EFFECTS OF HERPES-SIMPLEX VIRUS TYPE-1 INFECTION ON THE PLASMA- MEMBRANE AND RELATED FUNCTIONS OF HELA S3 CELLS

In this study we evaluated modifications of various structural and functional properties of the plasma membrane of HeLa S3 cells following infection by the lytic virus herpes simplex virus type 1 (HSV-1). Na+/K(+)-ATPase activity considerably decreased during the first few hours post-infection (p.i.), whereas Na+ and K+ concentrations were not significantly affected until a much later period. By 8 h p.i., a partial membrane depolarization in infected cells had occurred, as indicated by a small change in the transmembrane potential. HSV infection induced a time-dependent lipid peroxidation of HeLa cell plasma membranes temporally correlated with the progressive reduction in Na+/K(+)-ATPase activity. Moreover, a significant decrease of membrane fluidity appeared at a late phase of the viral replicative cycle probably representing cumulative membrane damage. These results demonstrate that HSV-1 infection induced the production of free radicals in non-phagocytic cells. Since lipid peroxidation begins at an early stage of the virus replicative cycle, it may be directly related to viral cytopathicity

Acyclovir resistance in Herpes simplex virus type 1: functional studies on the thymidine kinase of the highly resistant R100 strain.

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Endothelin-1 and its receptors A and B in human aldosterone-producing adenomas.

Endothelin-1 stimulates aldosterone secretion by interacting with specific receptors. Accordingly, we wished to investigate endothelin-1, endothelin-A (ETA) receptor, and endothelin-B (ETB) receptor gene expression, localization, and properties in aldosterone-producing adenomas and in the normal human adrenal cortex. We carried out 125I-endothelin-1 displacement studies with cold endothelin-1, endothelin-3, the specific ETA antagonist BQ-123, and the specific ETB weak agonist sarafotoxin 6 C and coanalyzed data with the nonlinear iterative curve-fitting program LIGAND. We also studied gene expression with reverse transcription-polymerase chain reaction with specific primers for endothelin-1, ETA, and ETB complementary DNA. Normal adrenal cortices from consenting kidney cancer patients (n = 2) and aldosterone-producing adenomas (n = 4) were studied; for the latter, surrounding normal cortex and kidney biopsy tissue served as controls. To further localize the receptor subtypes, tissue sections were studied by autoradiography in the presence and absence of 500 nmol/L BQ-123, 100 nmol/L sarafotoxin 6 C, and 1 mumol/L cold endothelin-1. In all tissues examined, endothelin-1, ETA, and ETB messenger RNAs were easily detected. However, in aldosterone-producing adenomas, both receptors' genes were expressed at a higher level than in the kidney

Investigation on the presence of polyomavirus, herpesvirus, and papillomavirus sequences in colorectal neoplasms and their association with cancer.

