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Purpose Epstein-Barr virus (EBV) is detected in a substantial subgroup of gastric adenocarcinomas worldwide. We have previously reported that these EBV-positive gastric carcinomas carry distinct genomic aberrations. In the present study, we analyzed a large cohort of EBV-positive and EBV-negative gastric adenocarcinomas for their clinicopathologic features to determine whether they constitute a different clinical entity. Patients and Methods Using a validated polymerase chain reaction/enzyme immunoassay–based prescreening method in combination with EBER1/2-RNA in situ hybridization, EBV was detected in the tumor cells of 7.2% (n = 41) of the gastric carcinomas from the Dutch D1D2 trial (N = 566; mean follow-up, 9 years). EBV status was correlated with clinicopathologic features collected for the Dutch D1D2 trial. Results EBV-positive gastric carcinomas occurred significantly more frequently in males (P < .0001) and in younger patients (P = .012). Most were of the intestinal type according to the Laurén classification (P = .047) or tubular according to the WHO classification (P = .006) and located in the proximal part of the stomach (P < .0001). A significantly lower tumor-node-metastasis system-stage (P = .026) was observed in the patients with EBV-carrying carcinomas, which was solely explained by less lymph node (LN) involvement (P = .034) in these cases. In addition, a better prognosis, as reflected by a longer disease-free period (P = .04) and a significant better cancer-related survival (P = .02), was observed for these patients, which could be explained by less LN involvement, less residual disease, and younger patient age. Conclusion EBV-carrying gastric adenocarcinomas are a distinct entity of carcinomas, characterized not only by unique genomic aberrations, but also by distinct clinicopathologic features associated with significantly better prognosis.

Epstein-Barr virus (EBV) is a herpesvirus that establishes a lifelong, persistent infection. It was first discovered in the tumor Burkitt's lymphoma (BL). Despite intensive study, the role of EBV in BL remains enigmatic. One striking feature of the tumor is the unique pattern of viral latent protein expression, which is restricted to EBV-encoded nuclear antigen (EBNA) 1. EBNA1 is required to maintain the viral genome but is not recognized by cytotoxic T cells. Consequently, it was proposed that this expression pattern was used by latently infected B cellsin vivo. This would be the site of long-term, persistent infection by the virus and, by implication, the progenitor of BL. We now know that EBV persists in memory B cells in the peripheral blood and that BL is a tumor of memory cells. However, a normal B cell expressing EBNA1 alone has been elusive. Here we show that most infected cells in the blood express no detectable latent mRNA or proteins. The exception is that when infected cells divide they express EBNA1 only. This is the first detection of the BL viral phenotype in a normal, infected B cellin vivo. It suggests that BL may be a tumor of a latently infected memory B cell that is stuck proliferating because it is a tumor and, therefore, constitutively expressing only EBNA1.

BACKGROUND AND OBJECTIVES: To investigate the angiogenic effect of CO2, Ho:YSGG, and XeCl excimer TMLR in a rat model with morphologic characteristics of chronic myocardial ischemia.STUDY DESIGN/MATERIALS AND METHODS: Two channels (200-320 microm) were created per rat heart. After 14 days, vessel numbers and densities in and around laser scars were assessed.RESULTS: Capillary densities in the laser scars were equal between the three lasers ( approximately 130 vessels/mm2) but much lower than in control areas ( approximately 2,100 vessels/mm2). Vessel densities excluding capillaries were significantly higher in Ho:YSGG and CO2 scars compared to excimer scars, while only Ho:YSGG scars contained significantly more large vessels (diameter > or = 20 microm) than control areas. Only rarely, extension of vascular growth into adjacent myocardium was observed in any of the three groups.CONCLUSIONS: These results indicate that the angiogenic response following TMLR is limited to the channel scar and related to the scar size rather than the specific laser type.

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with epithelial cell and B-cell malignancies. EBV infection of B lymphocytes is essential for acute and persistent EBV infection in humans; however, the role of epithelial cell infection in the normal EBV life cycle remains controversial. The rhesus lymphocryptovirus (LCV) is an EBV-related herpesvirus that naturally infects rhesus macaques and can be used experimentally to model persistent B-cell infection and B-cell lymphomagenesis. We now show that the rhesus LCV can infect epithelial cells in immunosuppressed rhesus macaques and can induce epithelial cell lesions resembling oral hairy leukoplakia in AIDS patients. Electron microscopy, immunohistochemistry, and DNA-RNA in situ hybridization were used to identify the presence of a lytic rhesus LCV infection in these proliferative, hyperkeratotic, or parakeratotic epithelial cell lesions. These studies demonstrate that the rhesus LCV has tropism for epithelial cells, in addition to B cells, and is a relevant animal model system for studying the role of epithelial cell infection in EBV pathogenesis.

Women with persistent HPV infections have increased risk of progressive CIN lesions. Transmission of HPV between sexual partners might maintain viral infection and, consequently, may influence the clinical course of CIN. We investigated the effect of condom use on regression of CIN lesions and on clearance of HPV. Women with CIN and their male sexual partners were randomized for condom use (condom group n = 72 and noncondom group n = 76). They were conservatively managed and followed every 3-6 months by colposcopy, cytology and HPV testing by GP5+/6+ PCR. Baseline cervical biopsy specimens were taken. Median follow-up time for women was 15.2 months (range 3.0-85.4). Outcomes of interest were clinical regression of CIN at colposcopy and clearance of HPV. Outcomes were assessed in 64 women of the condom group and 61 women of the noncondom group. Women in the condom group showed a 2-year cumulative regression rate of 53% vs. 35% in the noncondom group (p = 0.03). The 2-year cumulative rates of HPV clearance were 23% vs. 4%, respectively (p = 0.02). Although lower regression rates were found if women were HPV-positive and had > or =CIN2 lesions at baseline, effects of condom use were found both in women with CIN1 and in women with > or =CIN2 lesions. Condom use promotes regression of CIN lesions and clearance of HPV.

