Clinical and epidemiological features of patients with COVID-19 reinfection: a systematic review.

Recurrent positivity in a patient with COVID-19 may be due to various reasons, not necessarily reinfection. There is concern about the occurrence frequency of reinfection. Five databases and a preprint/preprint repository were searched. All case reports, case series, and observational studies were included. Bias was assessed for each study with the Newcastle-Ottawa Scale tool and reported according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA-2020). After eligibility, 77 studies were included for qualitative synthesis (52 case reports, 21 case series, and four case-controls; 1131 patients included). Of these, 16 studies described a second contact with the SARS-CoV-2 positive case, five studies described healthcare profession-related infection, ten studies described that the source of reinfection was likely to be from the community, one study described travel-related infection, nine studies described vulnerability-related infection due to comorbidity. The mean number of days from discharge or negative test to reinfection ranged from 23.3 to 57.6 days across the different included studies. The risk of bias for all case report/series studies was moderate/high. For observational studies, the risk of bias was low. Reinfection of patients with COVID-19 occurs between the first and second month after the first infection, but beyond, and 90 days have been proposed as a point to begin to consider it. The main factor for reinfection is contact with COVID-19 positive cases.

Better early outcome with enteral rather than parenteral nutrition in children undergoing MAC allo-SCT.

There is no consensus on the type of nutritional support to introduce in children undergoing allogeneic stem cell transplantation (allo-SCT) after myeloablative conditioning (MAC). This retrospective, multicenter, observational study compared the early administration of enteral nutrition (EN group, n = 97) versus parenteral nutrition (PN group, n = 97) in such patients with matching for important covariates. The primary endpoint was the study of day 100 overall mortality. The early outcome at day 100 was better in EN group regarding mortality rate (1% vs. 13%; p = 0.0127), non relapse mortality (1% vs. 7%; p = 0.066), acute GVHD grades II-IV (37% vs. 54%; p = 0.0127), III-IV (18% vs. 34%; p = 0.0333) and its gut localization (16% vs. 32%; p = 0.0136). Platelet engraftment was better in EN group than in PN group for the threshold of 20 G/L (97% vs. 80% p < 0.0001) and 50 G/L (92% vs. 78%, p < 0.0001). The length of stay was shorter in EN group (28 vs. 52 days, p < 0.0001). There were no differences between the two groups regarding the polynuclear neutrophil engraftment, infection rate or mucositis occurrence. These results suggest that, in children undergoing MAC allo-SCT, PN should be reserved to the only cases when up-front EN is insufficient or impossible to perform.

Lesiones etmoidales con extensión secundaria: estrategia quirúrgica y complicaciones / Ethmoidal lesions with secondary extension: surgical strategies and complications

Introduction: The removal of ethmoidal tumors with secondary extension to the cranial base and/or facial region involves a high complexity and it is associated to a high morbility. Objective: To determine the results of craniofacial surgery in patient with ethmoid extended tumors. Methods: It was carried out a traverse retrospective descriptive study. The sample was conformed by the patients intervened surgically of anterior cranial base lesions by means of a combined craneofacial surgery during the period: January of the 2009 to January of the 2012 in the National Institute of Oncology and Radiobiology with a 2 year pursuit. Descriptive statistical variables were used. Results: 20 patients were intervened. The age average was of 44,8 years. It prevail the masculine sex (65 percent). Nasal obstruction constitutes the most frequent presentation. Adenocarcinoma and epidermoid carcinoma constituted the most frequent malignant lesions. Inside the benign lesions prevail the invertedpapiloma. The techniques more employees were the bilateral frontal craneotomy and total etmoidectomy. The most frequent complication was the cerebrospinal fluid leak. The 2 years overall survival in patients with malignant lesions was 35 percent. Conclusions: A high number of complications was identified but they didn’t affect the survival neither the quality of life. An acceptable rate of survival was achieved in malignant lesions.

