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Lung transplantation (LTx) is a lifesaving procedure burdened with limited long-term survival. The most common cause of death after LTx is chronic lung allograft dysfunction (CLAD). Today, useful biomarkers for the detection of CLAD are lacking. Circulating cell-free DNA (cfDNA) is released during cellular decay and can be detected using polymerase chain reaction (PCR). Thus, donor-derived cfDNA in recipient serum indicates cellular decay in the transplanted organ. In the current study, we explore the possibility of using a novel PCR method to detect cfDNA as a biomarker for clinical events, especially CLAD.Four patients were retrospectively tested for levels of both donor and recipient-derived cfDNA using digital droplet PCR after targeted preamplification. The results were correlated to recorded clinical events.All available samples rendered results. Both patients that later developed CLAD showed a persistently elevated ratio between donor-and recipient-derived cfDNA. Also, the mean level of cfDNA was higher in the two patients who later developed CLAD than in patients who did not (p = .0015).This proof-of-concept study suggests that cfDNA quantified with PCR may be used as a biomarker of significant clinical events such as CLAD.

In chronic myeloid leukemia (CML) treatment response is determined by measurements of BCR-AB1L transcripts in peripheral blood by quantitative real-time PCR (qRT-PCR) and a 2-5 fold increase is considered a warning sign. The BCR-ABL1 gene is mainly expressed in myeloid cells whereas quantification of BCR-ABL1 is performed on the nucleated cell fraction of peripheral blood. Hence, leukocyte composition of the nucleated cell fraction may affect the result of BCR-ABL1 quantification. The aim of this study was to investigate if changes in leukocyte composition of peripheral blood had any effect on BCR-ABL1 transcript levels in CML patients. Six CML patients in complete cytogenetic remission (CCgR) performed a maximal physical exercise test. Blood samples were collected before exercise, at maximal exhaustion and after exercise. A biphasic increase in leukocyte count was observed and the relative proportion of granulocytes in peripheral blood changed significantly after exercise compared with baseline (p < 0.001). The BCR-ABL1 transcript level increased significantly following exercise, in nucleated cell fraction of peripheral blood (p < 0.05) but not in isolated granulocytes. In the nucleated cell fraction, the mean BCR-ABL1 transcript level was 3.3-fold (range 0.7-6.8) higher 180 min after exercise compared with baseline (p < 0.01). In conclusion, physical exercise induced significant increases in BCR-ABL1 transcript levels concomitant with changes in leukocyte content of peripheral blood. We therefore suggest that variations in leukocyte composition of peripheral blood, causing pre-analytic variations that affect BCR-ABL1 quantification, have to be accounted for. Consequently, small variations in BCR-ABL1 transcript levels should be interpreted cautiously in CML patients in CCgR.

This article describes a transplant recipient with underlying hypocomplementemic urticarial vasculitis syndrome who expressed persistently Epstein-Barr virus nuclear antigen 1 (EBNA1) in peripheral blood. The patient received a bilateral lung transplant and was subsequently followed with monitoring of EBV expression in peripheral blood. Evaluation of viral expression in peripheral blood, serum, and graft tissue was performed with RT-PCR, Q-PCR, indirect immunofluorescence, anti-peptide assays, and in situ hybridization; samples were collected at various time-points up to 91 days post-transplantation. The patient expressed EBNA1 in 8/10 (80%) of the peripheral blood samples tested during the post-transplantation period, and interestingly, even including the day of transplantation. After analyses of indicative EBV mRNA, EBNA1 expression was found mainly to be Qp-initiated EBNA1, known to be important for EBV maintenance. Anti-EBNA1 epitope mapping showed significantly higher and broader antibody responses to EBNA1 epitopes pre-transplantation when compared to normal controls and a matched lung transplant control. Post-transplantation this response was largely diminished but there were still epitopes significantly higher than controls. Our results show the presence of EBV-positive proliferating cells before onset of intensive immunosuppressive treatment. Although no previous connection between EBV and hypocomplementemic urticarial vasculitis syndrome has been reported, it is tempting to speculate that the continuous EBNA1 expression is not caused by immunosuppression or post-transplant lymphoproliferative disease, but may be a factor involved in the etiology of the autoimmune disease.

