During the last few years, cell-free DNA (cfDNA) has emerged as a possible non-invasive biomarker for prediction of complications after lung transplantation. We previously published a proof-of-concept study using a digital droplet polymerase chain reaction (ddPCR)-based method for detection of cfDNA. In the current study, we aimed to further evaluate the potential clinical usefulness of detecting chronic lung allograft dysfunction (CLAD) using three different ddPCR applications measuring and calculating the donor fraction (DF) of cfDNA as well as one method using the absolute amount of donor-derived cfDNA. We analyzed 246 serum samples collected from 26 lung transplant recipients. Nine of the patients had ongoing CLAD at some point during follow-up. All four methods showed statistically significant elevation of the measured variable in the CLAD samples compared to the non-CLAD samples. The results support the use of ddPCR-detected cfDNA as a potential biomarker for prediction of CLAD. These findings need to be validated in a subsequent prospective study.

Background: Heart transplantation (HTx) relies on an endomyocardial biopsy for rejection surveillance. Cell-free DNA (cfDNA) is released into the circulatory system from the cells of the recipient (recipient-derived rd-cfDNA) as well as from the graft (donor-derived dd-cfDNA) and is determined by the analysis of a blood test. There is growing evidence that the donor fraction (DF, the ratio of dd-cfDNA to all cfDNA) corresponds to the integrity of the graft. However, it is important that rd-cfDNA levels vary greatly in different, sometimes clinically silent circumstances, which in turn makes DF the sole measure of graft status vulnerable to bias. Methods: Using a digital PCR-based method that allowed us to quantify separate values of rd-cfDNA, dd-cfDNA, and DF, we investigated a prospective cohort of 52 patients during their first year after HTx. Results: We found median DF levels of approximately 0.1%, which is in accordance with the literature. We present patients with various clinical scenarios after HTx, including an uncomplicated clinical course, several infections (viral and bacterial), acute and chronic rejection, and false-negative and false-positive rejections. Conclusions: Quantification of circulating cfDNA reflects a live-view of cell turnaround in the recipient and graft. Measuring the DF alone may not be sufficient to fully understand the complex biology of cfDNA after HTx.

OBJECTIVE:Cell-free DNA (cfDNA) is used as a biomarker after transplantation to detect graft injury, relying on the donor fraction (DF). We have established a PCR-based approach allowing us to separately quantify absolute values of dd-cfDNA and recipient-derived cfDNA (rd-cfDNA). We aimed to present typical clinical scenarios after heart transplantation (HTx) to illustrate the advantages of absolute cfDNA values over DF. METHODS:We used the cfDNA results of our cohort (509 samples of 52 patients followed during the first year after HTx) as background and determined the trajectories of cfDNA in specific clinical situations. We profiled an uncomplicated clinical course, viral and bacterial infections, acute and chronic rejection, and false-negative and false-positive rejections in six patients (five adults, one child). RESULTS:There was a substantial discrepancy between relative (DF) and absolute cfDNA-levels in several clinical situations. Rd- and dd-cfDNA were independently elevated during episodes of rejection and infection and were better suited to depict treatment response than DF alone. CONCLUSIONS:Absolute quantification of cfDNA may offer clinically relevant information additive to DF in various situations after HTx and could be helpful for more accurate monitoring of diagnosis and treatment of rejection.

AbstractBackgroundLung 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.MethodsFour 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.ResultsAll 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).ConclusionsThis 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.

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.

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.

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 posttransplantation 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 posttransplant lymphoproliferative disease, but may be a factor involved in the etiology of the autoimmune disease.

Most patients with a chronic phase of chronic myeloid leukemia (CML) treated with imatinib mesylate achieve a cytogenetic remission, but in the majority, residual disease is detectable by RT-PCR. The mechanisms by which residual leukemic cells survive imatinib treatment are unresolved. However, induction of apoptosis in leukemic stem cells and immunotherapy are currently under investigation. We studied the mRNA expression of apoptosis-related genes in peripheral blood mononuclear cells from chronic-phase CML patients before imatinib treatment. It was found that their BCL2 and BAD expression was significantly different compared to the normal controls, and a lower BAD expression was associated with a better molecular response to imatinib treatment at 12 months.

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.

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.