Introducción: La remoción de lesiones etmoidales con extensión secundaria a la base cranealy/o región facial, entraña una elevada complejidad y se encuentra asociada a una elevada morbilidad. Objetivo: Determinar los resultados de la cirugía combinada craneofacial en pacientes con neoplasias etmoidales extendidas. Métodos: Se realizó un estudio descriptivo retrospectivo transversal. La muestra estuvo conformada por los pacientes intervenidos quirúrgicamente de lesiones de base craneal anterior mediante un abordaje combinado craneofacial durante el período: enero de 2009 a enero de 2012 en el Instituto Nacional de Oncología y Radiobiología con un seguimiento de 2 años. Se emplearon variables estadísticas descriptivas. Resultados: Fueron intervenidos 20 pacientes. El promedio de edad fue de 44,8 años. Predominó el sexo masculino (65 por ciento). La obstrucción nasal constituyó la presentación más frecuente. El adenocarcinoma y el carcinoma epidermoide constituyeron las lesiones malignas más frecuentes. Dentro de las lesiones benignas predominó el papiloma invertido. Las técnicas más empleadas fueron la craneotomía frontal bilateral y la etmoidectomía total. La complicación más frecuente fue la fístula de líquido cefalorraquídeo. La supervivencia global a los 2 años en los pacientes con lesiones malignas fue del 35 por ciento. Conclusiones: Se identificó un elevado número de complicaciones pero no afectaron la supervivencia ni la calidad de vida. Se logró una aceptable tasa de supervivencia en lesiones malignas.

[Superior vena cava thrombosis in patients with mediastinal large B-cell lymphoma: two pediatric cases]. / Thrombose de la veine cave supérieure et lymphome B à grandes cellules du médiastin: 2 cas pédiatriques.

We report two pediatric cases of superior vena cava thrombosis (VTE) in patients treated for primary mediastinal large B-cell lymphoma (PMBCL). PMBCL is a rare entity in children and adolescents and no thrombosis has been described in this population. Thrombosis in lymphoma is frequently asymptomatic, detected as an incidental finding in the first months following diagnosis. The thrombosis mechanisms are often multifactorial based on veinous compression by the mass, elevated risk of thrombosis in neoplasia, and/or presence of a central catheter. The risk factors of venous thromboembolism (VTE) in lymphoma are high-grade lymphoma, comorbidities, central nervous system lymphoma, and mediastinal mass. Because thrombosis has an impact on prognosis and treatment, it seems important to improve knowledge in order to improve the diagnosis and prevention of thrombosis in lymphoma.

[Cutaneous polymorph manifestations of familial Mediterranean fever in a child]. / Atteinte cutanée polymorphe de la fièvre méditerranéenne familiale chez un enfant.

We describe the case of a 4-year-old child with Mediterranean fever characterized by cutaneous features. Familial Mediterranean fever is an autosomal recessive disorder characterized by recurrent attacks of fever and polyserositis including peritonitis, pleuritis, and arthritis. Skin involvement is less common. In our case, the successively patient presented erysipelas-like erythema, edemas of the palmar and plantar regions, and purpuric lesions. From these clinical observations, several diagnoses were raised: infectious erysipelas, Kawasaki disease, Henoch-Schönlein purpura, and familial Mediterranean fever. Only the latter diagnosis was confirmed after exploration and then confirmed with genetic analysis, which found a M694V homozygous mutation. Erysipelas-like erythema is the most frequent cutaneous sign reported in the literature and the only one to be associated with the M694V homozygous mutation. The originality of this case is the dominancy and polymorphism of the skin lesions.

Patient characteristics and symptoms associated with perceived coercion during hospital treatment.

OBJECTIVE: Large numbers of psychiatric patients either are involuntarily admitted to hospital treatment or feel coerced despite a legally voluntary admission. For ethical and clinical reasons, their perceived coercion should be reduced as far as possible. There is however limited evidence on patient characteristics associated with perceived coercion during hospital treatment. This study aimed to identify i) sociodemographic and clinical characteristics associated with perceived coercion at admission and ii) changes in symptoms and global functioning associated with changes in perceived coercion over time. METHOD: Three thousand and ninety three in-patients who were involuntarily admitted or felt coerced to hospital treatment despite a legally voluntary admission were recruited in the European evaluation of coercion in psychiatry and harmonization of best clinical practice - EUNOMIA project in 11 European countries. Perceived coercion, global functioning and symptoms were assessed after admission and at a 3-month follow-up. RESULTS: Involuntary admission, female gender, poorer global functioning and more positive symptoms were associated with higher levels of perceived coercion at admission. Perceived coercion significantly decreased over time, and the improvements in global functioning and positive symptoms were associated with reduction in perceived coercion. CONCLUSION: Female patients perceive more coercion in psychiatric hospital treatment. Effective treatment for positive symptoms and improving patients' global functioning may lead to a reduction in perceived coercion.

How to improve clinical practice on involuntary hospital admissions of psychiatric patients: suggestions from the EUNOMIA study.