Background Cardiac allograft vasculopathy (CAV) limits survival after cardiac transplantation. Tumor necrosis factor-α (TNF-α) may be a key factor in the development of CAV. Two bi-allelic polymorphisms associated with high TNF-α production have been identified in the TNF gene locus, TNFA1/2, at position −308 and TNFB1/2 at +252. We hypothesized that recipient TNFA2 and TNFB2 homozygosity is associated with the development of CAV after heart transplantation. Methods TNF gene polymorphisms were analyzed by multiplex fluorescent solid-phase mini-sequencing in 70 cardiac transplant recipients. Recipients homozygous for TNFA2 or TNFB2 (Group A, n = 29) were compared with recipients heterozygous or homozygous for TNFA1 and TNFB1 (Group B, n = 41). Coronary arteriography was performed annually or when indicated. Cumulative freedom from CAV and survival was calculated according to the Kaplan-Meier test. Results Mean follow-up was 3.8 ± 0.3 years. In Group A, 11 of 29 recipients (38%) developed CAV compared with 9 of 41 (22%) in Group B (p = 0.12). Cumulative freedom from CAV at 3 years was 42% in Group A and 80% in Group B (p = 0.043). In Group A, 11 of 29 recipients (38%) died during follow-up compared with 4 of 41 (10%) in Group B (p = 0.006). Cumulative survival at 3 years was 72% in Group A and 93% in Group B (p = 0.003). Conclusions The results suggest that TNFA2 and TNFB2 allele homozygosity is associated with cardiac allograft vasculopathy and mortality in heart transplant recipients.

Cardiopulmonary bypass induces a systemic inflammatory response characterized by alterations in cardiopulmonary function. Mediators for this morbidity are the cytokines tumor necrosis factor (TNF)-alpha and interleukins. A genomic polymorphism within the TNF locus is associated with increased TNF-alpha levels and high mortality in severe trauma and sepsis. We assessed the relationship of biallelic polymorphisms of the TNF locus in patients undergoing elective cardiac surgery to release of proinflammatory cytokines and cardiopulmonary morbidity. TNF genotypes, plasma concentrations of TNF-alpha, interleukin-6, and cardiopulmonary morbidity were studied in 95 unselected, consecutive patients undergoing routine cardiac surgery. TNF genotypes were determined by the solid-phase minisequencing method. Patients homozygous for the TNFB2 allele (n = 42) displayed larger peak concentrations of TNF-alpha (11.3 +/- 1.3 versus 7.8 +/- 0.7 pg/mL; P = 0.013) and interleukin-6 (153 +/- 27 versus 87 +/- 7 pg/mL; P = 0.010) when compared with patients homozygous or heterozygous for TNFB1 (n = 53). The TNFB2 homozygotes had a higher incidence of left ventricular dysfunction (31% versus 9%; P = 0.029; odds ratio 3.84 [95% confidence interval, 1.40-24.3]), postoperative pulmonary dysfunction (24% versus 6%; P = 0.016; odds ratio 5.21 [95% confidence interval, 1.49-18.3]), and a lower pulmonary oxygenation index (29 +/- 1.9 versus 36.1 +/- 1.8; P = 0.013). Patients homozygous for the TNFB2 allele may develop an enhanced systemic inflammatory response with an increased risk of cardiopulmonary morbidity after cardiac surgery.The associations between tumor necrosis factor (TNF) gene polymorphism, plasma cytokines, and cardiopulmonary function after elective cardiac surgery were evaluated. Patients homozygous for the TNFB2 allele displayed larger concentrations of TNF-alpha and interleukin-6 and had an increased risk of developing left ventricular and pulmonary dysfunction compared with TNFB1 homo- or heterozygotes.

Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE epsilon2, epsilon3 and epsilon4) and has important functions in nerve development and repair. Inheritance of the 4 allele is a major risk factor for the development of Alzheimer's disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The epsilon4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of epsilon3 was significantly increased (P=0.005), suggesting that epsilon4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the epsilon4 allele in natural selection.

The pathogenesis of spontaneous abortion is complex, presumably involving the interaction of several genetic and environmental factors. The methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms are commonly associated with defects in folate dependent homocysteine metabolism and have been implicated as risk factors for recurrent embryo loss in early pregnancy. In the present study we have determined the prevalence of combined MTHFR C677T and A1298C polymorphisms in DNA samples from spontaneously aborted embryos (foetal death between sixth and twentieth week after conception) and adult controls using solid-phase minisequencing technique. There was a significant odds ratio of 14.2 (95% CI 1.78-113) in spontaneously aborted embryos comparing the prevalence of one or more 677T and 1298C alleles vs the wild type combined genotype (677CC/1298AA), indicating that the MTHFR polymorphisms may have a major impact on foetal survival. Combined 677CT/1298CC, 677TT/1298AC or 677TT/1298CC genotypes, which contain three or four mutant alleles, were not detected in any of the groups, suggesting complete linkage disequilibrium between the two polymorphisms. The present finding of high prevalence of mutated MTHFR genotypes in spontaneously aborted embryos emphasises the potential protective role of periconceptional folic acid supplementation.