Epstein-Barr virus (EBV)-specific immunoblot analysis was used to reveal the molecular diversity of immunoglobulin (Ig) G and IgA antibody responses against Epstein-Barr nuclear antigen (EBNA), early antigen (EA), and viral capsid antigen (VCA) in serum samples from patients with nasopharyngeal carcinoma (NPC) and control subjects, by use of immunofluorescence assay (IFA). Control donors (n=150) showed IgG responses to few EBV proteins--VCA-p18, VCA-p40, EBNA1, and Zebra--and sporadically weak IgA reactivity to EBNA1 and VCA-p18. Patients with NPC stage 1 (n=6) had similar response patterns. Patients with NPC stage 2-4 (n=132) showed significantly more diverse IgG and IgA responses to EA and VCA proteins--VCA-p18/-p40, EBNA1, Z-encoded broadly reactive activator, and EAd-p47/54, -DNAse, -thymidine kinase, and -p138. No correlation was found between IFA titers and the number of EBV proteins recognized by IgG or IgA. Our results reveal dissimilarity between EBV polypeptides recognized by IgG and IgA antibodies, which suggests independent B cell triggering events.

Background: Penile lesions and pearly penile papules (PPP) are frequently found in male sexual partners of women with cervical intraepithelial neoplasia (CIN). The former have been associated with human papillomavirus (HPV). Objectives: We estimated the prevalence of PPP in male sexual partners of women with CIN, and investigated the association between PPP and flat and papular penile lesions found in these men. We further evaluated a possible association between PPP and HPV, age, and CIN grade of the female partner. Methods: We evaluated by penoscopy the presence of HPV-associated penile lesions and PPP in 226 male sexual partners of women with CIN. HPV was tested by polymerase chain reaction-enzyme immunoassay and in situ hybridization. Results: The prevalence of PPP was 34% and was not associated with the presence of penile lesions or a positive HPV test. Age and CIN grade of the female partner were not related to the presence of PPP. Conclusion: The prevalence of PPP in male sexual partners of women with CIN is comparable with the prevalence described in men of more diverse populations. Our data do not support a causative role for HPV in the genesis of PPP. (J Am Acad Dermatol 2003;49:50-4.)

Circulating levels of C-reactive protein (CRP) constitute a cardiovascular risk marker. Immunohistochemical studies have revealed co-localization of CRP and activated complement in human infarcted myocardium suggesting CRP to enhance inflammation in ischemic myocardium by inducing local complement activation. The aim was to establish whether CRP activates complement in infarcted human myocardium and to assess the relationship between this activation and the duration of infarction. Myocardial tissue samples from 56 patients that had died from acute myocardial infarction were evaluated. Specimens were taken from infarcted as well as noninfarcted sites of the heart. CRP-mediated complement activation was assessed by immunohistochemistry and by measuring levels of complement, CRP, and CRP-complement complexes, specific markers for CRP-mediated activation, in homogenates of the heart. Infarctions of 12 hours to 5 days had significantly more extensive depositions of complement and CRP and contained significantly more CRP, activated complement, and CRP-complement complexes than infarctions that were less than 12 hours old. Levels of CRP complexes correlated significantly with CRP and complement concentrations in the infarctions, as well as with the extent of complement and CRP depositions as measured via immunohistochemistry. Specific activation products of CRP-mediated activation of complement are increased in infarcts of more than 12 hours in duration and correlate with the extent of complement depositions. Hence, CRP seems to enhance local inflammatory reactions ensuing in human myocardial infarcts of more than 12 hours duration.

The Epstein-Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane. Co-labelling of cells with both an LMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin's disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system.

The effect of long-term administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) on dendritic cell (DC) activation and survival in patients with locally advanced breast cancer (LABC) was studied. To this end, the number of activated DC (i.e. positive for the marker S100) in tumour-draining lymph nodes (TDLN) was determined and compared between LABC patients receiving neoadjuvant chemotherapy with GM-CSF (n=52) or without GM-CSF (n=11), and a control group of chemonaïve breast cancer patients (n=10). A significantly higher mean percentage of S100+ DC in the TDLN of the GM-CSF-treated patients (9.9%) was found compared with each of the respective control groups (5.3 and 5.1%, P=0.002). Moreover, intrapatient comparison before and after treatment showed that the percentage of S100+ DC significantly increased over the course of the GM-CSF treatment (P=0.018). In a univariate survival analysis with a median follow-up of 64 months, relatively high percentages of S100+ DC (> or =8%) were associated with a longer disease-free survival (DFS) (P=0.078). In patients with a high tumour load, where immunosuppressed conditions generally prevail, long-term administration of GM-CSF may thus contribute to survival through enhanced DC activation and consequently improved chances of effective antitumour immunity.

In the search for better protocols of preemptive therapy of human cytomegalovirus (HCMV) infection in hematopoietic stem cell transplant (HSCT) recipients, we conducted a randomized trial comparing antigenemia with the nucleic acid sequence–based assay (NASBA) for determination of HCMV immediate-early messenger RNA (IEmRNA) as the guiding assay for initiation of pre-emptive antiviral treatment. In the IEmRNA arm, antiviral therapy was started upon IEmRNA positivity confirmed the following day, whereas in the antigenemia arm, therapy was started in the presence of either at least 2 pp65-positive leukocytes/2 × 105 examined or a single positive leukocyte confirmed the following day. In both arms, treatment was stopped upon 2 consecutive negative results. All patients were monitored for 3 months after HSCT. The primary end point of the study was duration of anti-HCMV therapy. On the whole, 80 children (41 in the IEmRNA and 39 in the antigenemia arm), recipients of transplants from either a relative or an unrelated donor, completed the study. No patient developed HCMV disease. In the IEmRNA arm, the incidence of HCMV infection was higher compared to the antigenemia arm (80% vs 51%, respectively, P = .0069), as well as the percentage of treated patients (66% vs 44%, respectively, P = .045). However, the percentage of relapses and treated relapses was comparable in the 2 arms. There was no significant difference in median duration of therapy per patient. Although these data indicate that IEmRNA determination does not offer advantages in terms of treatment duration, it can safely replace antigenemia, while semiautomation is the major advantage of the NASBA procedure.

As an initial assessment of the feasibility of employing the adenovirus serotype 3 (Ad3) fiber knob as a locale for introducing a tropism-modifying motif, we generated an adenoviral vector containing a six-histidine tag genetically fused to the carboxy-terminus of the Ad3 fiber knob. The heterologous tag proved to be accessible for binding in the context of the virion and, moreover, had rendered the modified vector capable of mediating gene transfer through an artificial, non-Ad3 receptor.

Nucleic acid sequence-based amplification assays for detection of human cytomegalovirus (HCMV) immediate-early and pp67 mRNA in 65 amniotic fluid samples tested for prenatal diagnosis of congenital HCMV infection showed sensitivity, specificity, and negative and positive predictive values >90%.

