Blood Of Renal Transplant Recipients Research Articles

A Volumetric Absorptive Microsampling UPLC-MS/MS Method for Simultaneous Quantification of Tacrolimus, Mycophenolic Acid and Creatinine in Whole Blood of Renal Transplant Recipients.

(1) Background: Continuous monitoring of tacrolimus (TAC), mycophenolic acid (MPA), and creatinine (Cre) after renal transplantation is vitally important. In this study, we developed a new method based on volumetric absorptive microsampling (VAMS) combined with Ultra Performance Liquid Chromatography−Tandem Mass Spectrometry (UPLC-MS/MS) to simultaneously quantify three analytes including TAC, MPA, and Cre in whole blood. (2) Methods: The VAMS-based UPLC-MS/MS assay used a shared extraction and a single injection to simultaneously quantify the included TAC, MPA, and Cre. Development and validation were carried out following the Food and Drug Administration and European Medicines Agency guidelines for the validation of bioanalytical methods. Moreover, clinical validation for the three analytes was performed in both dried blood spot (DBS) and VAMS. Furthermore, a willingness survey was conducted using the system usability scale (SUS) for renal transplant recipients. (3) Results: The assay was successfully validated for all analytes. No interference, carryover, or matrix effects were observed, and extraction recoveries and process efficiencies were >90.00%. Analysis was unaffected by hematocrit (0.20~0.60, L/L) and anticoagulants (EDTA-2K). Dried VAMS samples were stable for 7 days at ambient temperature and stable for at least 1 month at −20 °C. During clinical validation, the measured TAC, corrected MPA, and Cre concentrations of VAMS samples met the analytical standards (95.00%, 88.57%, and 92.50%). When more stringent clinical acceptance criteria were set, the results obtained by VAMS (90.00%, 71.43%, and 85.00%) better than DBS (77.50%, 62.86%, and 70.00%). Compared with DBS, the survey found that renal transplant recipients are more inclined to use VAMS. (4) Conclusions: A robust extraction and UPLC-MS/MS analysis method in VAMS tips was developed and fully validated for the simultaneous quantification of TAC, MPA, and Cre concentrations. This method provides analytical support for the one-sample remote monitoring of both immunosuppressive drug concentrations and renal function in allo-renal recipients. Based on the good consistency between this method and the routine detection of venous blood samples and higher patient satisfaction than DBS, we believe that VAMS sampling can be a better alternative to venous whole-blood sampling.

Downregulation of Protein Inhibitor of Activated STAT (PIAS) 1 Is Possibly Involved in the Process of Allograft Rejection

BackgroundProtein inhibitors of activated STAT (PIAS) proteins are regarded as negative regulators of cytokine-signaling and potent immunosuppressive proteins. However, their role in the process of organ transplant rejection has not been elucidated. MethodsIn the current study, we compared transcript levels of PIAS1 to 4 in the peripheral blood of renal transplant recipients who experienced transplant rejection with those having normal transplant functions. Expression of PIAS1 was significantly higher in nonrejected group compared with the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other PIAS genes were not different between study groups. Significant pairwise correlations were found between expression levels of PIAS genes in all study subgroups. The current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.

Changes of NK cell subsets with time post-transplant in peripheral blood of renal transplant recipients

BackgroundThere is evidence that NK cells with low cytotoxicity but strong immunoregulatory characteristics contribute to good graft outcome. We attempted to investigate which NK cell subsets increase post-transplant and might affect graft function. MethodLymphocyte and NK cell subsets were determined in whole blood using eight-colour-fluorescence flow cytometry in patients pre-transplant and post-transplant. In total, 31 transplant recipients were studied. ResultsWhen cell numbers were compared in 9 patients pre- and 6 months post-transplant, post-transplant CD56dimCD16+ (p = 0.011) NK cells with the phenotype CD158a+ (p = 0.008), CD158e+ (p = 0.038), NKG2A+ (p = 0.008), NKG2D+ (p = 0.011), IFNyR+ (p = 0.008), perforin+ (p = 0.008), granzymeB+ (p = 0.008), perforin+granzymeB+ (p = 0.008) and perforin-granzymeB- (p = 0.021) were lower than those pre-transplant, indicating a post-transplant reduction of cytotoxic NK cells. In 28 patients NK cell subsets were analyzed with respect to time post-transplant (median 888 days post-transplant). CD56dimCD16+ NK cells co-expressing CD158a (p = 0.014), NKG2D (p = 0.047), IL4R (p = 0.038), IL10R (p = 0.008) and IFNy (p = 0.036) as well as CD56bright NK cells with the phenotype TGFß+ (p = 0.017), TGFR+ (p = 0.035), CD158a+ (p = 0.042) and perforin-granzymeB- (p = 0.048) increased with time post-transplant. ConclusionPost-transplant, cytotoxic NK cells were lower than pre-transplant and remained low, whereas NK cell subsets with potentially immunoregulatory properties increased.