There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

A polymorphism consisting of a deletion near the 5' splice site of exon 18 on the alpha2-macroglobulin (A2M) gene (A2M-2) has been suggested to be associated with Alzheimer's disease (AD) in family-based studies. We studied the A2M-2 allele together with the ApoE alleles in a large series on patients with AD (n = 449) and age-matched controls (n = 349). Neuropathologically confirmed diagnoses were available in 199 cases (94 AD and 107 control cases). We found no increase in A2M-2 genotype or allele frequencies in AD (27.5% and 14.6%) versus controls (26.4% and 14.9%). In contrast, a marked increase (p < 0.0001) in ApoE epsilon4 genotype or allele frequencies was found in AD (66.6% and 41.2%) as compared with controls (29.8% and 16.5%), suggesting sufficient statistical power in our sample. No relation was found between the A2M-2 and the ApoE epsilon4 allele. No change in A2M exon 17-18 mRNA size or sequence or A2M protein size was found in cases carrying the A2M-2 deletion, suggesting that there is no biological consequences of the A2M intronic deletion. No change in A2M protein level in cerebrospinal fluid was found in AD, suggesting that the A2M-2 allele does not effect the A2M protein expression in the brain. The lack of an association between the A2M-2 allele and AD in the present study, and the lack of abnormalities in the A2M mRNA or protein suggest that the A2M-2 allele is not associated with AD.

The purpose of this study was to investigate whether Epstein-Barr virus (EBV) is associated with acetowhite lesions of the portio cervix, demonstrating koilocytosis and/or cervical intraepithelial neoplasia (CIN) I-III. The study group comprised 37 women admitted to the Department of Gynaecology and Obstetrics, Sahlgrenska University Hospital, Go teborg because of pathological colposcopy or cytology of the portio cervix. Colposcopically, all exhibited acetowhite lesions of the portio cervix. Cells were sampled with a cytobrush for examination for EBV and human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) and a biopsy was taken for histopathology. Biopsies from 5 patients positive for EBV by PCR in cervical cell samples were examined by an in situ hybridization technique for EBER (Epstein-Barr virus encoded RNA), RNAs expressed in latent EBV infection. The control group consisted of women attending the Department of Dermato-Venereology at the same hospital for STD check-up. These had a normal cytology and no signs of acetowhiteness of the portio cervix. In the study group, EBV DNA was found in 30% and HPV DNA in 51%. In the control group 57% exhibited EBV DNA and 23% HPV DNA. EBV was not found to be a predictive factor in the development of koilocytosis and/or CIN I-III. HPV was a predictive factor in acetowhite, koilocytotic lesions. No expression of EBER was found in the 5 biopsies examined by in situ hybridization.

The HLA-related hemochromatosis mutation C282Y is thought to have originated in Ireland in a person with HLA-A3-B14 and was spread by Vikings. Irish people with two HLA-A3 alleles had a high risk of hemochromatosis. In this study, from west Sweden, we wanted to test these hypotheses. Methods: HFE mutations in controls, bone marrow donors with HLA-A3/A3 and patients with hemochromatosis. HLA haplotypes, extended haplotype analysis and pedigree studies. Results: The allelic C282Y frequency 0.04, (CI 0.01–0.07) was lower (P

In polycythemia vera (PV) and essential thrombocythemia (ET) the discovery of the presence of JAK2 V617F mutations indicates that tyrosine kinase activation is a common pathogenetic mechanism in Ph- MPD.[1][1] The main therapeutic goal is to diminish the risk of clinical complications without

The U leader exon in the 5' untranslated region of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site, the EBNA internal ribosome entry segment (IRES), which promotes cap-independent translation and increases the expression level of the EBNA1 protein. It was previously reported that immunosuppressed organ transplanted patients showed an alternatively spliced EBNA1 transcript, excluding the EBNA IRES element. To further investigate the function of the EBNA IRES, sequence analysis of the EBNA IRES mRNA was performed in samples from seven organ transplant patients. Two nucleotide changes, G --> A at position 67531 and C --> U at position 67585 were found in the EBNA IRES mRNA, relative to the corresponding genomic Epstein-Barr virus (EBV) sequence in all patients. Moreover, the patient derived EBNA IRES mRNA was shown to differ from the IRES mRNA derived from the cell line B95.8 at position 67531 and from the cell lines Rael and P3HR1 at positions 67531 and 67585. cDNA from the various EBNA IRES sequences were cloned into bicistronic vectors, respectively, and used in transient transfection experiments in six human cell lines. The patient specific sequence significantly decreased the IRES activity in T-cells, while the base changes had no significant impact on the activity in B- or in epithelial cells. The genetic mechanisms behind EBV-associated diseases are complex, involving gene regulation by alternative promoters, alternative splicing, and translational control. The nucleotide changes in the patient specific EBNA IRES transcript and its influence on the translational activity, might illustrate new strategies utilised by the EBV to adapt to the immune control in patients with EBV associated diseases.

