Gema Cerezo

Pretransplant Immediately Early-1-Specific T Cell Responses Provide Protection for CMV Infection After Kidney Transplantation

Cytomegalovirus (CMV) infection is still a major complication after kidney transplantation. Although cytotoxic CMV‐specific T cells play a crucial role controlling CMV survival and replication, current pretransplant risk assessment for CMV infection is only based on donor/recipient (IgG)‐serostatus. Here, we evaluated the usefulness of monitoring pre‐ and 6‐month CMV‐specific T cell responses against two dominant CMV antigens (IE‐1 and pp65) and a CMV lysate, using an IFN‐γ Elispot, for predicting the advent of CMV infection in two cohorts of 137 kidney transplant recipients either receiving routine prophylaxis (n = 39) or preemptive treatment (n = 98). Incidence of CMV antigenemia/disease within the prophylaxis and preemptive group was 28%/20% and 22%/12%, respectively. Patients developing CMV infection showed significantly lower anti‐IE‐1‐specific T cell responses than those that did not in both groups (p < 0.05). In a ROC curve analysis, low pretransplant anti‐IE‐1‐specific T cell responses predicted the risk of both primary and late‐onset CMV infection with high sensitivity and specificity (AUC > 0.70). Furthermore, when using most sensitive and specific Elispot cut‐off values, a higher than 80% and 90% sensitivity and negative predictive value was obtained, respectively. Monitoring IE‐1‐specific T cell responses before transplantation may be useful for predicting posttransplant risk of CMV infection, thus potentially guiding decision‐making regarding CMV preventive treatment.

Induction of suppressive allogeneic regulatory T cells via rabbit antithymocyte polyclonal globulin during homeostatic proliferation in rat kidney transplantation.

Experimental studies have shown that rabbit antithymocyte polyclonal globulin (ATG) can expand human CD4+CD25++Foxp3+ cells (Tregs). We investigated the major biological effects of a self‐manufactured rabbit polyclonal anti‐rat thymoglobulin (rATG) in vitro, as well as its effects on different peripheral T‐cell subsets. Moreover, we evaluated the allogeneic suppressive capacity of rATG‐induced Tregs in an experimental rat renal transplant model. Our results show that rATG has the capacity to induce apoptosis in T lymphocyte lymphocytes as a primary mechanism of T‐cell depletion. Our in vivo studies demonstrated a rapid but transient cellular depletion of the main T cell subsets, directly proportional to the rATG dose used, but not of the effector memory T cells, which required significantly higher rATG doses. After rATG administration, we observed a significant proliferation of Tregs in the peripheral blood of transplanted rats, leading to an increase in the Treg/T effector ratio. Importantly, rATG‐induced Tregs displayed a strong donor‐specific suppressive capacity when assessed in an antigen‐specific allogeneic co‐culture. All of these results were associated with better renal graft function in rats that received rATG. Our study shows that rATG has the biological capacity immunomodulatory to promote a regulatory alloimmune milieu during post‐transplant homeostatic proliferation.

Determination of ganciclovir in human plasma by ultra performance liquid chromatography-UV detection

OBJECTIVES Implement a sensitive UHPLC method for the assay of ganciclovir in human plasma. DESIGN AND METHODS We developed and validated a chromatographic method coupled to ultraviolet detection for quantification of ganciclovir, with a short run time using a small volume of human plasma. Comparison of system performance was made with respect to analysis time, efficiency and sensitivity. RESULTS Correlation coefficients (r) of the calibration curves ranged from 0.999744 to 0.999784. Within-day and between-day imprecision and inaccuracy, specificity and recovery were also evaluated for validation. The method was precise and accurate and the retention time was 0.7 min. The calibration curves were linear between 0.5 and 30 μg/mL. There was a good correlation between HPLC and UHPLC techniques. CONCLUSIONS We developed a method that is currently applied in a clinical study assessing GCV plasma concentration variability after ganciclovir and valganciclovir administration.

Impact of Small Molecules Immunosuppressants on P-Glycoprotein Activity and T-cell Function

PURPOSE P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several drugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T-cell subsets. METHODS Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 uptake, efflux and kinetic of extrusion in CD4+ and CD8+ subsets by flow cytometry. Antigen-specific memory T-cell responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. RESULTS Rho123 uptake in groups treated with CsA and CsA+Rapa was significantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8+ subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8+ T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8+ T-cell subset. CONCLUSIONS Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8+ T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens.