BACKGROUND/AIMS: Recent studies indicate the presence of reactive oxygen species (ROS) producing homologues of the enzymatic subunit (Nox2) of phagocytic NADPH oxidase in non-phagocytic cells. Interestingly, in these cells, ROS produced by the Nox2 homologue(s) was shown to play a role in various regulatory processes, including cell death, proliferation, and aging. The purpose of this study was to investigate whether human cardiomyocytes express Nox2.METHODS: The expression of Nox2 was studied in human cardiomyocytes using western blot and immunohistochemical analysis. To analyse the putative expression of Nox2 in human heart disease, cardiac samples from patients who had died subsequent to acute myocardial infarction (AMI) were studied.RESULTS: Both in western blot and immunohistochemical studies, Nox2 expression was found in normal human cardiomyocytes. In patients with AMI, a significant increase in Nox2 expression was found both in viable and in jeopardised cardiomyocytes in the infarcted area. In addition, in the "remote from infarction" area, Nox2 expression was present in cardiomyocytes, but was not increased.CONCLUSIONS: Nox2 or its homologue(s) is expressed in normal and jeopardised human cardiomyocytes. This expression is increased in patients with AMI, suggesting a role for this ROS producing Nox2 homologue(s) in the human heart after AMI.

Epstein–Barr virus (EBV) persistently infects B-cells in humans and can be shed into the saliva. The cellular source of infectious virus is uncertain. Hairy leukoplakia, an AIDS-associated lesion of the tongue, supports EBV replication in epithelial cells. However, the general significance of this observation has remained doubtful. Using immunohistochemistry and in situ hybridization, we demonstrate evidence of EBV replication in tongue epithelial cells in 4 of 168 samples from 84 autopsy cases. Thus, in patients who do not have AIDS, squamous epithelial cells of the tongue rarely support EBV replication. However, all individuals with evidence of EBV replication were either on immunosuppressive therapy or were terminally ill cancer patients, suggesting that an impairment of the immune system may have allowed EBV replication to occur at this site. Thus, our findings are consistent with the idea that EBV replication in oropharyngeal epithelial cells is an infrequent event.

The aim of this study was to develop a LightCycler-based real-time PCR assay for monitoring the Epstein-Barr virus (EBV) DNA load in unfractionated whole blood. This assay was compared with quantitative competitive PCR (Q-PCR) for EBV. The LightCycler-based assay was highly sensitive and reproducible when quantifying plasmid DNA in either the presence or absence of healthy donor blood DNA. Amplifying plasmid DNA in DNA backgrounds from different donors slightly increased the variation of quantification, indicating that clinical specimen DNA has an influence on quantification. In most transplant recipients, a good correlation was observed between EBV DNA load dynamics determined by LightCycler and Q-PCR in follow-up samples, although the correlation between absolute values of EBV DNA loads was weak and occasional samples were false negative in the LightCycler assay. In 253 cross-sectional blood samples from patients with Burkitt's lymphoma, infectious mononucleosis, or human immunodeficiency virus infection, a weak but significant correlation between the two methods was found ( r 2 = 0.37, P < 0.001). Our results indicate that the clinical specimen DNA background may influence the absolute values of EBV DNA load in LightCycler analyses but that this effect is rare. LightCycler PCR is very well suited for monitoring of EBV DNA load dynamics, and its diagnostic value is comparable to that of Q-PCR. To avoid false negativity or underestimation of viral load, future internal calibration of the LightCycler is recommended. This would also enhance EBV load assay standardization and interinstitute comparisons.

BACKGROUND: Confocal laser scanning microscopy (CLSM) presents the opportunity to perform three-dimensional (3D) DNA content measurements on intact cells in thick histological sections. So far, these measurements have been performed manually, which is quite time-consuming.METHODS: In this study, an intuitive contour-based segmentation algorithm for automatic 3D CLSM image cytometry of nuclei in thick histological sections is presented. To evaluate the segmentation algorithm, we measured the DNA content and volume of human liver and breast cancer nuclei in 3D CLSM images.RESULTS: A high percentage of nuclei could be segmented fully automatically (e.g., human liver, 92%). Comparison with (time-consuming) interactive measurements on the same CLSM images showed that the results were well correlated (liver, r = 1.00; breast, r = 0.92).CONCLUSIONS: Automatic 3D CLSM image cytometry enables measurement of volume and DNA content of large numbers of nuclei in thick histological sections within an acceptable time. This makes large-scale studies feasible, whereby the advantages of CLSM can be exploited fully. The intuitive modular segmentation algorithm presented in this study detects and separates overlapping objects, also in two-dimensional (2D) space. Therefore, this algorithm may also be suitable for other applications.

Background: Genital human papillomavirus infection (HPV) is causally associated with cervical carcinomas and premalignant lesions. Limited information is available about the prevalence of HPV and penile lesions in male sexual partners of women with cervical intraepithelial neoplasia (CIN). Objective: The aim of this study was to identify the presence of penile lesions and HPV in penile scrapings from male sexual partners of women with CIN. Methods: One hundred seventy-five male sexual partners of women with CIN were screened by peniscopy after acetowhite staining and HPV testing on penile scrapings. Results: Penile lesions were seen in 68% of the male sexual partners. More than one lesion type was diagnosed in 15%. Flat lesions, papular lesions, and condylomata acuminata were seen in 83%, 29%, and 4%, respectively. HPV was detected in 59% of the penile scrapings, containing mainly oncogenic HPV types. When penile lesions were present at peniscopy, 67% of penile scrapings were positive for HPV, whereas 37% were HPV-positive when no lesions were visible. Conclusions: Penile lesions are frequently found in sexual partners of women with CIN. Most of these lesions are subclinical (ie, only visible after acetowhite staining) and are often associated with the presence of high-risk HPV, indicating that male sexual partners of women with CIN might constitute a reservoir for high-risk HPV. (J Am Acad Dermatol 2002;47:351-7.)