A case of congenital toxoplasmosis is presented, where diagnosis by PCR amplification of Toxoplasma gondii DNA from peripheral blood led to early treatment of the infant and seemingly normal brain development despite the presence of intracranial calcifications at birth. The mother, who experienced a subclinical infection during pregnancy, was PCR-positive for toxoplasma DNA in a sample of peripheral blood drawn after delivery.

Point mutations within the ABL kinase domain of the BCR-ABL gene are associated with clinical resistance to imatinib mesylate in chronic myeloid leukemia (CML). To obtain more information about the association between BCR-ABL mutations and type of imatinib resistance, we studied 30 early chronic phase (CP) CML patients, commencing imatinib therapy, using a conventional sequencing technique. Seven patients treated in late CP and three patients treated in the accelerated phase were included for comparison. Blood samples were collected before and every third month during imatinib therapy. Mutations were not seen in any blood sample collected before start of therapy. During imatinib treatment, 2 of the 30 early CP patients acquired point mutations and both of them had other signs of imatinib resistance. None of the five early CP patients with a complete hematologic response (HR), but no cytogenetic response at 12 months, displayed any missense mutation. Likewise, none of 12 early CP patients with detectable BCR-ABL transcripts but in complete hematologic and cytogenetic remission at 12 months displayed any mutation. We conclude that screening early CP patients for BCR-ABL mutations before start of imatinib therapy is not cost-effective. BCR-ABL kinase domain mutations do not appear to explain cytogenetic or molecular (detectable BCR-ABL transcripts by polymerase chain reaction) disease persistence in patients otherwise in stable disease. However, in patients with signs of expanding disease burden, a search for BCR-ABL mutations is warranted.

In order to identify patients at risk for developing post-transplant lymphoproliferative disease (PTLD), a sensitive nested RT-PCR method for detection of EBNA1 gene expression in peripheral blood cells was used. EBNA1 expression in peripheral blood samples from 60 organ recipients was analyzed and compared with 24 healthy controls in a retrospective study. Overall, EBNA1-positive samples were detected at least once in 43% of the transplant patients with post-transplant lymphoproliferative disease, in 18% of the other transplant patients and in none of the healthy controls. The odds ratio for EBNA1 expression in patients with post-transplant lymphoproliferative disease was 3.42 (95% CI=1.02-11.54) compared to other transplant recipients. Together with normal EBV Q promoter initiated EBNA1 transcripts, an alternatively spliced form was expressed in peripheral blood cells in the above-mentioned transplant patients. This transcript lacks the U leader exon in the 5'-untranslated region (UTR). We have previously identified and characterized a functional internal ribosome entry site, the EBNA IRES, in the untranslated U leader exon of EBNA1. Transfection experiments with EBNA1 coding plasmids followed by Western blot showed that the EBNA IRES promotes cap-independent translation and increases the EBNA1 protein level. The alternative EBNA1 transcript lacking this function is expressed in the majority of the investigated EBNA1-positive patient samples as well as in some EBV-positive B-cell lines. Alternative splicing in this form gives EBV potential to regulate the translation of EBNA1 by modifying the 5' UTR. These findings indicate a new mechanism for EBNA1 expression in vivo.

The study was designed to investigate the influence of hydroxyurea (HU) treatment on PRV-1 expression. Eighteen newly diagnosed patients with essential thrombocythemia (ET) or polycythemia vera (PV) were included. HU significantly increased PRV-1 gene expression in the early stage of treatment.

