Analytical evaluation of a real-time PCR-based DNA demethylation assay to assess the frequency of naturally occurring regulatory T cells in peripheral blood.

OBJECTIVES: Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR). DESIGN AND METHODS: The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR). The assay was compared to conventional flow cytometry and the agreement of results between two laboratories using a comparable qPCR protocol was assessed. In addition, the effect of gender, age, and medications was investigated. RESULTS: Within and between series imprecision was <20% (n=6). Whole blood samples are suitable for analysis within 3days when stored at room temperature; both whole blood and DNA samples - within 12months when frozen at -80°C. A significant correlation between the qPCR results and flow cytometry was lacking both with samples from HI and KTR. qPCR results between laboratories showed a bias of 76% but correlated well (r=0.645; p=0.0002, n=29). nTregs determined by qPCR were significantly (p<0.05) higher in HI (0.73%±0.23%, n=60) than in KTR (0.45%±0.21%, n=60) and in female HI (1.0%±0.27%, n=30) than in male HI (0.45%±0.23%, n=27). No effect of drugs or age was observed. CONCLUSIONS: The qPCR assay for nTregs provides reproducible results and is of sufficient quality for working with patient samples although inter-laboratory differences can be encountered due to a lack of method standardization. It was confirmed that gender-specific reference ranges are required.

CD26/dipeptidyl peptidase IV: a comparative study of healthy persons and kidney transplant recipients before and early after transplantation.

OBJECTIVES: Human CD26 is co-stimulatory for lymphocytes, circulates in a soluble form in blood (sCD26), and has intrinsic dipeptidyl peptidase IV (DPPIV) activity. Associations between CD26 expression on the surface of T cells (CD26+/CD3+) and acute rejection and between (CD26+/CD3+)/DPPIV and clinical immunosuppression have been reported. These results encouraged the investigation of CD26 as a potential biomarker to optimize immunosuppressive therapy. To better understand the significance of CD26, a comparative study of CD26 expression on CD3+ cells, sCD26 concentration, and DPPIV activity in healthy persons (HP) and kidney transplant recipients (KTR) was performed. DESIGN AND METHODS: Thirty-one HP and 34 KTR were included in the study. CD26+/CD3+ was determined by FACS, sCD26 concentration was determined by ELISA, and DPP activity was determined by spectrophotometry. For KTR, these parameters were studied on the day before transplantation (preTx) and 7±1days after transplantation (postTx). RESULTS: There was no significant difference in the CD26+/CD3+, sCD26, and DPPIV data regarding gender, donor type (16 living donors), delayed graft function (n=8), or presence of ≥4HLA mismatches (n=16). Compared to the HP data, preTx CD26+/CD3+ was 4.5-fold higher, sCD26 was 1.2-fold higher, and DPPIV showed no significant difference. PostTx, CD26+/CD3+ was 3.8-fold higher, and sCD26 and DPPIV decreased significantly, reaching lower values than that observed in HP. Re-transplanted patients (n=5) showed significantly lower preTx CD26+/CD3+ expression than patients receiving their first transplant. Patients with preemptive transplantation (n=7) showed higher postTx CD26+/CD3+ expression than patients on dialysis. CONCLUSIONS: CD26 expression on CD3+ cells was strongly increased in patients with end stage kidney disease compared to HP and remained high early postTx. The differences in sCD26 and DPPIV behavior compared to that of CD26+/CD3+ postTx may reflect a regulatory response to the new immunological situation and the effects of therapy.

Association between adverse effects under azathioprine therapy and inosine triphosphate pyrophosphatase activity in patients with chronic inflammatory bowel disease.

BACKGROUND: Inosine triphosphate pyrophosphatase (ITPA) catalyzes the pyrophosphohydrolysis of inosine triphosphate to inosine monophosphate. Recently, single-nucleotide polymorphisms in the ITPA gene, associated with decreased enzyme activity, have been reported. Some clinical studies have demonstrated that the 94C>A mutation is linked to flu-like symptoms, rash, and pancreatitis during azathioprine (AZA) therapy and to early AZA discontinuation. In this study, we investigated whether the enzyme phenotype is also related to adverse effects (AEs). METHODS: Patients suffering from inflammatory bowel disease who were treated with AZA (N=160; age 43±12 years) were included. Data were categorized into quartiles according to the ITPA activity. Information about the therapeutic regimen, AEs [leucopenia, increased hepatic enzymes (alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase), flu-like symptoms, and pancreatitis], cotherapy, and comorbidity was obtained from the responsible clinicians and patients by using a standardized questionnaire. ITPA activity was measured by a validated high-performance liquid chromatography procedure. In patients with decreased ITPA activity, the 94C>A and IVS2+21A>C genotypes were determined. RESULTS: AEs were reported significantly more often for patients with low ITPA activity than for patients with high ITPA activity; the highest odds ratio for occurrence of AEs was found to be below a threshold of 59.9 µmol/(gHb·h) [hemoglobin (Hb)]. Decreased ITPA activities [particularly <89.2 µmol/(gHb·h)] were frequently accompanied by leucopenias, whereas very low enzyme activities [<37.3 µmol/(gHb·h)] were associated with a higher incidence of increased liver enzymes. CONCLUSIONS: The results demonstrate a relationship between low ITPA activity and AEs and support the idea that the determination of ITPA phenotype might be an appropriate alternative to genotyping.

