction of two principal signalling pathways involved in the enteric nervous system (ENS) formation: the RET-GDNF and the EDN3-EDNRB receptor systems. However, the NTF3/NTRK3 signalling pathway plays an essential role in the development of the ENS suggesting a potential role for those genes in the pathogenesis of HSCR. We have sought to evaluate the candidature of the NTRK3 gene, which encodes the TrkC receptor, as a susceptibility gene for Hirschsprung disease. Using dHPLC technology we have screened the NTRK3 coding region in 143 Spanish HSCR patients. A total of four previously described polymorphisms and 12 novel sequence variants were detected. Of note, the novel R645C mutation was detected in 2 affected siblings of a HSCR family also carrying a RET splicing mutation. Using bioinformatics tools we observed that the presence of an additional cysteine residue might implicate structural alterations in the mutated protein. We propose haploinsufficiency as the most probable mechanism for the NTRK3 R645C mutation. NTRK3 and RET mutations in this family only appear together in the HSCR patients, suggesting that they per se are necessary but not sufficient to produce the phenotype. In addition, it is quite probable that the contribution of other still unidentified modifier genes, may be responsible for the different phenotypes (length of aganglionosis) in the two affected members.

Allergic contact hobby dermatitis from turpentine.

INTRODUCTION: Turpentine is an oleoresin obtained from various species of pine. It contains a volatile oil (oil of turpentine) which is responsible for its properties and this is the form generally used. Opportunity for contact with turpentine is widespread. It is universally used as a solvent to dissolve and thin lacquers, varnishes and paints. It is also an ingredient in many liniments and cold remedies. Turpentine is regarded as both a local irritant and a sensitizer. Cases of allergic contact dermatitis in painters, mechanics, shoe repairers and home decorators have been reported. CASE REPORT: We report a case of a non-professional painter who developed a contact allergic dermatitis due to his exposure to turpentine while doing oil-painting as a hobby. DISCUSSION: Dermatitis is one of the biggest dangers of working with art materials and occupational contact dermatitis is often detected on the hands of the painters. Solvents are indispensable and turpentine is the most important and the traditional one used in oil-painting. Contact allergy to oil of turpentine was reported to have become rare in Europe but over the last few years, increased rates of turpentine sensitization have been reported.

Polymorphisms in the promoter regions of FAS and FASL genes as candidate genetic factors conferring susceptibility to endometriosis.

Although the pathophysiological mechanisms leading to endometriosis remain unknown, several hypothesis have been proposed, including a dysregulation of the normal apoptotic process which takes place in the endometrium. One of the apoptotic pathways playing a crucial role in the programmed cell death within the endometrium is the Fas-FasL system. In this study we have performed a case-control analysis in order to evaluate three polymorphisms located within FAS (-1377G>A and -670A>G) and FASL (-843C>T) genes, as susceptibility factors for endometriosis. We have analysed a series of women with endometriosis compared respectively to a group of women without symptoms of the disease, and to a group of confirmed unaffected women. The genotyping of the three variants was carried out by Fluorescence Resonance Energy Transfer (FRET) technology, and statistical analysis was performed using chi2 test with Yates correction. Our results show that the differences in the distribution of the polymorphic variants were not statistically significant when the group of patients was compared to the other groups. Thus, it seems to indicate that the variants here analysed are not involved in the pathogenesis of the disease in our population. However this does not let us to completely exclude such genes as potential candidates for the disease. A complete genetic analysis of the genes involved in the intricate regulatory system of the apoptosis may lead to the identification of susceptibility factors for the disease and a better understanding of its etiology.

Ancestral RET haplotype associated with Hirschsprung's disease shows linkage disequilibrium breakpoint at -1249.