Preemptive treatment based on the sensitive detection of CMV-DNA has helped to reduce HCMV-related mortality after allogeneic stem cell transplantation (SCT). Detection of active viral replication might help to better predict HCMV disease. In this study, 33 recipients at risk for HCMV infection after allogeneic SCT were prospectively monitored 1x/week for active HCMV infection by NASBA, whole blood DNA-PCR and virus culture assays. Preemptive antiviral therapy was initiated after the second positive PCR result, while NASBA results were not considered for clinical decision-making. Overall, a high agreement between PCR and NASBA on a per sample (85.3%) and per patient (87.9%) level was demonstrated. HCMV DNA titers in the blood were found to be higher in PCR(+)/NASBA(+) compared to PCR(+)/NASBA(-) samples (P < 0.01). None of the NASBA-negative patients developed HCMV disease. Sixteen of 18 patients receiving PCR-based preemptive therapy were also found NASBA positive. There was no difference between the assays for the time to the first positive test result. However, the time to the first negative test result upon initiation of antiviral therapy was significantly shorter for the NASBA assay (P = 0.002), indicating a high positive predictive value to assess the efficacy of antiviral therapy. Three patients developed late-onset HCMV disease, all of whom were found to be PCR and NASBA positive. In conclusion, the data presented clearly demonstrate the value of patient monitoring using the NASBA assay to early diagnose active HCMV infection and to assess the efficacy of antiviral therapy in high risk patients after allogeneic SCT. A prospective comparison of PCR-based vs NASBA-based preemptive therapy is ongoing.

Objective: To study Epstein-Barr virus (EBV) DNA loads in peripheral blood of HIV carriers to determine base-line values and diagnostic relevance of viral load in relation to quantitative serology; to compare EBV presence in parallel plasma and unfractionated whole blood samples; and to correlate EBV DNA load to HIV, CD4 T-cell counts and HAART. Design: One-hundred and nine random patients receiving highly active antiretroviral therapy (HAART) during 1999 and 99 patients on anti-HIV monotherapy during 1993-1996 were included. Methods: EBV DNA load was determined by quantitative competitive PCR. EBV serology was determined by immunoblot profile and quantitative enzyme-linked immunosorbent assay for responses against VCA-p18 and EBNA-1. Results: Twenty-two out of 109 patients receiving HAART and 28 out of 99 of patients on anti-HIV monotherapy showed elevated EBV DNA loads in whole blood (> 2000 copies/ml), without elevated loads in parallel plasma. EBV DNA load distribution did not differ between the two groups (P = 0.78) and did not correlate with HIV or CD4 T-cell count. In three patients with high EBV DNA loads EBV RNA was virtually absent. Patients with high EBV DNA loads (3610-89 400 copies/ml) had higher anti-VCA-p18 IgG levels than patients with undetectable EBV DNA (P < 0.0001) but lower anti-EBNA-1 IgG levels (P = 0.005), Conclusion: Absolute values of EBV DNA load may have poor diagnostic value for defining HIV patients at risk for developing EBV-associated disease. Elevated EBV DNA loads are cell-associated and are not influenced by HAART. Increased anti-pi 8-VCA and decreased anti-EBNA-1 IgG levels in patients with high EBV loads indicate impaired latency control and increased lytic replication suggesting disturbed overall immunosurveillance against EBV.

Epstein-Barr virus (EBV) is associated with several human malignancies that each show different viral gene expression profiles. In malignancies such as Hodgkin's disease and nasopharyngeal carcinoma only Epstein-Barr nuclear antigen 1 (EBNA1) and varying levels of latent membrane proteins 1 and 2 (LMP1 and -2) are expressed. Since endogenously expressed EBNA1 is protected from CTL recognition, LMP1 and LMP2 are the most likely target antigens for anti-tumor immunotherapy. Therefore, we sought to identify in a systematic way CD8(+) T-cell responses directed against eptitopes derived from LMP1 and LMP2. Using IFNgamma-ELISPOT assays of interferon-gamma release, peripheral blood mononuclear cells (PBMC) of healthy donors were screened with peptide panels (15 mer overlapping by 10) spanning the LMP1 and LMP2 sequences of the prototype EBV strain B95.8. When positive responses were found, CD4(+) or CD8(+) T cells were depleted from donor PBMC to determine the origin of the responder population. We detected CD8(+) T-cell responses to LMP1 in 9/50(18%) donors and to LMP2 in 15/28 (54%) donors. In addition to the already described epitopes, 3 new LMP1- and 5 new LMP2-derived CD8(+) epitopes were identified. In most donors LMP1- and LMP2-specific CD8(+) precursor frequencies were low compared with precursors against immunodominant EBV epitopes from latent (EBNA3A, -3B and -3C) and lytic cycle antigens. These results demonstrate that CD8(+) memory T cell responses to LMP1 and especially to LMP2 do exist in Caucasians, albeit at low levels and could potentially be exploited for therapeutic use.

BACKGROUND: The monitoring for HCMV mRNA expression in whole blood provides an accurate and informative diagnostic approach.METHODS AND MATERIALS: A multiplex real-time NASBA with three molecular beacons (MR-NASBA) was developed for the simultaneous detection and quantification of HCMV-encoded immediate early-1 (IE1) and late pp67 mRNA. The assay was evaluated using RNA from in vitro HCMV-infected cells and sequential whole blood samples (100 microl) of HCMV infected lung transplant recipients.RESULTS: The MR-NASBA showed equal performance compared with standard NASBA assays (sensitivity of 1-3 x 10(3) RNA molecules in 100 microl blood and a linear range of 10(3)-10(6) RNA molecules). The standard IE1 Q-RNA provides a reliable internal system control. No interference was observed between the individual beacon signals. The simultaneous 'one-tube' quantification of IE1-RNA levels combined with qualitative detection of pp67-RNA is feasible without loss of assay performance in clinical whole blood specimens. CONCLUSION AND COMMENTS: MR-NASBA may be suitable for monitoring HCMV-activity in transplant recipients to aid in fine-tuning of antiviral intervention in high risk populations employing a traffic light diagnostic approach: no HCMV RNA signal (green light: safe) reflects absent or fully latent infection requiring no antiviral intervention; an 'IE1-RNA only' signal (yellow light: alert) indicates an emerging or subclinical active infection, opting for preemptive treatment in high risk populations; simultaneous 'IE1-RNA plus pp67-RNA' detection (red light:danger) indicates disseminating productive infection requiring immediate antiviral treatment.