Number and procedures of involuntary hospital admissions vary in Europe according to the different socio-cultural contexts. The European Commission has funded the EUNOMIA study in 12 European countries in order to develop European recommendations for good clinical practice in involuntary hospital admissions. The recommendations have been developed with the direct and active involvement of national leaders and key professionals, who worked out national recommendations, subsequently summarized into a European document, through the use of specific categories. The need for standardizing the involuntary hospital admission has been highlighted by all centers. In the final recommendations, it has been stressed the need to: providing information to patients about the reasons for hospitalization and its presumable duration; protecting patients' rights during hospitalization; encouraging the involvement of family members; improving the communication between community and hospital teams; organizing meetings, seminars and focus-groups with users; developing training courses for involved professionals on the management of aggressive behaviors, clinical aspects of major mental disorders, the legal and administrative aspects of involuntary hospital admissions, on communication skills. The results showed the huge variation of involuntary hospital admissions in Europe and the importance of developing guidelines on this procedure.

Photodynamic inactivation of microorganisms as an innovative approach to kill mucocutaneous and skin microorganisms.

In 2002, the first documented case of a vancomycin-resistant S. aureus strain (MIC>or=32 microg/mL) was reported. Nowadays approximately 20% of S. aureus isolates in Europe are reported as methicillin-resistant. Besides bacteria infections, the emergence of fungal infections has increased considerably due factors such as immunosuppressive medications, broad-spectrum antibiotics, neutropenia and HIV infections. These tremendous effects underline the importance and the urgency to develop new alternative treatment approaches that are effective against infections caused by multi-resistant pathogens. Photodynamic inactivation of microorganisms (PDIM) is considered as a new approach, which utilizes a photoactive dye, oxygen and visible light to generate reactive oxygen species, which damage irreversible the pathogens during illumination. Cutaneous diseases caused by methicillin-resistant S. aureus or by fungal species are ideally suited to the treatment by PDIM for eradicating localized infections and for modulating wound healing due to the ability to deliver photosensitizer and light with topical application. The challenge of PDIM is to find a therapeutic window in vivo where multi-resistant microorganisms can be killed efficiently, thereby not harming the surrounding tissue or disturbing the residual bacteria-flora of the tissue. Different chemical classes of photosensitizers have demonstrate their potential to photoinactive Gram(+), Gram(-) and fungal cells. This review will focus on general photobiological and photochemical aspects of microbial inactivation by the photodynamic effect as well as to summarize the current knowledge about the possible application modalities of PDIM on localized infectious diseases in dermatology.

XF drugs: A new family of antibacterials.

The emerging increase of antibiotic resistance constitutes an important risk to human health. Two million patients acquire nosocomial infections in U.S. hospitals each year. Of these infections, 60% involve resistant bacteria. In the last decade, only a few new antibiotics with new mechanisms of action were approved by the FDA, but additional costs for preventing the spread of bacteria, side effects and resistance may limit their long-term usefulness. Therefore, the number of therapeutic options is limited and necessitates exploration of novel antibacterial agents/approaches to treat hospital- and community-acquired infections. The challenge in antibacterial research is to find appropriate structurally novel antibacterial agents inhibiting bacterial targets. The XF drug series, having a dicationic porphyrin structure, which is distinct from all other known antibiotic classes, are rapidly active against a broad range of bacteria. Another new strategy is called photodynamic inactivation of bacteria (PDIB), which utilizes visible light in combination with photosensitizing molecules to efficiently kill bacteria via reactive oxygen species. The XF drugs act additionally as photosensitizers to inactivate bacteria upon light activation. This review summarizes the efficacy of the XF series and describes it as a new class of antibacterial agents or as new photo-sensitizers.

EBSD: a powerful microstructure analysis technique in the field of solidification.

This paper presents a few examples of the application of electron back-scatter diffraction (EBSD) to solidification problems. For directionally solidified Al-Zn samples, this technique could reveal the change in dendrite growth directions from <100> to <110> as the composition of zinc increases from 5 to 90 wt%. The corresponding texture evolution and grain selection mechanisms were also examined. Twinned dendrites that form under certain solidification conditions in Al-X specimens (with X = Zn, Mg, Ni, Cu) were clearly identified as <110> dendrite trunks split in their centre by a (111) twin plane. In Zn-0.2 wt% Al hot-dip galvanized coatings on steel sheets, EBSD clearly revealed the preferential basal orientation distribution of the nuclei as well as the reinforcement of this distribution by the faster growth of <1010> dendrites. Moreover, in Al-Zn-Si coatings, misorientations as large as 10 degrees mm(-1) have been measured within individual grains. Finally, the complex band and lamellae microstructures that form in the Cu-Sn peritectic system at low growth rate could be shown to constitute a continuous network initiated from a single nucleus. EBSD also showed that the alpha and beta phases had a Kurdjumov-Sachs crystallographic relationship.