BACKGROUND: Hirschsprung disease (HSCR) is a complex disorder with traditional germline mutations in RET in up to 30% of familial cases and in 3% of sporadic cases in a population-based series. We have previously demonstrated that an ancestral haplotype at the 5' end of RET (haplotype 0) was strongly associated with a large subset of isolated HSCR cases and that a putative low penetrance susceptibility locus was encompassed within this ancestral haplotype, anchored by exon 2 SNP A45A. OBJECTIVE: To determine the 5' extent of the HSCR-associated ancestral haplotype by defining the linkage disequilibrium breakpoint in search for the low penetrance susceptibility locus. METHODS: Systematic screening of the region upstream of the anchoring A45A SNP, comprising RET intron 1, exon 1, and promoter in 117 population-based HSCR cases and 100 controls. Dual luciferase assay to determine differential activities between SNP combinations near a transcription start site. RESULTS: New SNP's were found which formed upstream haplotypes, anchored by A45A, in linkage disequilibrium with HSCR (2 = 76.96, p<0.00000001). Linkage disequilibrium appeared to break at the -1249C/T SNP. Further, the HSCR-associated genotype (00) was found in >60% of HSCR but only 2% of controls. Because only 2 variants, -200A>G and -196C>A, lie within the promoter region and are in proximity to the transcriptional start site (at -195), we modelled these combinations into constructs for luciferase reporter assay. The HSCR-associated SNP combination showed the lowest activity and the control-associated combination, the highest. CONCLUSIONS: Our observations seem to discard the existence of a HSCR-causing mutation as it is conceived in the traditional sense, but strengthen the idea of a specific combination of variants conferring susceptibility to the disease in a low penetrance fashion. The data derived from our functional "in vitro" studies would suggest that the HSCR-associated haplotype 0 may result in a lower level of expression of the RET gene [corrected]

[The elderly at risk in the province of Guadalajara]. / El anciano de riesgo en la provincia de Guadalajara.

OBJECTIVE: To study fragile elderly people in the population, their characteristics and their distribution according to fragility markers. DESIGN: Cross-sectional, descriptive study using a survey. SETTING: Primary care. Elderly people living in Guadalajara in January 2002. PARTICIPANTS: 434 people were interviewed (1.24% of population). There were 157 losses (26.6%). INTERVENTIONS: The questionnaire contained social and demographic variables (age, sex, setting), care variables (medication, pathologies, home care) and evaluative scales (Barthel, Folstein, Yesavage, Diaz-Palacios). MAIN MEASUREMENTS: Fragility markers: 3 or more pathologies, 6 or more drugs, dementia, and/or positive on one of the four scales used. RESULTS: Subjects lived in rural areas more (56.7%; CI, 52-61.3), but there were not more women. Fragile elderly, n=257 (59.2%; CI, 54.6-63.8), were more women (OR=1.8; CI, 1.5-2.2) and over 70 (OR=80-84, 5.2; CI, 3.7-7.5; OR=85-89, 8.2; CI, 5.3-12.8). Prevalence of markers was: 3 or more pathologies, 30.2% (95% CI, 25.8-34.5); cognitive deterioration, 22.6% (95% CI, 18.7-26.5); social risk, 20.8% (95% CI, 16.3-23.8); multi-medication, 18.7% (95% CI, 15-22.3); Barthel incapacity, 11.7 (95% CI, 8.7-14.8); depression, 5.3% (95% CI, 3.2-7.5); and dementia, 3.2% (95% CI, 1.6-4.9). Women suffered cognitive deterioration more. The over-80s suffered cognitive deterioration, social risk and incapacity more. CONCLUSIONS: There was high prevalence of fragility with cognitive deterioration, multi-medication and social risk; and lower presence of dementia. Women and the most elderly people were most affected.

Analysis of the involvement of CCR5-Delta32 and CCR2-V64I variants in the development of endometriosis.