Online haemodiafiltration improves inflammatory state in dialysis patients: A longitudinal study

Background Patients undergoing conventional hemodialysis (C-HD) present a greater immuno-inflammatory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompatibility, which could improve with a technique-switching to online hemodiafiltration (OL-HD). The antigen-independent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammatory state of patients in C-HD assessed by DC maturation. Methods 31 patients were submitted to C-HD and after 4 months switched to the OL-HD technique. Monocytes-derived DCs from HD patients were cultured in the presence of IL-4/GM-CSF. DC-maturation was evaluated by assessing the maturation phenotype by flow cytometry (FACs). DCs-functional capacity to elicit T-cell alloresponse was studied by mixed leucocyte reaction. Cytokine release was assessed by FACs and SNS was evaluated measuring renalase levels by ELISA. Results An up-regulation of maturation markers was observed in C-HD DCs which induced two fold more T cells proliferation than OL-HD DCs. Also, C-HD-mDCs presented with over-production of pro-inflammatory cytokines (IL-6, IL-1β, IL-8, IL-10 and TNF-α) compared with OL-HD-mDC (P<0·05). Results were correlated with clinical data. When SNS was evaluated, hypotension events and blood pressure were significantly lower and renalase levels were significantly higher after conversion to OL-HD. Diabetes mellitus type 2 patients also found beneficial reduction of mDC when converted to OL-HD compared to non-diabetics. Conclusions OL-HD could interfere with immuno-inflammatory state in HD patients with an improvement of renalase levels as potential key mediators in the mechanistic pathway of down-regulation of DC maturation.

Optimal sparse sampling for estimating ganciclovir/valganciclovir AUC in solid organ transplant patients using NONMEN.

Background: Ganciclovir and valganciclovir (GCV/VGCV) are used for the treatment and prophylaxis of cytomegalovirus in solid organ transplant (SOT) patients. An area under the time–concentration curve of 40–50 &mgr;g × h/mL is related to efficacy. Therapeutic drug monitoring could prevent suboptimal drug exposure and adverse events, but obtaining full concentration profiles is not feasible. Sampling optimization by developing a reliable and clinically applicable limited sampling strategy (LSS) may simplify dose adjustment. Methods: An LSS was developed using an original pharmacokinetic (PK) data set of 40 full profiles from 20 adult SOT patients. The LSS was developed based on population and Bayesian prediction approaches. Population PK parameters from a previous model were used for simulation or as priors (NONMEM version 7.2). Median percentage of prediction error and median of absolute percentage prediction error were calculated for plasma clearance (CL) and central compartment distribution volume (V2). Bias and precisions were compared using 1-way analysis of variance (SPSSv19.0). Results: Sampling windows were designed according to the PK profile previously observed with the entire set of data. The 4 windows selected were distributed from 0.5 to 1.5 hours, 2 to 3 hours, 4 to 5 hours, and 6 to 8 hours. Predose and concentrations beyond 8 hours were not considered in any case because simulated negative concentrations occurred in both cases. Predicted exposure using 3 sampling times (0.5–1.5, 4–5, and 6–8 hours) showed the best predictive performance, by either the population or Bayesian approaches. Bias and imprecision for CL and V2 were 0 and 0.60%, and −0.78% and 0.78%, respectively. Conclusions: GCV/VCG area under the time–concentration curve in SOT patients could be predicted with acceptable accuracy for clinical management and dose individualization using LSS. The estimator of GCV/VGC, using 3 concentrations measured at 0.5–1.5, 4–5, and 6–8 hours after drug intake, could be used for dose adjustment.

Measurement of ganciclovir concentration in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

BACKGROUND Ganciclovir/valganciclovir plays an important role in the treatment and prevention of cytomegalovirus disease after organ transplantation. MATERIAL AND METHODS We developed and validated a simple chromatographic method by ultra-performance liquid chromatography tandem mass spectrometry to measure plasma concentration of ganciclovir in human plasma. Chromatographic separation was achieved using an Acquity(®) UPLC(®) BEH™ (2.1×50mm id, 1.7μm) reverse-phase C18 column, with a water/methanol linear gradient containing ammonium acetate/formic acid at a 0.4mL/min flow rate. Ganciclovir and its internal standard (acyclovir) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode. RESULTS The limit of detection and quantification were 0.03 and 0.06mg/L, respectively, and linearity was observed between 0.06 and 30.0mg/L. Intra-day and day-to-day coefficients of variation and relative biases ranged from 3.6 to 5.4%, 4.2 to 6.2%, -2.6 to -1.1% and -4.0 to -2.8%, respectively. Recovery values were greater than 81.9%. Evaluation of the matrix effect showed ion suppression for ganciclovir and acyclovir. No carry-over was observed. CONCLUSIONS The validated method is useful for both therapeutic drug monitoring and pharmacokinetic studies. It could be applied to the daily clinical laboratory practice to measure the concentration of ganciclovir in human plasma.