OBJECTIVE: An increase of circulating secretory Phospholipase A(2) (sPLA(2)) is a risk factor for coronary artery disease. We hypothesized that this reflects participation of sPLA(2) in local inflammatory reactions ensuing in ischemic myocardium. Therefore, we studied the course of circulating sPLA(2), in patients with acute myocardial infarction (AMI) or unstable angina pectoris (UAP), and investigated the presence of sPLA(2) in infarcted myocardial tissue.METHODS: Plasma samples of 107 patients with AMI or UAP, collected on admission and at varying intervals thereafter, were tested for the presence of sPLA(2) and C-reactive protein (CRP). Cumulative release values of these parameters were calculated, which allowed for comparison of the results rearranged in time according to the onset of symptoms. By immunohistochemistry we studied the presence of sPLA(2) and CRP in myocardial tissue of 30 patients who died subsequent to AMI.RESULTS: Levels of sPLA(2) became elevated during the disease course in 66 of the 87 patients with AMI, and were higher than those of the patients with UAP of whom 8 of the 20 had elevated levels. By immunohistochemistry sPLA(2) was found to be localized in the infarcted myocardium, particularly in its borderzone, from 12 h after the onset of AMI. Positive staining for sPLA(2) was more extensive than that for CRP.CONCLUSIONS: The localization pattern of sPLA(2) in infarcted myocardium as well as its plasma course, in relation to those of CRP, are in line with a supposed pro-inflammatory role during AMI for sPLA(2) as a generator of lysophospholipids serving as ligands for CRP.

Human CD4+T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8+memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4+epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4+T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4+T-helper 1 cells (EBNA1, EBNA3C ≫ LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8+-T-cell responses (EBNA3C > EBNA1 > LMP2 ≫ LMP1). Furthermore, the range of CD4+memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8+-T-cell frequencies.

Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.

The c-met proto-oncogene, encoding the hepatocyte growth factor receptor, can be activated by various mechanisms. These include, among others, gene amplification with concomitant overexpression and the tpr-met oncogenic rearrangement. In the case of gastric cancer, contradictory results on the presence of the tpr-met oncogenic rearrangement have been published. The current study aimed therefore to assess the prevalence of tpr-met expression in Caucasian gastric adenocarcinomas, to evaluate the importance of this oncogene in their carcinogenesis. In addition, the level of c-met expression was determined, to evaluate the role of this alternative mode of activation of the proto-oncogene. A series of Caucasian gastric adenocarcinomas (n=43) and normal gastric mucosal samples (n=14) was analysed for tpr-met and c-met expression. Expression of tpr-met mRNA in the samples was performed by two reverse transcriptase polymerase chain reaction (RT-PCR) assays, with excellent correlation. The specificity of both methods was confirmed by direct sequencing of the PCR products of the MNNG-HOS cell line, which is known to contain the rearrangement. The level of c-met expression was assessed using semi-quantitative RT-PCR assays and immunohistochemistry (IHC). None of the normal gastric mucosal or gastric adenocarcinoma samples expressed tpr-met mRNA, as determined by both RT-PCR assays. Seventy per cent of the adenocarcinomas showed overexpression of c-met, according to elevated c-met mRNA levels, compared with the expression level of normal gastric mucosa. A significant correlation was found between the level of c-met mRNA and protein expression. In conclusion, these results strongly suggest that tpr-met activation does not play a role in Caucasian gastric carcinogenesis, while overexpression of the c-met gene occurs in the majority of Caucasian gastric adenocarcinomas.

Application of recombinant adenoviral vectors for cancer gene therapy is currently limited due to lack of specificity for tumor cells. For gastric and esophageal adenocarcinoma, we present here that the relative abundant expression of the primary adenovirus receptor, coxsackie/adenovirus receptor (CAR), on normal epithelium compared to carcinoma favors the transduction of the epithelium. As such, to achieve specific transduction of cancer cells, targeting approaches are required that ablate the binding of the virus to CAR and redirect the virus to tumor-specific receptors. By immunohistochemistry and reverse transcriptase polymerase chain reaction assays, we demonstrate a marked difference in expression of the human epithelial cell adhesion molecule (EpCAM) between normal and (pre)malignant lesions of the stomach and esophagus. Based on this, we explored the feasibility of using EpCAM to achieve gastric and esophageal adenocarcinoma selective gene transfer. Adenoviral vectors redirected to EpCAM using bispecific antibodies against the adenovirus fiber-knob protein and EpCAM specifically infected gastric and esophageal cancer cell lines. Using primary human cells, an improved ratio of tumor transduction over normal epithelium transduction was accomplished by the EpCAM-targeted vectors. This study thus indicates that EpCAM-targeted adenoviral vectors may be useful for gastric and esophageal cancer-specific gene therapy in patients.

BACKGROUND/AIMS: In Epstein-Barr virus (EBV) positive cell lines that are stably infected, three different promoters are known to direct the transcription of EBV nuclear antigen 1 (EBNA1). These are located in the BamHI-C, BamHI-Q, and BamHI-F regions of the viral genome (Cp, Qp, and Fp, respectively). Fp is activated upon induction of the viral lytic cycle. The aim of this study was to investigate the activity of Fp in EBV associated diseases.METHODS: Using reverse transcriptase polymerase chain reaction, a qualitative analysis of EBNA1 promoter usage in various EBV associated diseases was performed.RESULTS: Fp driven transcription was detected in the context of primary infection and/or lytic replication; at least a portion of the Fp driven transcripts encoded EBNA1. Qp driven EBNA1 transcripts were detected in most samples across the range of disorders tested. Cp driven EBNA1 transcripts were detected in the context of immune suppression and in samples containing EBV positive (non-neoplastic) lymphoid cells.CONCLUSIONS: These results confirm the previously proposed "housekeeping" function of the Qp promoter.

Posttransplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplantation recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per mL blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3.4% of the whole blood samples was above the cut-off value (P

This study was done to arrive at a fast and reliable protocol for assessment of fractional volumes of immunohistochemically stained dendritic cells in lymph nodes. Twenty axillary lymph nodes of patients with locally advanced breast cancer were immuno-histochemically stained with an S100 antibody. Fractional volumes of dendritic cells were assessed by stereology based quantitative immunohistochemistry using an interactive video overlay system including an automated microscope. The gold standard percentage of dendritic cells was the fractional volume of S100 stained cells in 500 fields systematically spread over the whole lymph node. Then, in a computer simulation, different sample sizes (1-200 fields of vision) were tested and the coefficient of variation (CV) for each sample size was calculated. The CV dropped with increasing sample size. A sample size of 100 fields of vision appeared to be optimal. Intra- and interobserver reproducibility appeared to be good (correlation coefficients of 0.95 and 0.86, respectively) when re-analyzing the cases with the established protocol. In conclusion, a fast and reliable assessment of the fractional volume of dendritic cells in lymph nodes is possible with semi-automated quantitative immuno-histochemistry. This method will form the base for further clinical studies into the immunological response in lymph nodes of patients with locally advanced breast cancer.

