RESUMEN
BACKGROUND AND AIMS: Epstein-Barr virus (EBV) is present in the malignant epithelial cells of 10% of all gastric adenocarcinomas; however, localization of the virus in normal gastrointestinal mucosa is largely unexplored. In the present study, we measured EBV DNA and localized viral gene products in gastritis specimens (n = 89), normal gastric and colonic mucosa (n = 14), Crohn's disease (n = 9), and ulcerative colitis (n = 11) tissues. METHODS: A battery of sensitive and specific quantitative polymerase chain reactions targeted six disparate regions of the EBV genome: BamH1 W, EBNA1, LMP1, LMP2, BZLF1, and EBER1. EBV infection was localized by EBV-encoded RNA (EBER) in situ hybridization and by immunohistochemical stains for viral latent proteins LMP1 and LMP2 and for viral lytic proteins BMRF1 and BZLF1. B lymphocytes were identified using CD20 immunostains. RESULTS: EBV DNA was essentially undetectable in normal gastric mucosa but was present in 46% of gastritis lesions, 44% of normal colonic mucosa, 55% of Crohn's disease, and 64% of ulcerative colitis samples. Levels of EBV DNA exceeded what would be expected based on the numbers of B lymphocytes in inflamed tissues, suggesting that EBV is preferentially localized to inflammatory gastrointestinal lesions. Histochemical staining revealed EBER expression in lymphoid cells of some PCR-positive lesions. The viral lytic viral proteins, BMRF1 and BZLF1, were expressed in lymphoid cells of two ulcerative colitis tissues, both of which had relatively high viral loads by quantitative PCR. CONCLUSION: EBV-infected lymphocytes are frequently present in inflamed gastric and colonic mucosa. Active viral replication in some lesions raises the possibility of virus-related perpetuation of gastrointestinal inflammation.
Asunto(s)
Colitis Ulcerosa/epidemiología , Enfermedad de Crohn/epidemiología , Infecciones por Virus de Epstein-Barr/epidemiología , Gastritis/epidemiología , Mucosa Intestinal/virología , Adolescente , Niño , Preescolar , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/virología , Comorbilidad , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/virología , ADN Viral/metabolismo , Infecciones por Virus de Epstein-Barr/diagnóstico , Gastritis/metabolismo , Gastritis/virología , Herpesvirus Humano 4/genética , Humanos , Lactante , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Linfocitos/metabolismo , Linfocitos/patología , Linfocitos/virología , Reacción en Cadena de la Polimerasa , Prevalencia , Sensibilidad y Especificidad , Transactivadores/metabolismo , Adulto JovenRESUMEN
Negative-staining electron microscopy (EM) has played a pivotal role in diagnostic virology. It is a rapid technique for viral detection in the urine and can provide an easy means for monitoring viral activity and productive infections. EM of urine for the detection of polyomaviruses has hitherto not been systematically evaluated as a screening tool for renal transplant patients at risk for BK polyomavirus nephropathy (BKN). Here, the authors discuss technical aspects of negative-staining EM of urine (n = 76 samples) and present a simple and rapid protocol for the semiquantitative evaluation of patient samples. In two patient populations (either with (n = 15 samples) or without (n = 15 samples) an established diagnosis of BKN), EM results were compared with two previously established techniques for monitoring polyomavirus activation: (1) cytology for the quantitation of decoy cells, and (2) quantitative PCR assays for the detection of BK virus DNA load levels. In both patient groups, the dynamics of decoy cell shedding by urine cytology closely paralleled free viral particle shedding by EM, and viral load levels as measured by PCR. A trend toward higher readings was observed in patients with BKN (median values, control versus BKN groups: decoy cells 21 versus 50/slide; free virions by EM: 32 versus 66 viral particles/10 high-power fields; PCR: 3.5 x 10(8) versus 5.4 x 10(8) BK virus copies/ml; all differences not statistically significant). The authors conclude that negative-staining EM and the semiquantitative assessment of free viral particles in the urine can be a useful clinical method to identify patients at increased risk for BKN. EM can be used alone or in combination with urine cytology or PCR assays.
Asunto(s)
Microscopía Electrónica de Transmisión/métodos , Infecciones por Polyomavirus/orina , Poliomavirus/ultraestructura , Orina/virología , Citodiagnóstico/métodos , ADN Viral/ultraestructura , ADN Viral/orina , Humanos , Poliomavirus/aislamiento & purificación , Infecciones por Polyomavirus/virología , Coloración y Etiquetado/métodosRESUMEN
Polyploidy is a profound phenotype found in tumors and its mechanism is unknown. We report here that when B-cell lymphoma gene-2 (Bcl-2) was overexpressed in a Chinese hamster ovary cell line that was deficient in CTP:phosphocholine cytidylyltransferase (CT), cellular DNA content doubled. The higher DNA content was due to a permanent conversion from diploid cells to tetraploid cells. The mechanism of polyploid formation could be attributed to the duplication of 18 parental chromosomes. The rate of conversion from diploid to tetraploid was Bcl-2 dose dependent. The diploid genome was not affected by Bcl-2 expression or by CT deficiency alone. Endogenous CT or expression of recombinant rat liver CTalpha prior to Bcl-2 expression prevented the formation of polyploid cells. This conversion was irreversible even when both initiating factors were removed. In this study, we have identified Bcl-2 as a positive regulator and CTalpha as a negative regulator of polyploid formation.