Dear Sir, Studies in literature suggest that some human viruses, including JC polyomavirus (JCV),1\u20133 human cytomegalovirus (HCMV),4 human papillomaviruses (HPVs),5\u20138 and Epstein-Barr virus (EBV),9, 10 have a pathogenic role in starting or promoting colorectal cancer. However, a revision of the literature indicates that this association between colorectal tumors and viral infection is still matter of debate11\u201315 and further studies using robust methods to detect viral infection are required. In fact, the prevalence of viral infection in colorectal cancer samples varies widely among series, and this variability appears to be mainly related to technical aspects, such as in the case of JCV, for which the prevalence in colorectal tumors has been reported to range from 011 to 90%1\u20133, 16 in different studies. In our study, we used sensitive quantitative real-time PCR analysis and PCR and sequencing to investigate the prevalence of genome sequences not only of JCV, but also of other polyomaviruses which have been involved in human infection, i.e., BK virus (BKV), Simian virus 40 (SV40), and the newly discovered Washington University polyomavirus (WUV), Karolinska Institute polyomavirus (KIV) and Merkel cell carcinoma polyomavirus (MCV),17 in a large series of colorectal adenocarcinomas, adenomas and normal mucosa samples. Moreover, in the same samples, we investigated the presence DNA sequences of the herpesviruses HCMV and EBV and HPVs, which have been also suggested to be associated with colorectal tumorigenesis. A detailed description of methods is reported in the supporting information table. Patients gave informed consent to the study, which was approved by the local ethics committee and performed according to Declaration of Helsinki guidelines. Originally, the aim of our study was to investigate the prevalence of JCV infection in colorectal cancers from different age groups. To this aim, we first analyzed 144 archival samples of formalin-fixed paraffin-embedded tissues (group 1, Table I), collected at the Department of Medical Diagnostic Sciences and Special Therapies of the University of Padova in the period ranging from 1998 to 2007. Group 1 included colorectal adenocarcinomas and adjacent mucosa, from 72 patients, who were selected on the basis of age (36 patients were 6445 years, age range 32\u201345 years; 36 patients were 6575 years, age range 75\u201389 years) and matched for gender (14 males and 22 females in both subgroups), tumor site (28 in sigmoid, 30 in ascendent, 11 in descendent, and 3 in rectal colon in both subgroups), and stage (11 stage I-II, 17 stage III, 8 stage 4 in both subgroups). Quite unexpectedly, all samples of group 1 tested negative for JCV DNA at real-time PCR analysis. So, we tried other 3 validated primers and probe sets to detect JCV DNA and investigated whether genome sequences of other polyomaviruses, including the newly discovered viruses, were detectable in colorectal samples. But, no BKV, SV40, KIV, WUV and MCV DNA was detectable in any sample (Table I). Regarding the study of herpesviruses, 3 cancers and 1 cancer-adjacent mucosa from group 1 were positive for EBV DNA; likewise, 2 cancers and 2 cancer-adjacent mucosa samples were HCMV DNA-positive, without significant differences between age groups and among tumor sites and stages (Table I). Analysis of HPV infection demonstrated that 2 sigmoidal cancers from a male and a female patient <45 years of age were positive for HPV-16 DNA (Table I). The low frequency of viral DNA detection in this group of archival tumor samples prompted us to subsequently investigate fresh-frozen tissue samples to obtain better quality DNA. In particular, we investigated colorectal biopsies and tissue samples, which were consecutively collected during colonoscopy or surgery performed at the Department of Surgical and Gastroenterological Sciences of the University of Padova in the period ranging from January 2007 to July 2007 (group 2, Table II). Group 2 samples were obtained from 22 patients with colorectal adenomas (2 hyperplastic, 15 tubular, 5 tubulovillous) and 28 patients with colorectal adenocarcinoma (1 stage 0, 5 stage I, 8 stage II, 10 stage III and 4 stage IV). Mean age was 71.6 years (range, 41\u201392 years), 24 were males and 26 females. Overall, 5 tumors were located in caecum, 8 in ascendent, 1 in transvers, 4 in descendent, 17 in sigmoid colon and 15 in rectum. From each patient, pathologic tissues and tissues at about 3 and 10-cm distance from the lesions were collected. In addition, we analyzed biopsies of rectal, ascending and transverse colonic mucosa, obtained from 15 control subjects without medical history of colorectal pathology and without detection of lesions at colonoscopy (mean age, 58 years; age range, 36\u201382 years; 10 males, 5 females) (group 3). Like in group 1, no polyomavirus DNA sequences were detected in any sample of groups 2 and 3 (Table II). Regarding herpesviruses, EBV-DNA was detected in 11 colorectal cancers (including 2 with positive adjacent mucosa), in 5 adenomas (including 4 with positive adjacent mucosa), in 2 samples of 3-cm-adjacent mucosa, and in 1 sample of 10-cm-adjacent mucosa (with negative associated cancer or adenoma samples); HCMV-DNA was detected in 3 carcinomas (including 1 with positive adjacent mucosa) and in 1 sample of 10-cm-adjacent mucosa, but not in adenoma samples (Table II). EBV and HCMV-DNA load in tissue samples, calculated as the number of viral genome equivalents per cell, was very low in both tumor samples and adjacent mucosa (median EBV DNA load, 50 copies/106 cells; range, 10\u201310,000 copies/106 cells; median HCMV DNA load, 70 copies/106 cells; range, 10\u201330000 copies/106 cells) and consistent with lymphocyte/macrophage infiltration, rather than infection of tumor cells. The rate of EBV-DNA detection in carcinoma and adenoma samples was significantly higher than in adjacent mucosa (\u3c72-test, p < 0.05) and was explained by a greater inflammatory cell infiltration in neoplastic tissues than in normal mucosa. In the control group (group 3), EBV-DNA was detected in 5 out of 15 subjects (in the rectal mucosa in 2 cases, in the ascending colon in 2, and in all 3 colon samples in one subject); HCMV-DNA was detected in the rectal mucosa from a patient who was also EBV-positive. High-risk HPV-16 DNA was detected by both nested PCR/sequencing and real-time PCR in 2 rectal carcinomas and in a sigmoid adenoma of group 2, but not in control group 3 samples (Table III). In the 3 positive cases, the adjacent mucosa was also HPV-16 DNA-positive. In the literature, JCV genome sequences were detected by PCR amplification and Southern blot hybridization in 90% of colorectal cancers and in the adjacent normal colonic epithelium, with a 10-fold higher viral load in cancers than in adjacent mucosa,1, 2 as well as in 82% of sporadic adenomatous polyps.18 In colorectal mucosa, JCV was suggested to promote tumorigenesis through a direct interaction of its large T antigen (Tag) with \u3b2-catenin and p53.2, 3 Since a variant of the JCV Mad-1 strain was preferentially detected in colon cancers, it was hypothesized that this strain might be selectively activated in colonic epithelial cells.19 It cannot however be excluded that detection of a unique JCV strain represented a laboratory contamination, since the same Mad-1 strain was used as positive control in the laboratory.11 In our study, we used real-time PCR to detect viral sequences. This method is very sensitive and specific, but much less susceptible to amplicon contamination than PCR followed by Southern-blot, which was employed in the other studies. A study on large series of colorectal cancer/normal tissue pairs,11 which also employed real-time PCR, failed to demonstrate the presence JCV-DNA sequences in all samples, in agreement with our results. Immunostaining for SV40 TAg has been reported to give problems of false positive results,20 so it cannot be excluded that reported JCV TAg detection in colorectal tissues is also a technical artefact, since the same antibodies against SV40 TAg were employed to investigate JCV expression, exploiting their cross-reactivity with JCV Tag.2, 18 Reports regarding detection of BKV and SV40 in colorectal cancers are conflicting, with negative results in 1 study,21 in agreement with our data, but positive findings in a recent Italian report which demonstrated BKV and SV40 DNA in 6 and 10 out of 66 colorectal carcinomas, respectively.22 Problems of PCR contamination cannot be excluded even in this study, since the primers employed could amplify SV40 DNA sequences which are present in some laboratory plasmids.22 Our data seem also to exclude WUV, KIV and MCV polyomavirus have any role in colorectal tumorigenesis. HCMV has also been implicated in colorectal tumorigenesis because of its detection in about 80% of tumors, but not in adjacent non-neoplastic mucosa,4, 23 and because of the demonstration that HCMV infection of colorectal cancer cell lines in vitro resulted in induction Bcl2 and cycloxygenase-2 proteins, both of which are involved in important pathways of colorectal cancer progression.4 In contrast, other studies failed to detect HCMV DNA sequences in tumour samples.12\u201314 Regarding EBV, it has been associated with gastric adenocarcinoma and gastric lymphoepithelioma, whereas its involvement in colorectal tumorigenesis seems to be excluded by several studies which, generally, did not detect EBV-positive cancer cells, even though some EBV-positive tumor infiltrating B lymphocytes were frequently found.9, 24\u201326 However, we do not rule out the possibility that the occasional presence of EBV and HCMV in infiltrating lymphocytes/monocytes in colorectal mucosa may promote the production and release of cytokines and growth factors or deregulate tumor suppressor genes and thus cooperate with cancer development. Among candidate oncogenic viruses for colorectal tumorigenesis, HPV is the most reliable, as indicated by the epidemiological evidence of high-risk HPV infection in a relatively high percentage of colorectal tissues from patients with cancer, but not in control subjects without cancer.5\u20138 Accordingly, we also identified HPV-16 DNA in some cases of distally localized colorectal cancers. Persistent high-risk HPV infection has been definitely demonstrated to be a necessary cause for development of cervical cancer and other anogenital cancers27, 28 and might also be responsible for some cases of colorectal cancer. While waiting for further epidemiological data and for experimental studies to confirm this hypothesis, we can predict anti-HPV vaccination might have an impact on colorectal cancer prevention. In conclusion, our study, which for the first time systematically investigated the presence of genomic sequences of several putative oncogenic viruses in a large series of colorectal neoplasms, seems to exclude JCV, BKV, SV40, WUV, KIV, MCV, HCMV and EBV have a prominent role in the pathogenesis of colorectal cancer, whereas it suggests high-risk HPV could be involved in some cases of distally localized colorectal carcinoma. Yours sincerely, Valentina MILITELLO, Marta TREVISAN, Laura SQUARZON, Maria Angela BIASOLO, Massimo RUGGE, Carmelo MILITELLO, Giorgio PAL a and Luisa BARZO