Nucleic acid sequence-based amplification (NASBA) was used for detection of the human cytomegalovirus (CMV) immediate early-1 (IE) and the late pp67 mRNA in 353 blood samples collected from 34 liver transplant patients. The diagnostic value of these assays was compared to that of the pp65 antigenemia assay. Overall, 95 and 42% of the antigenemia-positive samples were IE NASBA and pp67 NASBA positive, respectively. Although the results from pp67 NASBA and the antigenemia assay appeared to correspond poorly, a clear correlation was seen between pp67 NASBA-negative results and low numbers of pp65 antigen-positive cells. Twenty patients (59%) were treated with ganciclovir after the diagnosis of symptomatic CMV infection. Before initiation of the antiviral therapy, the antigenemia assay detected the onset of symptomatic infection in all patients, whereas 95 and 60% of these patients were IE NASBA and pp67 NASBA positive, respectively. Although the sensitivity of IE NASBA was very high, the positive predictive value (PPV) of this assay for the onset of a symptomatic infection was only 63%. The PPV of the antigenemia assay as well as pp67 NASBA was considerably higher (80 and 86%, respectively). Thus, the detection of IE mRNA using NASBA appears to be particularly useful as a marker for early initiation of antiviral therapy in patients at high risk for the development of a symptomatic infection. Also, IE NASBA was found to be more sensitive than the antigenemia assay for monitoring CMV infection during antiviral therapy. On the contrary, pp67 NASBA did not appear to have additional diagnostic value compared to the antigenemia assay.

The development of the nucleic acid sequence-based amplification (NASBA) technology has allowed qualitative determination of human cytomegalovirus (HCMV) immediate-early (IE) and late (pp67) transcripts for monitoring of HCMV infections in the post transplantation period. pp67-mRNA NASBA was shown to be less sensitive than pp65 antigenemia and leukoDNAemia, yet more sensitive than viremia in (i) detecting HCMV infection in both patients and blood samples and (ii) anticipating diagnosis of HCMV infection in solid organ (heart, lung) transplant recipients (SOTR). Use of pp67-mRNA NASBA, as a parameter for initiation of pre-emptive therapy, could be employed as an alternative to detecting antigenemia or DNAemia in SOTR, whereas in bone marrow transplant recipients (BMTR) its use would be too risky because of the delayed detection of HCMV infection. On the other hand, IE-mRNA NASBA was shown to be largely superior to the other assays both in detecting HCMV infection in patients and blood samples and in anticipating diagnosis of HCMV infection. This appears particularly useful in BMTR, where early initiation of antiviral treatment is mandatory in order to prevent the appearance of HCMV interstitial pneumonia. Pre-emptive therapy in BMTR, however, if based upon IE-mRNA NASBA would imply treatment of a greater number of patients as compared with antigenemia- or DNAemia-guided treatment. The clinical usefulness of this approach should be evaluated in prospective trials in the near future, pp67-mRNA NASBA in SOTR with reactivated HCMV infections and IE-mRNA NASBA in BMTR could represent two new virologic parameters to be used as a cutoff for pre-emptive therapy control of HCMV infections in the post-transplant period. Viral transcripts are more direct markers of viral replication in vivo and their disappearance indicates block of the replication process.

Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is expressed in malignancies with latency type II and III and is an important transforming protein. To further study this protein LMP1 was expressed by and purified from recombinant baculovirus infected Sf9 cells. Expression levels of LMP1 in EBV transformed B cell lines and Sf9 cells were analyzed using a newly developed quantitative LMP1-capture ELISA. Highest expression was found in the cell line X50/7 (6.2 ng/10(7) cells), whereas expression levels of recombinant LMP1 (bLMP1) in Sf9 cells reached 506 ng/10(7) cells. Surprisingly bLMP1 could also be detected in the culture medium as a stable full-length protein. Highest expression in Sf9 cells (506 ng/10(7) cells) was observed at 48 h post infection and in the culture medium (1590 ng/ml) at 96 h post infection. Before purification bLMP1 was solubilised using 0.22 m octyl-beta-glucoside at pH 6.0. Purification of bLMP1 using Q-Sepharose FF yielded 10-80 times enriched bLMP1 fractions, indicating that Q-Sepharose can be used for pre-purification. A one-step monoclonal antibody based immunoaffinity chromatography yielded highly purified bLMP1. Although the overall yields (20 microg purified LMP1 from 100 ml culture supernatant) and protein concentrations were low, higher concentrations of >95% purified BLMP1 could be reached after freeze drying.

BACKGROUND: Human cytomegalovirus (CMV) infection is a major cause of morbidity in transplant patients. Early diagnosis and treatment have been shown to improve outcome. We evaluated the suitability of CMV immediate early, early, and late gene expression detected by nucleic acid sequence-based amplification (NASBA) as markers of CMV infection.METHODS: Blood samples were taken immediately before transplant and every one to two weeks after transplantation for 12 weeks from 50 patients undergoing thoracic organ transplantation. CMV-NASBA was performed and results compared with serology, CMV pp65 antigenaemia (CMV-AG) and the development of clinical CMV infection. Patients received "preemptive" anti-CMV therapy with ganciclovir based on the CMV-AG results.RESULTS: CMV immediate early and early gene expression were detected in 87 and 47%, respectively, of patients without other evidence of CMV infection. CMV late gene expression had a sensitivity of 97% for infection (compared with 83% for CMV-AG P=0.06) and a specificity of 93% (compared with 100% P=NS). Late gene expression occurred at the same time as CMV antigenaemia but 1.1 weeks earlier than the threshold of antigenaemia (CMV-AG>10) used to initiate preemptive therapy.CONCLUSION: NASBA provided a standardized tool for the detection of CMV transcripts with a greater sensitivity than the standard antigenemia test. Detection of immediate early and early gene transcripts was not specific for subsequent infection. CMV late gene expression determined by NASBA was an accurate and early marker of CMV infection. Detection of CMV late gene expression could be used to trigger "preemptive" anti-CMV therapy.

While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.