Association between pharmacodynamic biomarkers and clinical events in the early phase after kidney transplantation: a single-center pilot study.

INTRODUCTION: Strategies based on monitoring pharmacodynamic effects are increasingly evaluated to individualize immunosuppressive therapy. In the present investigation, both drug-specific and general pharmacodynamic biomarkers were assessed and their association with clinical events early after kidney transplantation was examined. METHODS: Thirty-five de novo kidney transplant patients receiving basiliximab, enteric-coated mycophenolate sodium (2×720 mg/day), steroids, and tacrolimus (target: 6-8 µg/L) were included. Blood was drawn on days 7(±1) and 21(±2) after transplantation. Mononuclear leucocytes were isolated and the following parameters were investigated: inosine monophosphate dehydrogenase activity (high-performance liquid chromatography-diode array detection), cell proliferation (bromodeoxyuridine test), and CD marker cell surface expression (CD25, CD71, CD26) on stimulated (phytohemagglutinin 2.5 µg/10(6) cells) and nonstimulated CD3 cells. Acute rejection, gastrointestinal adverse effects, leucopenia, and infections were monitored over 3 months. RESULTS: There was no association between clinical events and inosine monophosphate dehydrogenase activity apart from patients with diarrhea showing a significantly higher inosine monophosphate dehydrogenase activity 2 hours after the enteric-coated mycophenolate sodium dose (P<0.05). Cell proliferation was significantly reduced in patients with leucopenia (P<0.05). CD71 expression was less inducible in patients with infections (P<0.05). A lower CD26 expression on non stimulated CD3 cells predicted freedom from rejection (Day 7; negative predictive value 100%). No associations were found between CD25 expression and events. CONCLUSIONS: A potential benefit of pharmacodynamic monitoring to optimize immunosuppressive combination therapy has been demonstrated. In particular, CD26 and CD71 may be promising biomarkers to assess adequate immunosuppression in the early phase after kidney transplantation. The results of this pilot study require verification in further trials with more patients and events as well as with different graft types.

Simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR assay on the lightcycler instrument.

BACKGROUND: Clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C and they are, under normal circumstances, performed as separate assays. DESIGN AND METHODS: In order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler Instrument (Roche Diagnostics). RESULTS: The quantification assay was calibrated against WHO Standard 96/790. The detection limit was 30 IU/ml, the dynamic range up to 500,000,000 IU/ml. Intra- and interassay imprecisions were 1.2% and 1.9% (n = 10), respectively. The HCV RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HCV monitor test (r = 0.992; p < 0.001). CONCLUSIONS: The genotyping was performed by means of the melting temperature analysis. The concordance between our new genotyping method and the Trugene HCV 5'NC Kit was at the level of genotypes 100%. This rapid (3 h) and convenient assay is suitable for HCV genotyping, HCV detection and disease monitoring.

Two-step real-time PCR quantification of all subtypes of human immunodeficiency virus type 1 by an in-house method using locked nucleic acid-based probes.

Current HIV-1 viral-load assays are too expensive and time-consuming for small sample quantity or resource-limited setting. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. We developed an internally controlled, two-step, reverse transcription-initiated real-time PCR protocol on the LightCycler instrument achieving a favourable detection limit with an extended quantification range, detecting all HIV-1 subtypes suitable for laboratories with low sample throughput. The detection limit was found to be 100 copies/ml, the dynamic range up to 500,000,000 copies/ml. Intra and inter assay imprecision were 1.6% and 2.0% (n=10), respectively. The assay was calibrated against WHO Standard 97/656. The HIV-1 RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HIV-1 Monitor (r = 0.954; p < 0.001). This rapid (3 h) and cost effective assay is suitable for both HIV-1 detection and disease monitoring with the ability to detect and quantify all HIV-1 subtypes including O1 and O2.