Expression of T lymphocyte cytokines in peripheral blood of renal transplant recipients

To evaluate the clinical values of T-lymphocyte cytokines in renal transplant acute rejection. A total of 31 recipients with renal transplantation and 15 healthy volunteers from January 2010 to January 2012 were enrolled and divided into acute rejection group (n = 11) and stable renal allograft function group (n = 20) according to the inclusion criteria. Peripheral blood was collected from the patients before transplantation, 1, 7, 14, 28 day after transplantation and acute rejection onset. Cytometric bead array (CBA) was used to monitor the levels of interleukin-17 (IL-17), interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin(IL)-10, IL-6, IL-4 and IL-2. The associations of the changes and levels of cytokines in 3 groups were examined with Pearson correlation analysis. The levels of IL-17A, TNF, IL-10 and IL-2 in recipients before transplantation were (3.40 ± 1.29) , (1.79 ± 0.53) , (2.73 ± 0.65) and (1.79 ± 1.02) ng/L respectively and decreased significantly compared to healthy volunteers ((4.52 ± 2.01), (3.36 ± 1.09) , (3.91 ± 0.42) , (3.12 ± 1.07) ng/L respectively, all P < 0.05). However the levels of IFN-γ, IL-6 and IL-4 showed no significant changes between two groups (all P > 0.05). In acute rejection group after transplantation, the levels of IL-17A, IFN-γ, IL-10 and IL-6 were (9.47 ± 4.75) , (5.01 ± 2.23) , (12.20 ± 5.79) , (6.55 ± 3.45) ng/L respectively and increased significantly compared to the renal allograft function group ((4.39 ± 1.26), (2.90 ± 0.87),(5.68 ± 2.25) and (2.10 ± 0.70) ng/L respectively, all P < 0.05); the level of IL-17A was correlated with those of IFN-γ and IL-4 (Pearson r = 0.519, 0.395, both P < 0.01), the level of IFN-γ was correlated with those of IL-4 and IL-2 (r = 0.276, 0.335, all P < 0.05) , the level of TNF was correlated with that of IL-4 (r = 0.423, P < 0.05) and the level of IL-10 was correlated with that of IL-6 (r = 0.361, P < 0.05). Cytokines play an important role in acute rejection of renal transplant. And further understanding of its mechanism may provide experimental and preventive rationales.

Calcineurin Free Protocol Is Associated With Increase in FOXP3 Expression in the Peripheral Blood of Recipients of Extended Criteria Donor Kidneys.