Survival among chronic myelogenous leukemia (CML) patients can be linked to the reduction in leukemic cell burden. Treatment with imatinib mesylate results in a high frequency of complete cytogenetic response, which can be further stratified using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). We have serially monitored peripheral blood and bone marrow BCR-ABL transcripts using qRT-PCR in CML patients commencing imatinib therapy, and compared the results with bone marrow cytogenetics. Seventeen patients (aged 25-74 yr) with Philadelphia chromosome positive CML in first chronic phase were treated with imatinib targeting a dose of 400 mg/d. The median follow up is 30 mo (range 9-33 mo). Every third month the product of the BCR-ABL fusion gene was evaluated in both blood and bone marrow specimens by real-time RT-PCR using the TaqMan probe system. In 113 simultaneously obtained blood and bone marrow samples, the BCR-ABL transcript values agreed well with cytogenetic data. Blood and bone marrow specimens gave comparable values for BCR-ABL transcripts. Before start of imatinib therapy there was a considerable variation in BCR-ABL transcripts among the patients, ranging approximately one log (base 10). Similarly, patients with a complete cytogenetic response following imatinib therapy had variable BCR-ABL transcript levels, ranging at least three logs (base 10). The major decline in BCR-ABL transcripts occurred within 6 mo after start of imatinib therapy. The decline in BCR-ABL transcripts, following imatinib therapy, appears to level off at 12-15 mo. Two late responders were identified with a still decreasing level in BCR-ABL transcripts after 24 mo of treatment. It is concluded that BCR-ABL mRNA quantification in peripheral blood is suitable for routine monitoring of the response to treatment and long-term disease status in CML, especially in patients who have achieved a complete cytogenetic response. A plateau in BCR-ABL transcripts seems to have been reached after 12-15 mo of imatinib treatment; however, some "late responders" are seen.

In the absence of pathognomonic markers, the diagnosis of the two chronic myeloproliferative disorders polycythemia vera (PV) and essential thrombocythemia (ET) has relied on a set of clinical and laboratory criteria (1)(2)(3)(4)(5). The cloning of the cell surface receptor polycythemia rubra vera-1 (PRV-1) has recently been described (6), and the consistent overexpression of PRV-1 mRNA observed in PV patients indicates that this might constitute a new diagnostic marker for the disease. In the initial cohort examined by Northern blot analysis, PRV-1 expression was increased in all PV patients examined as well as in some ET patients, but not in healthy controls (6). These results have also been verified and extended using a quantitative reverse transcription-PCR (RT-PCR) assay. All PV as well as 50% of ET patients displayed increased PRV-1 expression (7)(8). Patients with secondary erythrocytosis and healthy controls tested showed PRV-1 concentrations within the reference interval. Interestingly, the observed increase in PRV-1 mRNA expression does not lead to a corresponding increase in protein expression on the cell surface (9). Erythropoietin-independent colony growth and PRV-1 overexpression seem to go hand in hand in both PV and ET patients (7), raising the hope that RT-PCR for PRV-1 could replace the need for the technically demanding erythropoietin-independent colony assay, although a recent report suggests that the erythropoietin-independent colony growth assay is a more reliable method (10). The aim of the present work was to develop a quantitative RT-PCR method to measure PRV-1 transcripts in whole-blood leukocytes and to determine the potential usefulness of the method in the differential diagnosis of polycythemias and thrombocytosis. The assay was compared with a method using isolated granulocytes (7)(9) to assess whether granulocyte purification is necessary before RNA extraction. Granulocyte fractionation is cumbersome to standardize, and …

Essential thrombocythaemia (ET) is a heterogeneous disorder with respect to plasma erythropoietin concentration at diagnosis and clonality of haematopoiesis. Polycythaemia rubra vera-1 (PRV-1) positivity, i.e. PRV-1 mRNA overexpression, is known to be present in the vast majority of patients with polycythaemia vera and also in some patients with ET. In the present study, PRV-1 expression was quantified by real-time polymerase chain reaction in 70 ET patients; 17 of them (24%) were found to be PRV-1 positive. Ten of the 17 PRV-1 positive ET patients had experienced thromboembolic complications compared with 14 of 53 PRV-1 negative patients, the difference between the two groups being statistically significant (P=0.02). In addition, the frequency of total vascular complications, thromboembolic events and major bleedings, was significantly higher in the group of PRV-1 positive as compared with PRV-1 negative ET patients (P=0.03). The time from diagnosis of ET to the requirement of platelet-lowering therapy was significantly shorter in PRV-1 positive compared with PRV-1 negative ET patients (P=0.014). It can be concluded that PRV-1 positive patients appear to suffer from a more aggressive disorder with increased risk for vascular complications and a greater need for platelet-lowering therapy, compared with PRV-1 negative ET patients.

