Quantitative polymerase chain reaction (Q‐PCR) studies of urine sediments have demonstrated an increased expression of cytotoxin genes during episodes of acute rejection of renal allografts. To compensate for differences in initial sample size and cDNA preparation, standard Q‐PCR experiments involve normalization to a reference gene. Although stable expression of reference genes is a prerequisite for any Q‐PCR analysis, commonly used reference genes have demonstrated a varying expression across tissues and various stimuli. In this study, cellular expression of several reference genes was investigated in a mixed lymphocyte reaction as a model of gene expression during alloreactive T‐lymphocyte activation and acute rejection. Gene expression was quantified using Q‐PCR, normalized to cell counts obtained by DNA quantification and corrected for cell polyploidy using flow cytometry. Examined reference genes were 18S rRNA, β‐actin (ACTB), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1) and peptidylprolyle isomerase B (PPIB). This study also examined two novel T‐lymphocyte‐specific reference genes: CD3E and CD8B. HMBS and HPRT were 18.8‐ and 7.4‐fold upregulated, respectively, ACTB was 5.3‐fold upregulated, PPIB was 3.2‐fold upregulated while 18S rRNA remained stably expressed. The T‐lymphocyte‐specific reference gene CD3E remained stable while CD8B was upregulated 2.3‐fold. In conclusion, several commonly used reference genes were actively regulated during alloreactive T‐lymphocyte activation. Additionally, we introduce two stable T‐lymphocyte‐specific reference genes that might be useful in a Q‐PCR analysis of T‐lymphocyte‐specific cytotoxin genes in urine sediments, as they overcome the contribution of reference gene mRNA from cells irrelevant for diagnosis.

Acute rejection remains an important cause of renal allograft dysfunction and the need for accurate diagnosis is essential to treat transplant recipients successfully. Molecular markers in urine may serve as a diagnostic tool in acute rejection, but controversy still exists regarding the uniqueness of these biomarkers. We measured mRNA of perforin (PRF), granzyme B (GZMB) and granulysin (GNLY) normalized to cyclophilin B in urine specimens from 24 renal allograft recipients with acute rejection, 12 with bacteriuria, 11 with cytomegalovirus (CMV) infections and 17 controls with stable graft function. Measurements were performed using a real‐time polymerase chain reaction assay. mRNA levels (means [95% CI]) for all three markers were significantly higher in recipients with acute rejection compared with controls: PRF (0.23 [0.12–0.42] versus 0.04 [0.02–0.07] P < 0.001), GZMB (0.14 [0.09–0.23] versus 0.05 [0.03–0.08] P = 0.003), GNLY (0.24 [0.14–0.41] versus 0.06 [0.03–0.11] P = 0.001). GZMB and GNLY levels during acute rejection were significantly higher when compared with bacteriuria (P = 0.011 and P = 0.005 respectively), and PRF level during acute rejection was significantly elevated compared with CMV infection (P = 0.015). No significant difference was found when comparing marker levels during bacteriuria and CMV infection to controls. Urinary mRNA levels of PRF, GZMB and GNLY are significantly elevated during acute rejection but not during bacteriuria or CMV infections when compared with recipients with stable graft function. The ability to differentiate acute rejection from bacteriuria and CMV infections was only present for some of the markers, that is why careful consideration should be given before applying this technique to clinical practice.

Cyclosporine A (CsA) is metabolized into a vast spectrum of metabolites. The potential immunosuppressive action of CsA's metabolites has been studied extensively in the early 1990's, with conflicting and inconclusive results. Since then, the pharmacological and clinical consensus guidelines recommend the use of specific monoclonal assays for measurement of CsA's concentrations thus avoiding metabolite interference. Nevertheless, clinical benefit or superiority of these assays was never convincingly demonstrated. We provide a review of the performed in vivo, in vitro and animal studies and their conclusions. During the last years, many transplantation centres have employed the C(2) monitoring of CsA (2 hours post-dose concentration). The metabolites / parent drug ratio two hours post dose is completely different from trough levels (predose concentration). Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase (CaN). Currently, our laboratory, among others, is working on determination of the enzyme's inhibition and its potential use as a pharmacodynamic biomarker. Utilizing this novel pharmacodynamic approach, we have performed in vitro and in vivo studies investigating the immunosuppressive impact of CsA's metabolites on C2 monitoring and on various monitoring assays (mono-/polyclonal). Interestingly, even though we have estimated in vivo that the potential immunosuppressive action of metabolites is less than 10% of the parent drug, we have found assays that take metabolites into consideration to correlate stronger with calcineurin phosphatase inhibition. We believe that the old controversial issue of metabolite induced immunosuppressive action examined under the light of newer pharmacodynamic approaches is still intriguing. Instead of a priori neglecting CsA's metabolites maybe we should investigate the potential of utilizing them as an additional tool towards better therapeutic drug monitoring of cyclosporine.

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Aim: To report a case of a pheochromocytoma secreting vasoactive intestinal peptide (VIP). Methods: We studied a 77-year-old woman who had suffered from persistent diarrhea and episodes of sweating and palpitations. Results: She had neither previous or current anamnesis of hypertension nor any known familial dispositions to endocrine diseases. Initially gastrointestinal investigations were carried out based on longstanding diarrhea with hypopotassemia, but radiological imaging revealed a unilateral adrenal mass. Biochemical testing showed increased levels of catecholamine and VIP, and ¹²³I-metaiodobenzylguanidine scintigraphy confirmed the adrenal origin as well as the diagnosis of a VIP-producing pheochromocytoma. The patient underwent surgical removal of the tumor which led to relief of symptoms and normalized laboratory values. Conclusion: This case report focuses on the protean mode of presentation seen in pheochromocytomas as well as their capacity to produce several neuropeptides, ultimately intensifying the need for early examination for this condition despite unrelated symptoms.