Background: The significance of the factor forkhead box (FOXP3) expression in the peripheral blood of renal transplant recipients (RTx) of extended (ECD) or standard (SCD) criteria donor kidneys is yet to be determined. Aim: To investigate the clinical significance of FOXP3 level in relation to the type of kidney donor, immunosuppressive protocol (ISS) and graft function in stable ECD RTx. Methods: Prospective, open label study with 36 stable RTx randomized according to the type of donor to receive tacrolimus (TAC; n= 19) or everolimus (EVL; n=17) based protocols combined to mycophenolate sodium, prednisone and induction therapy with basiliximab. Using qRT-PCR we measured FOXP3 expression in the peripheral blood of patients (pts) at the 3rd month after Tx. Results: Greater expression of FOXP3 was observed in recipients of ECD when compared to SCD (ECD=1.98 ± 2.8 vs SCD= 0.63 ± 0.71; p=0.02). EVL based immunosuppression (EVL= 1.49 ± 2.1 vs TAC= 0.51 ± 0.57; p=0.03) and absence of DGF post-Tx (DGF- = 1.94 ± 2.5 vs DGF+ = 0.54 ± 0.6; p=0.014) were also associated with high levels of FOXP3 expression. Cold ischemia time, acute rejection episodes and serum creatinine (sCr) at 3rd month were not associated with changes in FOXP3 expression. Multivariate logistic regression analysis showed that ECD (p= 0.025) absence of DGF (0.015) were independently associated with high levels of FOXP3. Correlation between sCr and FOXP3 was inverse in TAC treated pts and positive in those taking EVL (r= 0.74, P = 0.007). FOXP3 expression was low in kidneys with mild or no chronic lesions (IFTA score < 2) regardless the ISS. Patients treated with EVL presenting IFTA score ≥ 2 had increased expression of FOXP3 (p=NS vs TAC). Conclusions: Results suggest that recipients of ECD kidneys without having undergone DGF, treated with EVL presented an increased expression of FOXP3 in their peripheral blood. The higher FOXP3 levels associated with more chronic histological lesions may represent an anti-inflammatory response elicited by ongoing chronic allograft damage. DISCLOSURES:Abbud-Filho, M.: Grant/Research Support, Novartis. Dias, C.: Grant/Research Support, Novartis. Caldas, H.: Grant/Research Support, Novartis. Nunes, R.: Grant/Research Support, Novartis. Mazeti, C.: Grant/Research Support, Novartis. Fernandes-Charpiot, I.: Grant/Research Support, Novartis. Baptista, M.: Grant/Research Support, Novartis. Abbud-Filho, M.: Grant/Research Support, Novartis.

BK Virus Encoded MicroRNAs Are Present in Blood of Renal Transplant Recipients With BK Viral Nephropathy

BK viral infection is an important cause of renal transplant dysfunction and failure. Current strategies utilize surveillance for infection with DNA polymerase chain reaction assays and modulation of immunosuppression. Many viruses including polyomaviruses encode microRNAs (miRNAs). We have detected BK virus (BKV) encoded miRNAs in the blood of infected renal transplant recipients, and see a strong correlation between BKV encoded miRNA and BKV DNA in blood and a relationship between levels of bkv-miR-B1-5p and the presence of biopsy-proven BK viral nephropathy. Further research is needed to determine whether the detection of this and other virally encoded miRNAs may be useful in the diagnosis of active viral replication.

Early Microchimerism in Peripheral Blood Following Kidney Transplantation

BackgroundThe role of microchimerism found in the peripheral blood of renal transplant recipients remains a matter of debate. We assessed the frequency of microchimerism after kidney transplantation and examined its influence on clinical courses over a 12-month follow-up period. Patients and MethodsTen single-kidney recipients underwent microchimerism detection at 2 days, 2 weeks, and 1, 3, 6, and 12 months after transplantation, with mismatch human leukocyte antigen (HLA)-A, -B, and -C used as markers. ResultsMicrochimerism was detected in 8 (80%) patients at 2 days after kidney transplantation. In 3 of those, microchimerism became negative within 3 months after transplantation, whereas it remained present for up to 12 months in 3 patients (33 %). There was 1 acute rejection episode in a patient in whom microchimerism became negative within 3 months. Protocol renal graft biopsy specimens obtained 3 months after transplantation revealed no acute cellular-mediated rejection (ACMR) or acute antibody-mediated rejection (AAMR) in the 5 patients positive for microchimerism at 3 months. ConclusionsMicrochimerism was frequently detected after kidney transplantation. Microchimerism that remained for more than 3 months post-transplantation might be correlated with a lower incidence of rejection, thus its monitoring may help identify recipients with a low rejection risk.