Gene expression, localization, and characterization of endothelin A and B receptors in the human adrenal cortex.

Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 125I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (Bmax) of the ETA and ETB subtype was 2.6 +/- 0.5 pmol/mg protein (m +/- SEM) and 1.19 +/- 0.6, respectively. The dissociation constant (Kd) of ET-1, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells

A prospective study of BK-virus-associated haemorrhagic cystitis in paediatric patients undergoing allogeneic haematopoietic stem cell transplantation

We investigated the incidence, risk factors and outcome of haemorrhagic cystitis (HC) in paediatric patients undergoing HSCT and the predictive value of BK viruria and viraemia for developing HC. Over a period of 54 months, 74 patients were recruited. The cumulative incidence of HC was 22%. Among 15 patients prospectively monitored for BK viruria and viraemia, four patients developed HC of grade > or =II. This group, which had two consecutive BK positive samples, showed a sensitivity of 100%, a specificity of 82%, a positive predictive value of 67%, and negative predictive value of 100% for developing HC. Analysed by a receiver-operator characteristic curve (ROC), a urine BK load >9 x 10(6) genomic copies/ml had a sensitivity of 95% and specificity of 90%; while a blood BK load >1 x 10(3) genomic copies/ml had a sensitivity of 40% and a specificity of 93% for HC, respectively. In univariate analysis, BK positivity was the only factor significantly associated with HC. After a median follow-up of 1.8 years, patients with HC showed a lower overall survival, 40 vs 65%, P 0.01, and a lower event-free survival, 42 vs 62%, P 0.03, compared to patients without HC. We conclude that BK detection in urine and/or plasma is a specific predictor for developing HC