References 1. Carpenter KH, Wiley V. Application of tandem mass spectrometry to biochemical genetics and newborn screening. Clin Chim Acta 2002;322:1–10. 2. Carreiro-Lewandowski E. Newborn screening: an overview. Clin Lab Sci 2002;15:229–30. 3. Jones PM, Bennett MJ. The changing face of newborn screening: diagnosis of inborn errors of metabolism by tandem mass spectrometry. Clin Chim Acta 2002;324:121–8. 4. Chace DH, Kalas TA, Naylor EW. The application of tandem mass spectrometry to neonatal screening for inherited disorders of intermediary metabolism. Annu Rev Genomics Hum Genet 2002;3:17–45. 5. Wilcken B, Wiley V, Hammond J, Carpenter K. Screening newborns for inborn errors of metabolism by tandem mass spectrometry. N Engl J Med 2003; 348:2304–12. 6. Chamoles NA, Blanco M, Gaggioli D. Fabry disease: enzymatic diagnosis in dried blood spots on filter paper. Clin Chim Acta 2001;308:195–6. 7. Chamoles NA, Blanco MB, Gaggioli D, Casentini C. Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper. Clin Chem 2001;47:2098–102. 8. Umapathysivam K, Hopwood JJ, Meikle PJ. Determination of acid -glucosidase activity in blood spots as a diagnostic test for Pompe disease. Clin Chem 2001;47:1378–83. 9. Chamoles NA, Blanco M, Gaggioli D, Casentini C. Gaucher and Niemann-Pick diseases—enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards. Clin Chim Acta 2002;317: 191–7. 10. Chamoles NA, Blanco M, Gaggioli D, Casentini C. Tay-Sachs and Sandhoff diseases: enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards. Clin Chim Acta 2002;318: 133–7. 11. Galvin-Parton PA. Screening for GALC to make neonatal diagnosis and initial neonatal stem cell treatment with umbilical cord blood. Pediatr Transplant 2003;7:83–5. 12. Parvathy MR, Ben-Yoseph Y, Mitchell DA, Nadler HL. Detection of Krabbe disease using tritiated galactosylceramides with medium-chain fatty acids. J Lab Clin Med 1987;110:740–6. 13. Mei JV, Alexander JR, Adam BW, Hannon WH. Use of filter paper for the collection and analysis of human whole blood specimens. J Nutr 2001;131:1631S–6S.

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 …

Eukaryotic translation can be initiated either by a cap-dependent mechanism or by internal ribosome entry, a process by which ribosomes are directly recruited to structured regions of mRNA upstream of the initiation codon. Here we report the finding of an internal ribosome entry site (IRES) in the untranslated region of the Epstein–Barr nuclear antigen 1 (EBNA1) gene. EBNA1 is the only nuclear protein expressed in all known states of Epstein–Barr virus (EBV) latency and in the virus lytic cycle, and is required for the maintenance of the EBV episome. Using cDNA reporter constructs and in vitro transfection assays, we found that sequences contained in the 5′ untranslated region (UTR) of the Fp and Qp initiated EBNA1 mRNA increased the expression level 4–14- fold in different Burkitt lymphoma cell lines. The U leader exon, located within the 5′ UTR, included in all known EBNA1 transcripts and also contained in the EBNA3, 4 and 6 mRNAs, was demonstrated by bicistronic expression analyses to contain an IRES. The EBNA IRES initiates translation more efficiently than the encephalomyocarditis virus IRES in EBV-positive lymphoma cells. We propose that the EBNA IRES constitute a novel mechanism, whereby EBV regulates latent gene expression.

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.

Monocytes, recovered directly from peripheral blood by counter-current centrifugal elutriation (CCE), were shown to provide two regulatory signals for induction of interferon-gamma (IFN-gamma) in natural killer (NK) cells in response to interleukin-2 (IL-2): an upregulating signal and an inhibitory signal. The inhibitory signal was time-dependent, irreversible, and operating on a pretranslational level, as indicated by the inability of enriched NK cells to accumulate IFN-gamma mRNA in the presence of elutriated monocytes. Monocyte-induced inhibition of IFN-gamma production was abrogated by the biogenic amine serotonin, acting via the 5-hydroxytryptamine, or serotonin (5-HT1A), subset of serotonin receptors (5-HTR). Thereby, serotonin effectively promoted IFN-gamma production in the presence of monocytes. We conclude that serotonergic 5-HT1A receptors transduce signals that are required for NK cells to produce IFN-gamma in response to IL-2.