Comparison of Regulatory T Cells and FoxP3-Positive T-Cell Subsets in the Peripheral Blood of Renal Transplant Recipients With Sirolimus Versus Cyclosporine: A Preliminary Study

BackgroundSirolimus-based regimens have recently been introduced as nephron-sparing strategies to avoid the calcineurin inhibitors–induced nephrotoxicity. MethodsTo investigate the different effects of these 2 immunosuppressants on CD4+CD25hiFoxP3+ regulatory T cells (Tregs) and the newly defined subsets of FoxP3+ T cells, including CD45RA−FoxP3lo cytokine-secreting T cells (csT cells), CD45RA−FoxP3hi activated Treg (aTregs), and CD45RA+FoxP3+ resting Treg (rTregs), 52 cases of renal transplant recipients who have received a maintenance immunosuppressive regimen comprised either cyclosporine (n = 34) or sirolimus (n = 18) were collected and proportion of Tregs and each subset of FoxP3+ T cells in peripheral blood was detected by flowcytometry. ResultsSirolimus significantly prompted the proportion of Tregs (5.92 ± 0.40 vs 2.19 ± 0.18; P < .001) and both of the CD45RA-negative subsets, including csT cells (4.92 ± 0.50 vs 1.83 ± 0.18; P < .001) and aTregs (1.04 ± 0.15 vs 0.23 ± 0.05; P < .05) when compared with cyclosporine. However, the rTregs were not remarkably affected (0.36 ± 0.09 vs 0.79 ± 0.28; P = .063). ConclusionSirolimus plays its immune regulatory role in renal transplantation partly by increasing the proportion of Tregs. However, the increasing of both pro- and anti-inflammatory subsets of FoxP3+ T cells also indicates the potential compromising of the effects of sirolimus on immune regulation.

CD4+CD25+Foxp3+IFNg+CD178+ Human Induced Treg (Itreg) Contribute to Suppression of Alloresponses by Apoptosis of Responder Cells

Recently, a new subset of induced immunoregulatory T cells (iTreg) that produced IFNg was reported. In vivo, iTreg with the phenotype CD4+CD25+Foxp3+IFNg+ were shown to be associated with good long-term graft outcome in renal transplant recipients. In vitro, CD4+CD25+Foxp3+IFNg+ were shown to inhibit allogeneic T cell responses antigen-unspecifically as well as antigen-specifically. Inhibition was characterized by decreased activation of responder T cells. Others reported that iTreg initiate immunosuppression by apoptosis induction in effector cells. In the present study, we investigated whether apoptosis and Fas/FasL-dependent pathways contribute to the inhibition of T cell activation mediated by IFNg-producing CD4+CD25+Foxp3+ iTreg. Early apoptosis and necrosis rates as well as co-expression of immunostimulatory and immunosuppressive proteins in/on CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+ and CD4+CD25+IFNg+ PBL were analyzed using cells from healthy controls and four-color flow cytometry, PMA/Ionomycin-stimulated PBL, and MLC. 16h PMA/Ionomycin stimulation induced iTreg subsets with the phenotype CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+, and CD4+CD25+IFNg+ co-expressing CD178, CD95, CD152, CD279, Granzyme A, Granzyme B, Perforin, IL-10, and TGFβ1 (all p< 0.05). Using, in addition, five-color fluorescence flow cytometry, approximately 1% of stimulated CD4+ PBL were CD25+Foxp3+IFNg+CD178+ (unstimulated 0%). CD178+ iTreg increased within 3h after PMA/Ionomycin stimulation in parallel to early apoptotic Annexin+/PI− PBL, suggesting CD178-mediated apoptosis of responder cells by CD4+CD25+Foxp3+IFNg+CD178+ iTreg. Apoptosis was inhibited dose-dependently in the presence of increasing concentrations of monoclonal antibody against CD178, CD95, or IFNg (all p< 0.05). CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary cell cultures and added to autologous PMA/Ionomycin stimulated secondary cell cultures induced apoptosis immediately. Early apoptosis was not antigen-specific as shown with CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary MLC and added to secondary MLC with autologous or third-party cells as stimulator. In summary, polyclonal or allogeneic stimulation of PBL induces an iTreg subpopulation with the phenotype CD4+CD25+Foxp3+IFNg+CD178+. These iTreg, when isolated from primary cell cultures, enable immediate apoptosis in stimulated responder and effector cells. We speculate that CD4+CD25+Foxp3+IFNg+ iTreg detected in the blood of renal transplant recipients with good long-term graft outcome induce immunosuppression in part by Fas/FasL-dependent mechanisms. Because CD4+CD25+Foxp3+IFNg+ iTreg express IFNg receptors and are induced by IFNg, they represent the first line of iTreg during an initiating immune response. IFNg-activated effector cells with strongly up-regulated CD95 expression might be the perfect target for this early apoptosis-inducing iTreg subset.