Approximately 45% of newly diagnosed patients with essential thrombocythaemia (ET) demonstrate subnormal plasma erythropoietin (EPO) concentrations, which constitutes a risk factor for occlusive vascular events. In 58 ET patients, a possible association between polycythaemia rubra vera-1 (PRV-1) overexpression and subnormal plasma EPO was investigated, which was always measured prior to the institution of platelet lowering agents. At the time when PRV-1 expression was measured, 28 of 58 (48%) ET patients had received platelet lowering treatment. PRV-1 expression was measured by quantitative real-time reverse transcription-polymerase chain reaction assay of mRNA extracted from purified peripheral blood buffy coat. The cycle threshold (CT) value of PRV-1 was determined and was divided with the CT value for the housekeeping GAPDH gene transcript. A quotient0.93 was defined as PRV-1 positive. Of the ET patients 12 of 58 (21%) were PRV-1 positive and 19 of 58 (33%) demonstrated subnormal plasma EPO. In the 58 ET patients there was a significant association between low plasma EPO and PRV-1 positive results (P = 0.001). The 30 ET patients who had not received any platelet lowering treatment showed a significant (P = 0.005) relation between PRV-1 positivity and subnormal plasma EPO. No such relationship was present in the 28 ET patients who had received prior treatment with the above drugs (P = 0.147).

In a previous report we demonstrated Epstein-Barr virus expression in cutaneous squamous cell carcinomas from heart transplant recipients. In a comparative study, skin lesions from renal allograft recipients were investigated for the presence of Epstein-Barr virus. Thirty cutaneous squamoproliferative lesions from 10 kidney transplant recipients were examined for Epstein-Barr virus-specific gene expression. The techniques used for detection were polymerase chain reaction, in situ hybridization and immunohistochemistry. Epstein-Barr virus DNA was not detected by polymerase chain reaction in the neoplasias, and only single Epstein-Barr virus-positive carcinoma cells were shown by in situ hybridization in three cases of infiltrative squamous cell carcinomas. Immunohistochemistry for Epstein-Barr virus latent membrane protein 1 showed a negative result in all samples. These findings differ from our earlier investigations of cutaneous squamous cell carcinomas from heart transplant recipients where Epstein-Barr virus expressions were common. This may indicate that the part Epstein-Barr virus plays in the development of post-transplant, cutaneous squamoproliferative disorders is related to type of organ transplantation and/or grade of immunosuppression.

of progression in male patients who otherwise do not have a major burden of documented and important Background. The insertion/deletion (I/D) polymorph- ism of the angiotensin-converting enzyme (ACE ) gene prognostic factors for progressive renal insuYciency. determines the concentration of ACE in serum and local tissues. The role of this polymorphism in pro- progression gressive chronic renal disease is still not fully clear. Methods. We analysed the impact of the D/D poly- morphism on the rate of decline in renal function in patients with non-diabetic, chronic progressive renal Introduction insuYciency. Seventy non-diabetic patients, aged 21-69 years at baseline, with moderately advanced Polymorphism of the angiotensin I-converting enzyme renal insuYciency due to primary chronic renal (ACE) gene was identified in 1990 (1). The insertion/ disease were followed for an average of 3 years with deletion (I/D) polymorphism of the ACE gene has repeated measurements of their glomerular filtration been shown to determine the concentration of ACE in rate (GFR). Their mean GFR at baseline was serum and in local tissues (2,3). The pathophysiological 41 ml/min/1.73 m2 body surface area (BSA). The poly- role of this polymorphism has been discussed and it merase chain reaction (PCR) amplification method has been proposed that the DD genotype of the ACE was used to detect the I/D polymorphism of the ACE gene may be an independent risk factor for cardiac gene. GFR was measured as the clearance of 51Cr- disease (4-8). Whether the I/D polymorphism plays EDTA and the individual rate of progression was an important role in renal disease is still controversial calculated using linear regression. (9). Results. The distributions of the genotypes were: D/D In diabetic nephropathy there are conflicting results 30%, I/D 49%, and I/I 21%. The rates of progres- regarding whether patients with the DD genotype sion in the three ACE genotype groups were an progress in their renal insuYciency at a higher rate annual decline in renal function of '4.2 (SD (10-14). In the meta-analysis of Staessen et al. (8) it 4.6) ml/min◊1.73 m2 BSA in the D/D group, '2.7 was concluded that the deletion polymorphism sets the (SD 3.4) in the I/D group and '1.7 (SD 3.4) in the stage for renal microvascular complications. Studies in I/I group (ANOVA P=0.12). In patients with pro- patients with IgA nephropathy have suggested that the teinuria below 3.5 g/24 h, the D/D group had a signi- I/D polymorphism may play a role in the progression ficantly higher rate of progression than patients with of this particular renal disease (15-20). In three recent, the I allele. The same was found in a separate analysis but rather small, studies it was reported that the decline when only patients with normal apoliprotein B (below in renal function was also more rapid in patients with 155 mg/dl ) levels were analysed. Furthermore, the D/D the DD genotype in other non-diabetic renal patient genotype was a significant predictor of a more rapid categories (21-23). Furthermore, it has also been decline in renal function in male, but not female, discussed whether the therapeutic response to ACE- patients. inhibition is dependent on the ACE gene polymorph- Conclusion. The results in this study in non-diabetic ism (9,15,17,21,24,25). patients with chronic renal disease indicate that the As pointed out in two recent reviews (9,26 ), a presence of the D allele in the ACE genotype may be common problem in addressing the question of whether of particular importance as a predictor of a high rate gene polymorphism plays a role in various renal dis- eases is the limited size of the patient samples, with