Human herpesviruses are characterized by distinct states of infection. Typically in permissive herpesvirus infection, abundant virus production results in cell lysis. In latent transforming Epstein-Barr virus (EBV) infection, viral proteins that induce cell growth are expressed. The immunodeficiency-associated hairy leukoplakia (HLP) lesion is the only pathologic manifestation of permissive EBV infection; however, within HLP, viral proteins characteristic of latent infection have also been detected. In this study, we further analyzed expression of EBV latent genes and investigated their contribution to the unique histologic phenotype of HLP. Coexpression of lytic and transforming viral proteins was detected simultaneously within individual HLP keratinocytes. LMP1 has now been shown to be uniformly expressed in the affected tissue, and it is associated and colocalizes with tumor necrosis factor receptor-associated factor (TRAF) signaling molecules. Effects induced by activated TRAF signaling that were detected in HLP included activation of NF-κB and c-Jun terminal kinase 1 (JNK1) and upregulated expression of epidermal growth factor receptor (EGFR), CD40, A20, and TRAFs. This study identifies a novel state of EBV infection with concurrent expression of replicative and transforming proteins. It is probable that both replicative and latent proteins contribute to HLP development and induce many of the histologic features of HLP, such as acanthosis and hyperproliferation. In contrast to other permissive herpesvirus infections, expression of EBV transforming proteins within the permissively infected HLP tissue enables epithelial cell survival and may enhance viral replication.

In neoplastic cells of EBV-positive lymphoid malignancies latent membrane protein (LMP1) is expressed. Because no adequate cellular immune response can be detected against LMP1, we investigated whether LMP1 had a direct effect on T lymphocyte activation. In this study we show that nanogram amounts of purified recombinant LMP1 (rLMP1) strongly suppresses activation of T cells. By sequence alignment two sequences (LALLFWL and LLLLAL) in the first transmembrane domain of LMP1 were identified showing strong homology to the immunosuppressive domain (LDLLFL) of the retrovirus-encoded transmembrane protein p15E. The effects of rLMP1 and LMP1-derived peptides were tested in T cell proliferation and NK cytotoxicity assays and an Ag-induced IFN-gamma release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides showed strong inhibition of T cell proliferation and NK cytotoxicity, while acetylated LALLFWL peptides had an even stronger effect. In addition, Ag-specific IFN-gamma release was severely inhibited. To exert immunosuppressive effects in vivo, LMP1 has to be excreted from the cells. Indeed, LMP1 was detected in supernatant of EBV-positive B cell lines (LCL), and differential centrifugation in combination with Western blot analysis of the pellets indicated that LMP1 is probably secreted by LCL in the form of exosomes. The amount of secreted LMP1 in B cell cultures is well below the immunosuppressive level observed with rLMP1. Our results demonstrate direct immunosuppressive properties of LMP1 (fragments) and suggest that EBV-positive tumor cells may actively secrete LMP1 and thus mediate immunosuppressive effects on tumor-infiltrating lymphocytes. Moreover, we demonstrate, for the first time, that transmembrane protein-mediated immunosuppression is not solely restricted to RNA tumor viruses, but can also be found in DNA tumor viruses.

AIM: We studied the expression of TCR zeta-chain on tumour-infiltrating lymphocytes in EBV-positive and EBV-negative cases of Hodgkin's disease (HD), to assess whether downregulation of TCR zeta-chain on tumour-infiltrating lymphocytes might be a mechanism for immune escape of the neoplastic cells.METHODS AND RESULTS: By immunohistochemistry we investigated tissue of 27 cases of primary HD, both paraffin embedded and frozen, for the presence of T-cell receptor complex zeta-chain and other T-cell markers on the reactive cells. Strong membranous staining of TCR zeta-chain was present in all cases in frozen tissue. In contrast, in paraffin-embedded material substantial loss of TCR zeta-chain was detected in old (> 6 years) tissues. However, no differences in either the number of positive cells or their staining intensity were observed in EBV-positive and negative cases of HD as detected in frozen tissue. Storage of paraffin-embedded tissue leads to a rapid and substantial loss of TCR zeta-chain reactivity compared to frozen material of the same HD cases. Staining reactivity of other T-cell markers (CD3, CD4 and CD8) on paraffin-embedded material remained unaffected. Immunofluorescent double-staining confirmed colocalization and coexpression of TCR zeta-chain and CD3.CONCLUSIONS: In frozen biopsies of primary HD TCR zeta-chain was expressed on all reactive CD3-positive cells, both in EBV-positive and EBV-negative cases. This suggests that zeta-chain downregulation is not a likely mechanism whereby neoplastic cells of HD can escape immune surveillance.

Approximately 10% of gastric adenocarcinomas worldwide are associated with human EBV. These carcinomas generally do not express the latent membrane protein 1 (LMP1), the major known EBV oncogene. Recently, another EBV gene [ie., BARF1 (BamHI A rightward open reading frame)] was shown to have transforming and immortalizing capacities. Therefore, in this study, we investigated the expression of BARF1 in EBV-carrying gastric adenocarcinomas in relation to the expression of other latent EBV transcripts. In the present study, 10 of 132 gastric adenocarcinomas tested positive for EBV using EBER1/2-RNA in situ hybridization. We demonstrate BARF1 gene transcription in nine EBV-carrying gastric adenocarcinomas (with sufficient RNA quality) using the BARF1-specific nucleic acid sequence-based amplification assay. In addition, we also detected other latent EBV transcripts (ie., BARF0-, LMP2A-, and Q/K-driven EBNA1 transcripts in these carcinomas using reverse transcription-PCR analysis. No expression of LMP1, EBNA2, and ZEBRA (either at transcription or protein level) was found. In addition, two cases were positive for BHRF1 transcripts, the viral bcl-2 homologue. Thus, together with BARF1 transcription, a unique and distinct EBV latency type has been found in EBV-associated gastric adenocarcinomas. Because BARF1 exerts immortalizing effects on human epithelial cells in vitro and EBV-carrying gastric adenocarcinomas lack the expression of LMP1, the BARF1 gene might act as the viral oncogene in EBV-carrying gastric carcinomas. The BARF1 gene offers an alternative way for EBV-mediated oncogenesis other than LMP1.