CD4+CD25+Foxp3+IFNg+CD178+ Human Induced Treg (Itreg) Contribute to Suppression of Alloresponses by Apoptosis of Responder Cells

Recently, a new subset of induced immunoregulatory T cells (iTreg) that produced IFNg was reported. In vivo, iTreg with the phenotype CD4+CD25+Foxp3+IFNg+ were shown to be associated with good long-term graft outcome in renal transplant recipients. In vitro, CD4+CD25+Foxp3+IFNg+ were shown to inhibit allogeneic T cell responses antigen-unspecifically as well as antigen-specifically. Inhibition was characterized by decreased activation of responder T cells. Others reported that iTreg initiate immunosuppression by apoptosis induction in effector cells. In the present study, we investigated whether apoptosis and Fas/FasL-dependent pathways contribute to the inhibition of T cell activation mediated by IFNg-producing CD4+CD25+Foxp3+ iTreg. Early apoptosis and necrosis rates as well as co-expression of immunostimulatory and immunosuppressive proteins in/on CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+ and CD4+CD25+IFNg+ PBL were analyzed using cells from healthy controls and four-color flow cytometry, PMA/Ionomycin-stimulated PBL, and MLC. 16h PMA/Ionomycin stimulation induced iTreg subsets with the phenotype CD4+CD25+Foxp3+, CD4+IFNg+Foxp3+, and CD4+CD25+IFNg+ co-expressing CD178, CD95, CD152, CD279, Granzyme A, Granzyme B, Perforin, IL-10, and TGFβ1 (all p< 0.05). Using, in addition, five-color fluorescence flow cytometry, approximately 1% of stimulated CD4+ PBL were CD25+Foxp3+IFNg+CD178+ (unstimulated 0%). CD178+ iTreg increased within 3h after PMA/Ionomycin stimulation in parallel to early apoptotic Annexin+/PI- PBL, suggesting CD178-mediated apoptosis of responder cells by CD4+CD25+Foxp3+IFNg+CD178+ iTreg. Apoptosis was inhibited dose-dependently in the presence of increasing concentrations of monoclonal antibody against CD178, CD95, or IFNg (all p< 0.05). CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary cell cultures and added to autologous PMA/Ionomycin stimulated secondary cell cultures induced apoptosis immediately. Early apoptosis was not antigen-specific as shown with CD4+CD25+IFNg+ and CD4+CD25+CD178+ PBL separated from primary MLC and added to secondary MLC with autologous or third-party cells as stimulator. In summary, polyclonal or allogeneic stimulation of PBL induces an iTreg subpopulation with the phenotype CD4+CD25+Foxp3+IFNg+CD178+. These iTreg, when isolated from primary cell cultures, enable immediate apoptosis in stimulated responder and effector cells. We speculate that CD4+CD25+Foxp3+IFNg+ iTreg detected in the blood of renal transplant recipients with good long-term graft outcome induce immunosuppression in part by Fas/FasL-dependent mechanisms. Because CD4+CD25+Foxp3+IFNg+ iTreg express IFNg receptors and are induced by IFNg, they represent the first line of iTreg during an initiating immune response. IFNg-activated effector cells with strongly up-regulated CD95 expression might be the perfect target for this early apoptosis-inducing iTreg subset.

Foxp3+ T cells in peripheral blood of renal transplant recipients and clinical correlations