Four promoters, Cp, Wp, Fp and Qp, are known to participate in transcription of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene in EBV-infected cell lines. The promoters are used differentially during the different phases of infection and establishment of the stages of latency. This has raised questions about the regulation of the promoters and the molecular mechanisms underlying the switches between them. To obtain a measure of the activity of the different EBNA1 transcription units in EBV-transformed cell lines of different phenotypes, RNA probes were constructed that allowed the detection and relative quantification, by RNase protection analysis, of EBNA1 transcripts initiated at Fp and Qp and, in an indirect manner, Cp/Wp. RNase protection and PCR assays were performed with cytoplasmic RNA from B-lymphoid cell lines in latency stages I, II-III and III and after induction of the virus lytic cycle. The experiments demonstrated that, in addition to previously identified EBNA1 transcripts, cell lines of all latency types also contained different mRNAs that carried sequences from the EBNA1-encoding K exon. Induction of the virus lytic cycle resulted in low levels of an FpQ/U/K-spliced transcript. However, there was a large increase of FpQ- and FpQ/U-spliced transcripts with unknown 3' sequences. Furthermore, a new transcript, initiated at an unidentified site 5' of the BamHI f/K cleavage site and continuing through BamHI K into the EBNA1-encoding K exon without interruption, was produced in substantial amounts in the lytic cycle.

Summary: Oral hairy leukoplakia (OHL), thought to be caused by Epstein-Barr virus (EBV), shows similar histological and clinical features to human papillomavirus (HPV)-related acetowhite lesions of the vulva. We thus aimed to investigate the role of both HPV and EBV in men with acetowhite lesions of the penis. HPV but not EBV was significantly associated with penile acetowhite lesions showing koilocytosis compared with normal penile skin (12/20 versus 5/20, P 0.02). HPV (5/20) and EBV (6/20) was detected in oral mucosa of some of these individuals. These results confirm an aetiological association between HPV and acetowhite penile lesions showing koilocytosis. HPV and EBV carriage in the oral mucosa is relatively common in young sexually active men. <

Detection of cytomegalovirus (CMV) DNA by the polymerase chain reaction (PCR) in samples of cerebrospinal fluid (CSF) has been shown to be a sensitive method of diagnosing CMV disease in the central nervous system. Since CMV causes latent infection in white blood cells, an unanswered question is whether detection of latent CMV DNA in the cell fraction of CSF samples by PCR is possible in seropositive patients. In a prospective study, the finding of CMV DNA in CSF of CMV seropositive patients with suspected viral infection of the central nervous system (CNS) was evaluated clinically. Fractionation of 64 CSF samples from seropositive patients was carried out before analysing the samples for CMV DNA by PCR. In four of the five patients who had CMV DNA in the cell pellet and/or supernatant, the clinical data suggested CMV-associated neurological disease. The remaining 59 samples were negative in both pellet and supernatant. In addition, 11 CSF samples with high cell counts from patients with bacterial meningitis were examined for CMV DNA and found to be negative in 10 patients and positive in 1. One hundred thirty two uncentrifuged CSF samples were used as negative controls. The results of the study indicate that detection of CMV DNA in CSF samples by PCR correlated well with disease and was not due to latent CMV infection.

A group of 91 women attending the STD-clinic, Department of Dermato-venereology, Sahlgrenska Hospital, Gothenburg, were screened for EBV DNA and HPV DNA of the cervix with the PCR-technique. Presence of EBV DNA was demonstrated in 35 (38%) women and HPV DNA in 30 (33%) women. Fourteen (15%) women had both EBV DNA and HPV DNA present. Without the colposcope 20 of these women had macroscopic signs of HPV infection on the vulva and/or vagina and 71 had no signs of infection. Presence of EBV DNA was not correlated to clinical signs of HPV infection.