Several arguments support the proposal that the cytokine network plays a critical role in the aetiology of endometriosis. Among various chemokines, regulated-on-activation, normal-T-cell-expressed and -secreted (RANTES) and monocyte chemotactic protein 1 (MCP-1) concentrations have been shown to be increased in the peritoneal fluid of women with endometriosis. Some studies have demonstrated that, in the context of endometriosis, these chemokines are involved in apoptosis, angiogenesis and/or chemotaxis. Since the chemokines exert their effects by binding to their receptors, it would be plausible that factors affecting such interactions might play a role in the pathogenesis of endometriosis. Thus we postulated that the genes encoding CCR5 and CCR2, which are the receptors for RANTES and MCP-1 respectively, could be good candidate genes for the disease. We have used real-time PCR and FRET technologies to genotype and evaluate the variants CCR5-Delta32 and CCR2-V64I, as susceptibility factors in a cohort of Spanish women with endometriosis. No differences have been found in the frequencies of the two polymorphisms nor in the haplotype/genotype distribution between cases and controls. These data would suggest the lack of association between these polymorphisms and endometriosis in our population, although they do not permit us to discard completely a possible role of other variants within CCR5 and CCR2 genes in this pathology.

Evaluation of germline sequence variants within the promoter region of RANTES gene in a cohort of women with endometriosis from Spain.

The RANTES (regulated upon activation normal T cells expressed and secreted) chemokine, is known to be expressed in endometriotic lesions in a concentration correlating with the severity of endometriosis. Since it has been widely demonstrated that endometriosis has a genetic basis, we postulated that the gene encoding RANTES could be a good candidate gene for the disease. We have used fluorescence resonance energy transfer (FRET) technology to genotype and evaluate the role of the variants -403G-->A and -28C-->G, located within the promoter region of the gene, as susceptibility factors in a cohort of Spanish women with endometriosis. No differences have been found in the allelic frequencies of both variants nor in the haplotype/ genotype distribution between patients and controls. These data are consistent with the lack of association between these polymorphisms and endometriosis in our population. They do not exclude completely a possible role of other variants within RANTES gene in this pathology.

Germline sequence variant S836S in the RET proto-oncogene is associated with low level predisposition to sporadic medullary thyroid carcinoma in the Spanish population.

OBJECTIVE: The molecular basis of sporadic medullary thyroid carcinoma (MTC) remains elusive. While germline gain-of-function mutations in the RET proto-oncogene cause hereditary MTC, somatic activating RET mutations and loss of heterozygosity of markers in various chromosomal regions representing deletions of tumour suppressor genes, have been described in a variable number of sporadic MTC. A previous report suggested that the presence of a germline variant at RET codon 836 (S836S) was associated with the development of sporadic MTC and, furthermore, that the presence of S836S was highly correlated with somatic RET M918T mutation in the MTC. Thus, we sought to determine if the S836S variant would be associated with sporadic MTC from a completely different population base, that of Andalucia. DESIGN: This is a case-control study to determine whether the presence of RET germline S836S is correlated with sporadic MTC in Andalucia. PATIENTS: Thirty-two patients with sporadic MTC from the Andalucia region of Spain, serviced by our University Hospital, were ascertained throughout the period 1995-99. Sporadic MTC was defined as a lack of personal or family history suggestive of multiple endocrine neoplasia type 2 (MEN 2) and lack of germline RET mutations which define any MEN 2 subtype. A region and race matched cohort of 250 controls was also obtained. MEASUREMENTS: The frequency of the S836S allele was determined in cases and controls and compared using the standard chi-squared statistic and Fisher's exact test. RESULTS: The polymorphic allele frequency at codon 836 in the control population (18/500 chromosomes, 3.6%) differed significantly from the MTC case cohort, 9.3% of case chromosomes (six of 64 alleles, Fisher's exact test, two-tailed, P = 0.043). CONCLUSIONS: Germline RET S836S variant is associated with a two- to three-fold risk of sporadic MTC in the Spanish population, in accordance with a previous study based on German cases. Our observations suggest that this phenomenon might be universal and not limited to Germany.