Immunophenotype peripheral blood T cells from renal transplant recipients (RTR) using cellular markers of regulatory T cells (Tregs) and flow cytometry, including Foxp3, and correlate these findings with clinical parameters. Expression of phenotypic markers of Tregs was assessed by flow cytometric analysis of peripheral blood lymphocytes (PBL) from (i) RTR (n = 95); (ii) patients with end-stage renal failure (ESRF) awaiting transplantation (n = 17); and (iii) normal healthy controls (n = 18). The percentage of CD4(+) CD25(+) Foxp3(+) cells within the CD4(+) cell population did not significantly alter at different time points post-transplant. However, the percentage of CD4(+) CD25(+) Foxp3(+) cells within the CD4(+) population was significantly lower in RTR compared with patients with ESRF. In contrast, RTR and ESRF had a similar percentage of CD4(+) CD25(+) cells expressing Foxp3. Multivariate analysis of PBL and clinical parameters demonstrated (i) a positive linear relationship between the percentage CD4(+) CD25(+) cells expressing Foxp3 and estimated glomerular filtration rate and (ii) a higher percentage of CD4(+) CD25(+) cells in the CD4(+) cell population in patients with malignancy (the majority were skin cancers). Malignancy also correlated strongly with time post-transplant and age of the RTR. Immune monitoring of the PBL phenotype in RTR using CD4, CD25 and Foxp3 may stratify RTR and predict graft outcome and function, and risk of complications from immunosuppression. Longitudinal and functional studies of Tregs are essential to extend the findings of the present study.

Cyclosporin but not everolimus inhibits chemokine receptor expression on CD4+ T cell subsets circulating in the peripheral blood of renal transplant recipients

The peripheral chemokine receptors chemokine receptor 3 (CXCR3) and CC chemokine receptor 5 (CCR5) have been reported to be associated with allograft rejection. The impact of the expression of immunosuppressive drugs on peripherally circulating CD4(+) T cell subsets after renal transplantation is unknown. Expression of CXCR3 and CCR5 was investigated by flow cytometry in 20 renal allograft recipients participating in a prospective, randomized trial (NCT00514514). Initial immunosuppression consisted of basiliximab, cyclosporin A (CsA), mycophenolate sodium and corticosteroids. After 3 months, patients were treated either with CsA, mycophenolate sodium (MPA) plus corticosteroids (n = 6), CsA and everolimus plus corticosteroids (n =8) or CsA-free (CsA(free)) receiving everolimus, MPA and corticosteroids (n = 6). After initial reduction of CD4(+) forkhead box protein 3 (FoxP3)(+) and CD4(+) CD25(hi) FoxP3(+) regulatory T cells (T(regs)) (P < 0.05; P < 0.01), 3-month post-transplant percentages of T(regs) were reconstituted in CsA(free) and CsA(lo) arms compared to CsA(reg) 12 months post transplant. Expression of CCR5 and CXCR3 on CD4(+) FoxP3(+) and CD4(+) FoxP3(-) T cells 12 months post transplant was increased in CsA(free) versus CsA(reg). Increase in CCR5(+) CXCR3(+) co-expressing CD4(+) FoxP3(-) cells between 3 and 12 months correlated negatively with the glomerular filtration rate (GFR) slope/year [modification of diet in renal disease (MDRD); r = -0.59, P < 0.01]. CsA, but not everolimus, inhibits both T(reg) development and expression of CXCR3 and CCR5 on CD4(+) T cell subsets. Increase in CCR5(+) CXCR3(+) co-expressing CD4(+) FoxP3(-) T cells is associated with early loss in allograft function.

Quantification of Plasmacytoid Dendritic Cells and Type 1 Myeloid Dendritic Cells in Peripheral Blood of Renal Transplant Recipients With and Without Squamous Cell Carcinoma

Organ transplant recipients are at high risk of developing squamous cell carcinoma (SCC) (1–3). These viralassociated skin cancers cause severe morbidity and may also be life-threatening (4, 5). Thus, regular follow-up by a dermatologist is recommended. The time intervals of the examinations should be determined by the individual’s risk for SCC development (6–8). One of the main risk factors is long-term immunosuppression (3, 9), leading to severe deficits in immunosurveillance. Dendritic cells (DC) play an important role in immuno surveillance, even though they constitute only 0.5% of the leukocytes in blood (10). Plasmacytoid DC (pDC) are a subset of DC. They produce interferon-alpha (IFN-α) and are considered to play a critical role in antiviral immunity (11). Type 1 myeloid DC (mDC1), another DC subset, are responsible for induction of Tcell responses (12). On the basis of the potential of DC to prevent cancer development, we wanted to determine whether renal transplant recipients (RTR) who develop SCC have a reduced quantity of these two DC subgroups in blood compared with RTR who do not develop SCC.