Acetowhite lesions in the vulva disclosing koilocytosis have been related to infection by human papilloma virus (HPV). Because of the clinical resemblance of these lesions to oral hairy leukoplakia (OHL), and EBV-manifestation, 23 women with acetowhite koilocytotic lesions in the vulva were examined. The PCR-technique was used to detect EBV DNA as well as HPV DNA in 17% of 23 patients examined. In a control group of 19 patients EBV DNA was detected in 11% and HPV DNA in 42% of cell samples from normal vulvar mucosa. EBV DNA has not previously been demonstrated in the vulvar mucosa, and this virus might be related to subclinical acetowhite lesions.

The high sensitivity of nested polymerase chain reaction (PCR) offers the possibility of rapid detection of cytomegalovirus (CMV) DNA in serum. Five consecutive serum samples were examined from 52 human immunodeficiency virus (HIV)-seropositive patients (19 of whom had clinically presumed diagnosis of CMV chorioretinitis). Presence of CMV DNA in serum was shown to precede development of clinical disease. Eleven patients who developed chorioretinitis were positive for CMV DNA in serum samples obtained 3 months before clinical disease, and 3 retinitis patients who initially were negative for CMV DNA became positive with the onset of clinical retinitis. In contrast, 29 of 33 HIV-seropositive subjects without clinical CMV chorioretinitis and matched with respect to age and CD4 T cell numbers were negative for CMV DNA in all 5 serum samples. Thus, the presence of CMV DNA in serum analyzed by PCR is a good predictive marker of CMV retinitis in HIV-seropositive subjects. A positive PCR results supports the clinical diagnosis and may be useful for monitoring response to antiviral treatment.

An Epstein-Barr virus (EBV)-negative human lymphoid B-cell line, DG75, was stably transfected with recombinant selection vectors that carry a subfragment of the BamHI WYH region (nucleotides 44664 to 50628), the BamHI K fragment, or a subfragment of the EcoRI D region (nucleotides 166614 to 170149) of B95-8 EBV DNA. These fragments contain the coding exons for the EBV-determined nuclear antigens EBNA2 and EBNA1, and the membrane antigen LMP, respectively. Antigen expression of the cells was detected by immunofluorescence. EBNA2 was expressed in 80-100% of the transfected cells, in contrast to EBNA1 which was expressed in only 25%, and LMP in only about 5% of the cells. Humoral antibody responses were measured by immunofluorescence and compared to cellular immunity as determined by the leukocyte migration inhibition (LMI) technique. Extracts from transfected cell lines expressing EBNA1, EBNA2 or LMP elicited an LMI response with cells from healthy EBV-seropositive individuals whereas the extract from the parental DG75 cell line did not. The results demonstrate the value of stably transfected cell lines expressing a defined EBV antigen for the monospecific analysis of host responses to the EBV-encoded antigen complex in growth-transformed cells.

Recombinant vectors carrying DNA fragments from the BamHI E region of the B95-8 Epstein-Barr virus (EBV) genome were transfected into COS-1 cells, and the transient expression of EBV-encoded nuclear antigens (EBNAs) was analyzed by using polyvalent human antisera and rabbit antibodies to synthetic peptides. Vector DNA containing two rightward open reading frames in the BamHI E fragment, BERF2a and BERF2b, induced the expression of a nuclear antigen identical serologically and with respect to size to the larger of the two polypeptides previously designated as EBNA4 in B95-8 cells. An antigen corresponding to the smaller polypeptide was induced in cells transfected with constructs that contained two neighboring reading frames, BERF3 and BERF4. This antigen also reacted with a rabbit antiserum to the synthetic peptide 203, deduced from BERF4. Thus, the findings show that the two components of the EBNA4 doublet in B95-8 cells are encoded by separate genes. The antigen encoded by BERF2a and/or BERF2b has been designated as EBNA4 and the antigen encoded by BERF3 and/or BERF4 has been designated as EBNA6. Polyvalent human antisera detected EBNA4 and EBNA6 in 9 of 11 lymphoid cell lines carrying independent EBV isolates. In the remaining two lines, either EBNA4 or EBNA6 was not detectable.

Six non-compromised patients with cytomegalovirus (CMV) associated meningoencephalitis are described. CMV was isolated from the cerebrospinal fluid (CSF) in 2/4 cases, while the diagnosis was based on an 8-fold rise in CMV-specific serum IgG antibodies and intrathecal antibody production against CMV in one case. By the polymerase chain reaction (PCR) CMV DNA was detected in the CSF in 5/5 cases and in serum in 3/4 cases. In one patient who had an Influenza A infection, both CMV and Epstein-Barr virus DNA were detected by PCR in the CSF. In 4 patients possible triggering events could be identified. Symptoms and signs indicating a multifocal brain involvement were present in 4 patients. The outcome was generally favourable except for sequelae in form of slight dysphasia in one case.