BACKGROUND: Previous glioma studies have described separate grading systems for oligodendrogliomas and astrocytomas. Many of these gliomas contain mixtures of neoplastic astrocytes and oligodendrocytes. Prognosis may be related to the percentages of these neoplastic components. Previous survival/grading studies have been limited to histopathological features but have not evaluated the importance of percentages of neoplastic components. This study attempted to perceive the relative importance of percentages of neoplastic astrocytes and oligodendrocytes for definition of astroglial, oligodendroglial and mixed oligoastroglial tumors. After determination of these limits we explored the possibility to develop a grading system for common supratentorial gliomas based on reproducible histopathological features.METHODS: A retrospective study was performed of 362 cases of unselected supratentorial glioma. One hundred and thirty-eight binary and nine continuous histopathological variables, amongst which percentages of neoplastic astrocytes and oligodendrocytes, were scored and related to survival. Only well reproducible histological features were accepted in Cox regression to define glioma grades.RESULTS AND CONCLUSIONS: Supratentorial gliomas appeared to be composed of variable percentages of neoplastic oligodendrocytes and astrocytes, but this spectrum did not correspond to a continuous change in prognosis. Gliomas containing 30% or more neoplastic oligodendrocytes had a slightly better outcome (p < 0.0432) but higher percentages did not further improve prognosis. Percentages of neoplastic astrocytes were not correlated to survival. We therefore propose to designate gliomas containing 30% or more neoplastic oligodendrocytes as oligodendroglial tumors, and others as astroglial tumors. From a prognostic point of view there is no need to recognize mixed oligoastrocytomas. An interesting finding was the recognition of a low grade glioma group with Rosenthal fibers, which had the longest postoperative survival. Another prognosticator of interest concerns the mitotic rate as a continuous variable. Atypical mitoses indicated the worst survival, after necrosis. It was possible to develop a grading system for all supratentorial gliomas using six reproducible histological parameters: necrosis, atypical mitoses, the mitotic rate, endothelial proliferative activity, percentage of neoplastic oligodendrocytes and Rosenthal fibers. This resulted in four grades for astroglial tumors (p < 0.002) and three grades for oligodendroglial tumors (p < 0.008) which differed significantly within each group with respect to survival.

The autooxidation of L-Dopa, a catecholamine used in the symptomatic treatment of Parkinson's disease, generally yields reactive oxygen species and neurotoxic quinones. NAD(P)H:quinone oxidoreductase (NQO) is a flavoenzyme that is implicated in the detoxication of quinones, including those formed during L-Dopa autooxidation. Through the action of this enzyme, deleterious redox-labile quinones are turned into less toxic and more stable hydroquinones that are amenable to further detoxication and/or cellular excretion. In the present study, using primary rat astrocytes and C6 astroglioma as a model to evaluate the neuroprotective response of astroglial cells upon exposure to L-Dopa, we demonstrate that this compound, or more correctly its quinone (auto)oxidation products, up-regulates astroglial NQO in a time- and concentration-dependent way as assessed at the level of mRNA expression, protein level, and enzymatic activity. Moreover, under similar conditions cellular glutathione content was enhanced. It is concluded that, similar to glutathione, the oxidative stress limiting NQO is likely to contribute to the capacity of astroglial cells to protect dopaminergic neurons against L-Dopa, and, hence, may be considered as a potential target for the development of neuroprotective strategies for Parkinson's disease.

Studies on relationships between angiogenesis and tumour cell proliferation have provided conflicting results. This study has therefore investigated the relationships between the number and location of fully automatically identified CD31-positive microvessels and interactively segmented mitoses and necrotic compartments by image processing. These features were studied in ten invasive breast cancers, in the 'hot spots' and in whole tumour sections. Microvessel and mitosis hot spots were topographically close or overlapping and were always located at the periphery of the tumour. The numbers of mitoses and microvessels per mm(2) in the hot spot were strongly correlated with the respective numbers in the whole tumour section, as well as mutually. The ratio of mitoses in the hot spot to the whole tumour section was significantly higher than the corresponding microvessel ratio. Mitoses were preferentially located at a distance of 50-150 microm from microvessels. No significant difference was found between the average distance between mitoses and microvessels in the whole tumour sections and in the hot spot (79 vs. 72 microm), although considerable inter-tumour differences were found (hot spot 43-101 microm, tumour 47-111 microm). The presence of necrotic areas correlated with the number of mitoses per mm(2) and necrosis was in general observed at a distance of more than 150 microm from the microvessels, suggesting that necrotic areas have outgrown their vascular system. These results indicate the usefulness of image processing of whole tumour sections for the identification of proliferation and vascularization hot spots, which are strong prognostic factors in breast cancer. The results also support a close relationship between tumour necrosis and microvessels.

A competitive quantitative PCR (Q-PCR) assay combined with simple silica-based DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competitive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentration. The IS has the same amplicon length and base composition as the wild-type (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Competitive coamplification yields two PCR products that are quantified by enzyme immunoassay or by electrochemiluminescence detection, with probes specific for the 23 differing internal nucleotides. The Q-PCR has a sensitivity of 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quantification of known amounts of plasmid containing the WT EBNA-1 target. Furthermore, we determined EBV genome copy numbers in different cell lines. For EBV quantification in clinical samples, DNA was isolated from lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor peripheral blood samples were positive by Q-PCR. In most of these samples, viral load was less than 2,000 EBV copies/ml of blood. In peripheral blood samples from two AIDS-related non-Hodgkin's lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy. In Burkitt's lymphoma patients, up to 4,592,000 EBV genome copies/ml of blood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship between EBV load and clinical parameters.

OBJECTIVE: To evaluate discrepancies between flow cytometry (FCM) and image cytometry (ICM), ploidy incidence and relation between DNA ploidies and survival in distal bile duct carcinomas (DBDCs).STUDY DESIGN: Forty-four archival tumor samples from patients with DBDC who underwent subtotal pancreatoduodenectomy from 1985 to 1996 were examined for DNA ploidy using FCM and ICM.RESULTS: Overall, 59% (26/44) of the tumors were aneuploid by at least one of the two techniques. We detected more cases of aneuploidy with ICM than FCM in formalin-fixed, paraffin-embedded DBDCs, 62% (21/34) versus 33% (13/40), respectively. When results could be compared, moderate strength of agreement (kappa = .45) was demonstrated. No correlation was found between DNA ploidy by FCM, ICM or combined FCM-ICM and survival time (P = .80, P = .35, and P = .54, respectively).CONCLUSION: Approximately 59% of DNA histograms contained aneuploid cell populations. Although ICM, as compared to FCM, is more sensitive in assessing the ploidy status of DBDC, both methods were complementary. Most discrepancies between FCM and ICM were due to the dilution of aneuploid populations by non-neoplastic diploid cells. DNA ploidy assessment in DBDC did not offer the possibility of improving the ability to predict survival.