Following Anti-CD25 Treatment, A Functional CD4+CD25+ Regulatory T-Cell Pool Is Present in Renal Transplant Recipients

Daclizumab, a humanized antibody directed against the alpha-chain of the interleukin-2 receptor (CD25), has shown efficacy in the prevention of acute rejection following organ transplantation. However, anti-CD25 therapy can be expected to affect not only alloreactive effector T cells, but also CD4(+)CD25(+) regulatory T (Treg) cells that are shown to play an important role in the induction of transplantation tolerance. Therefore, the size and function of the Treg pool in human renal allograft recipients after single-dose daclizumab administration was investigated in this study. Approximately 8 weeks after administration, daclizumab was cleared from the circulation and the Treg population then present appeared not different from that observed before transplantation. Functional analysis revealed that the Treg possessed a normal capacity to suppress mixed lymphocyte reactions in vitro. These data indicate that after daclizumab therapy a Treg population, normal in number and function, is present in the peripheral blood of renal transplant recipients.

Calcineurin Inhibitors Affect Circulating Regulatory T Cells in Stable Renal Transplant Recipients

Introduction Immunosuppression, although crucial for short-term management, has been described in renal transplantation to be a major hurdle for long-term graft survival. Efforts have been directed at achieving a true state of allotolerance, thereby reducing the load of immunosuppression. Recently, increased frequencies of CD4 +CD25 high regulatory T cells (Tregs) have been described as an additional mechanism to induce alloimmune tolerance. Materials and methods We assessed 64 renal transplant recipients with stable renal function for at least 1 year, divided into two groups: one composed of patients receiving rapamycin but not calcineurin inhibitors (CNIs), and another, of those receiving CNIs but not rapamycin. Results We demonstrated that T cells with a regulatory phenotype were decreased in peripheral blood of renal transplant recipients under CNI therapy compared to those who were CNI-free. The Tregs in our patients showed a modest association with renal function as measured by the delta serum creatinine, which was not significant. Conclusions CNIs, but not rapamycin, reduce the frequencies of circulating Tregs in renal transplant recipients. The use of rapamycin might be further exploited in strategies reducing immunosuppression in renal transplantation. Furthermore, quantification of blood Tregs may be a suitable tool to identify those recipients who are candidates for reducing immunosuppression.

Anti-vimentin Antibody Detection in Recipients of Heart-Beating and Non–Heart Beating Donor Kidneys

Alternative donor sources include non–heart-beating donors (NHBDs). There donors have been exposed to significant ischemia, so that it is common to utilize machine perfusion to either improve the organs or at least assess their viability. Both prolonged warm ischemia and machine perfusion can potentially damage the vascular endothelium, thereby exposing vimentin to antigenic recognition. The aim of this study was to determine whether anti-vimentin antibodies could be detected in the blood of renal transplant recipients at specific time points after transplant and whether they could be related to the donor source. Fifty-one recipients of NHBD kidneys were compared to 52 recipients of heart-beating donor (HBD) kidneys. All recipients had similar anti-vimentin levels pretransplant. However, at 1 month those kidneys from Maastricht category II NHB donors showed significantly higher levels. At 6 months both Maastricht category II and category III NHB donor recipients displayed significantly higher levels than recipients of HBD kidneys.

Use of cytokine enzyme-linked immunosorbent spot (ELISPOT) assay as an immune monitoring tool in solid organ transplantation

The development of surrogate markers to predict outcome and guide therapeutic interventions after solid organ transplantation is sorely needed by the transplant community. The cytokine enzyme-linked immunosorbent spot (ELISPOT) assay is one such candidate marker that can accurately detect the frequency and cytokine profile of peripheral alloantigen-reactive T lymphocytes in human allograft recipients at high resolution. Published studies provide strongly suggestive evidence that the frequency of donor-reactive interferon γ ELISPOTs in the the peripheral blood of renal transplant recipients directly correlates with the future risk of acute rejection and chronic graft injury. The assay may additionally have utility for directing minimization of toxic immunosuppressants and identifying patients tolerant to their allografts. It is envisioned that ELISPOT-based immune monitoring, complemented by use of several additional surrogate markers, will ultimately provide clinicians with necessary tools to optimize therapy so as to prolong transplant survival with minimal toxicity.