Evaluación de los efectos antioxidante, antibacteriano y antifúngico de Calophyllum Brasilense Cambes (Lagarto Caspi) / Evaluation of antioxidant effects, antibacterial and antifungal Brasilense Calophyllum Cambes (Caspi Lizard)

Objetivo: Evaluar el efecto antibacteriano, antifúngico y antioxidante de diferentes extractos del Calophyllum brasiliense Cambess. Material y Métodos: El efecto antioxidante fue determinado por captación de radicales libres, midiendo la decoloración de una solución de 2,2-difenil-1-picril hidrazilo (DPPH); La actividad antibacteriana y antifúngica, in Vitro, se determinó mediante la prueba de dilución. El efecto antibacteriano se evaluó en cepas de E. coli ATCC25922 y Staphylococcus aureus ATCC25923, utilizando medios de cultivo: Caldo y Agar Mueller Hinton. Para evaluar el efecto antifúngico, se utilizó cepas de Cándida albicans en medio de Agar Sabouraud. Resultados: La actividad antioxidante de los extractos acuoso, metanólico y etanólico fue muy satisfactoria, siendo de 110.56 por ciento, 99.17 por ciento y 99.57 por ciento, respectivamente, a una concentración de 100 ug/mL, en comparación con la Vitamina C que presentó 86,5 por ciento. Asimismo, observamos un buen efecto antifúngico para los extractos acuoso y etanólico al 20 por ciento p/v a los volúmenes de 3, 3.5 y 4mL. y en el caso del extracto etanólico también presentó un buen efecto a una concentración del 10 por ciento a un volumen de 1.6mL. Conclusiones: Los extractos: acuoso, metanólico y etanólico, presentaron un buen efecto antioxidante y antifúngico, en las cepas estudiadas.

Objetive: To evaluate, in vitro, the antibacterial, antifungal andantioxidant effects of different extracts of Calophyllum brasiliense Cambess. Material and Methods: The antioxidant effect was tested by free radicals capture, measuring discoloration of the 2,2-diphenyl-1-pycryl hydrazyle solution (DPPH). We used a dilution to measure antibacterial and antifungal in vitro activity. Strains of E. coli ATCC25922 and Staphylococcus aureus ATCC25923 were used to evaluate antibacterial effect; using Broth and Mueller Hinton Agar as culture medium. Strains of Candida albicans and Agar Sabouraud as culture medium were usedto evaluate antifungal effect. Results: Aqueous, methanolic and ethanolic extracts, showed good antioxidant activity having 110.56 per cent, 99.17 per cent and 99.57 per cent of antioxidant activity at 100 ug/mL concentration, respectively. This is superior to the vitamin C referencepattern that showed 86,5 per cent activity. Good antifungal effect for aqueous and ethanol extracts at a 20 per cent concentration for 3, 3.5 and 4 ml of volume was determined. Ethanol extracts had the same antifungal effect at a 10 per cent concentration for 1.6mL. of volume. Conclusions: The aqueous, methanolic and ethanolic extracts showed a good antioxidant and antifungal activity on the strains studied.

What works for whom in a computer-mediated communication intervention in community psychiatry? Moderators of outcome in a cluster randomized trial.

OBJECTIVE: An intervention to structure patient-key worker communication has been tested in a randomized controlled trial. The aim of this paper was to investigate effectiveness of the intervention in terms of moderators of effectiveness. METHOD: A total of 507 patients with schizophrenia were included. Moderators of effectiveness were investigated using two-way anovas. RESULTS: Patients with a better relationship with their key worker and a shorter duration of illness at baseline benefited more from the intervention in terms of quality of life. Patients who received the intervention who were in competitive employment or had a shorter duration of illness showed greater reduction of unmet needs. Older patients receiving the intervention had better treatment satisfaction. CONCLUSION: Outcome of the intervention was moderated by patient characteristics. Moreover, the moderating characteristics varied depending on the specific outcome. Evidence on moderators is very limited, even though, they are significant for understanding, targeting and implementing complex interventions.