Onset and duration of cytomegalovirus immediate early 1 mRNA expression in the blood of renal transplant recipients.

Human cytomegalovirus (CMV) messenger (m) RNA expression in circulating leukocytes reflects directly viral activity in the human host. In this study, sixty-nine patients were monitored prospectively for CMV infection and mRNA expression during the first year after renal transplantation. Of the 69 recipients, 58 (84%) recipients were positive for CMV immediate early 1 (IE1) mRNA as detected by nucleic acid sequence-based amplification. The median onset of IE1 expression started at day 22 after transplantation and continued for a median duration of 82 days. IE1 mRNA expression started significantly earlier in recipients who developed an active CMV infection (P = 0.001) and in mycophenolate mofetil (MMF) treated recipients (P = 0.002). The duration of IE1 mRNA expression was significantly longer in recipients that had previously an early onset of IE1 mRNA expression (P = 0.001) and in recipients with active CMV infection (P = 0.007). Remarkably, longer prednisolone intake was correlated with a significantly (P = 0.02) shorter duration of IE1 expression compared to a longer duration of IE1 expression in recipients with only a short prednisolone intake. In recipients infected with glycoprotein B (gB) type 1 CMV strains, the duration of IE1 expression was significantly (P = 0.04) shorter compared to recipients infected with non-gB type 1 CMV strains (64 days vs. 150 days). The study indicates that multiple factors play a role in the onset and/or duration of CMV IE1 mRNA expression, for example, MMF treatment, prednisolone intake, and gB type of the specific CMV strain. The clinical significance of these correlations remains to be studied in more detail.

Cytomegalovirus infection in transplant recipients resolves when circulating gammadelta T lymphocytes expand, suggesting a protective antiviral role.

gammadelta T cells undergo massive expansion in the peripheral blood of renal transplant recipients who are infected with cytomegalovirus (CMV). In a 3-year prospective study, the relationship between the evolution of CMV infection and the kinetics of gammadelta T cell amplification was followed for 10 months after transplantation. Patients with late gammadelta T cell expansion (>/=45 days) had significantly longer (P<.0001) and higher (P<.0003) pp65 antigenemia and more-symptomatic CMV disease than did patients with early expansion. Analysis of data for each patient showed that gammadelta T cell expansion is concomitant with the resolution of CMV infection and disease, regardless of the CMV serologic status of donor and recipient before transplantation. These observations point to gammadelta T cell percentage determination as a new, rapid, and reliable prognosis factor to predict the resolution of CMV infection and strongly suggest that gammadelta T cells play a protective role against CMV infection.

Early metabolic changes in peripheral blood cells of renal transplant recipients treated with Cyclosporine A

We histochemically examined (phosphatase acid-AcP, phosphatase alkaline-ALP, succinate dehydrogenase-SDH, lactate dehydrogenase-LDH) the peripheral blood of renal transplant recipients and controls before (day 0) and after Cyclosporine A (CsA) treatment (days 1, 2, 7 and 30). We wanted to detect the metabolic changes induced in the CsA resistant cells (leucocytes) by CsA and to evaluate the early effects determined by the drug. There was no difference in enzyme activities between the control group and renal patients before CsA treatment (day 0). AcP and ALP activity increased 1 day after CsA administration and became similar to the control by the day 30. LDH activity increased one day after CsA treatment and remained high during the treatment period (30 days), while SDH activity did not change. These enzymatic variations may suggest that the LDH enzyme is involved in the drug degradation as are other phosphatase and oxidoreductase enzymes (i.e. cytochrome P450). Moreover, the high activity of LDH, the enzyme responsible for interconversion of pyruvate in lactic acid, would indicate that anaerobic glycolysis is preferentially used in the pyruvate pathway. However, SDH did not seem to be directly involved in CsA metabolism. Our findings showed that the CsA treatment induced clear variations of the activity of the cellular phosphatase and oxidoreductase enzymes from the first days of drug administration. The variation of the enzymes studied and the appearance time and duration of the metabolic changes, may be markers of the cellular stress due to CsA internalization.