A mutation in human VAP-B--MSP domain, present in ALS patients, affects the interaction with other cellular proteins.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset Motor Neuron Disease (MND), characterized by motor neurons death in the cortex, brainstem and spinal cord. Ten loci linked to Familial ALS have been mapped. ALS8 is caused by a substitution of a proline by a serine in the Vesicle-Associated Membrane Protein-Associated protein-B/C (VAP-B/C). VAP-B belongs to a highly conserved family of proteins implicated in Endoplasmic Reticulum-Golgi and intra-Golgi transport and microtubules stabilization. Previous studies demonstrated that the P56S mutation disrupts the subcellular localization of VAP-B and that this position would be essential for Unfolded Protein Response (UPR) induced by VAP-B. In the present work we expressed and purified recombinant wild-type and P56S mutant VAP-B-MSP domain for the analysis of its interactions with other cellular proteins. Our findings suggest that the P56S mutation may lead to a less stable interaction of this endoplasmic reticulum protein with at least two other proteins: tubulin and GAPDH. These two proteins have been previously related to other forms of neurodegenerative diseases and are potential key points to understand ALS8 pathogenesis and other forms of MND. Understanding the role of these protein interactions may help the treatment of this devastating disease in the future.

Analysis of the Saccharomyces cerevisiae exosome architecture and of the RNA binding activity of Rrp40p.

The exosome is a complex of eleven subunits in yeast, involved in RNA processing and degradation. Despite the extensive in vivo functional studies of the exosome, little information is yet available on the structure of the complex and on the RNase and RNA binding activities of the individual subunits. The current model for the exosome structure predicts the formation of a heterohexameric RNase PH ring, bound on one side by RNA binding subunits, and on the opposite side by hydrolytic RNase subunits. Here, we report protein-protein interactions within the exosome, confirming the predictions of constituents of the RNase PH ring, and show some possible interaction interfaces between the other subunits. We also show evidence that Rrp40p can bind RNA in vitro, as predicted by sequence analysis.

Enumeration of waterborne Escherichia coli with petrifilm plates: comparison to standard methods.

Escherichia coli is often monitored in environmental waters as an indicator of the possible presence of human pathogens associated with feces. Petrifilm E. coli/coliform count plates (3M, Minneapolis, MN), previously validated for enumerating E. coli in food, were tested for monitoring E. coli in environmental water. Escherichia coli counts in environmental water samples enumerated with Petrifilm were significantly correlated (R > 0.9; slope = 0.9-1.0; p < 0.001) with counts obtained with three commonly used methods, mTEC (Becton Dickinson, Sparks, MD), m-ColiBlue (Hach, Loveland, CO), and Colilert-18/IDEXX Quanti-Tray 2000 (IDEXX, Westbrook, ME). Blue colonies on Petrifilm plates were most reliably identified as E. coli when accompanied by gas formation, as determined by characterization of the colonies on MacConkey agar plates (PML Microbiologicals, Mississauga, ON, Canada) and by polymerase chair reaction (PCR) with E. coli-specific primers. The main disadvantage of Petrifilm plates for environmental water testing is the small volume (1 mL per sample) that can be tested; however, the plates appear to be suitable for screening and locating sites that exceed criteria for total body and partial body contact. Simplicity of use and storage, reliability, and relatively low cost make Petrifilm plates suitable for volunteer-based and educational water quality monitoring applications, particularly when used as a preliminary screening method to identify problem sites.

PPAR alpha is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue.

Adenovirus-induced hyperleptinemia causes rapid disappearance of body fat in normal rats, presumably by up-regulating fatty acid oxidation within white adipocytes. To determine the role of peroxisomal proliferation-activated receptor (PPAR)alpha expression, which was increased during the rapid loss of fat, we infused adenovirus-leptin into PPAR alpha(-/-) and PPAR alpha(+/+) mice. Despite similar degrees of hyperleptinemia and reduction in food intake, epididymal fat pad weight declined 55% in wild-type but only 6% in PPAR alpha(-/-) mice; liver triacylglycerol fell 39% in the wild-type group but was unchanged in PPAR(-/-) mice. Carnitine palmitoyl transferase-1 mRNA rose 52% in the wild-type mice but did not increase in PPAR alpha(-/-) mice. PPAR gamma coactivator-1 alpha rose 3-fold in the fat and 46% in the liver of wild-type mice but was unchanged in PPAR alpha(-/-) mice. Although AMP-activated protein kinase could not be implicated in the lipopenic actions of hyperleptinemia, acetyl CoA carboxylase protein was reduced in the liver of wild-type but not in PPAR alpha(-/-) mice. Thus, in PPAR alpha(-/-) mice, up-regulation of carnitine palmitoyl transferase-1 mRNA in fat, down-regulation of acetyl CoA carboxylase in liver, and up-regulation of PPAR gamma coactivator-1 alpha mRNA in both tissues are abolished, as is the reduction in their triacylglycerol content.

Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer.

The methylation status of binding sites of the insulator protein, CTCF, in the H19 promoter has been suggested as being critical to the regulation of imprinting of the H19/IGF2 locus located in chromosome 11p15. In this study, we have analyzed the methylation of all of seven potential CTCF-binding sites in the human H19 promoter since the methylation status of these sites has not been reported. We found that all the binding sites except the sixth were hypermethylated whereas only the sixth binding site showed allele-specific methylation in normal human embryonic ureteral tissue. We also analyzed the methylation status of these sites in human-mouse somatic-cell-hybrid clones containing a single copy of human chromosome 11 and which were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) to yield clones which expressed human IGF2 and H19 mutually exclusively of each other. In most of the clones, a correlation between methylation of the sixth CTCF-binding site and expression of IGF2 was observed. Therefore, we analyzed the methylation status of this site in human bladder cancer and found hypomethylation of the paternal allele in two of six informative cases. These results demonstrate that only the sixth CTCF-binding site acts as a key regulatory domain for switching between H19 or IGF2 expression, whereas the other sites are not subject to allele-specific methylation. Loss of methylation imprinting of H19 is linked to hypomethylation of the paternal allele in human bladder cancer, unlike the situation in Wilms' tumor and colon cancer where the maternal allele becomes hypermethylated.

Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation.

Silencing of tumor-suppressor genes by hypermethylation of promoter CpG islands is well documented in human cancer and may be mediated by methyl-CpG-binding proteins, like MeCP2, that are associated in vivo with chromatin modifiers and transcriptional repressors. However, the exact dynamic between methylation and chromatin structure in the regulation of gene expression is not well understood. In this study, we have analyzed the methylation status and chromatin structure of three CpG islands in the p14(ARF)/p16(INK4A) locus in a series of normal and cancer cell lines using methylation-sensitive digestion, MspI accessibility in intact nuclei and chromatin immunoprecipitation (ChIP) assays. We demonstrate the existence of an altered chromatin structure associated with the silencing of tumor-suppressor genes in human cancer cell lines involving CpG island methylation, chromatin condensation, histone deacetylation and MeCP2 binding. The data showed that MeCP2 could bind to methylated CpG islands in both promoters and exons; MeCP2 does not interfere with transcription when bound at an exon, suggesting a more generalized role for the protein beyond transcriptional repression. In the absence of methylation, it is demonstrated that CpG islands located in promoters versus exons display marked differences in the levels of acetylation of associated histone H3, suggesting that chromatin remodeling can be achieved by methylation-independent processes and perhaps explaining why non-promoter CpG islands are more susceptible to de novo methylation than promoter islands.

The endothelin receptor B (EDNRB) promoter displays heterogeneous, site specific methylation patterns in normal and tumor cells.

The 5' region for the endothelin receptor B (EDNRB) gene is a complex CpG island giving rise to four individual transcripts initiating within the island. Here, for the first time, we analyze the relationship between methylation and gene expression in a CpG island located in the 5' region of a gene with multiple transcription start sites. The CpG island was unmethylated in normal prostate and bladder tissue, whereas it became methylated in apparently normal colonic epithelium. Tumors derived from these tissues were frequently hypermethylated relative to the respective normal tissues. Analysis of 11 individual CpG sites located throughout the CpG island showed that specific sites with high methylation levels in several tumors were also methylated in normal tissues, suggesting that they might serve as foci for further de novo methylation. This region also had high levels of methylation in several cancer cell lines, and we found that a low methylation level in a small region within the 5' region correlated with expression of the 5'-most transcript. Interestingly, almost complete methylation 200-1000 bp downstream of the transcriptional start site did not block expression of this transcript. Finally, we show that treatment with 5-aza-2'-deoxycytidine can induce transcriptional activation of the four EDNRB transcripts. Our results show the existence of differential, tissue-dependent methylation at the EDNRB 5' region, suggest the existence of a spreading mechanism for de novo methylation, starting from methylation hotspots, and show that hypermethylation immediately 3' to a transcriptional start site does not prevent initiation.