Type 1 Diabetes Mellitus Donor Mesenchymal Stromal Cells Exhibit Comparable Potency to Healthy Controls In Vitro.

: Bone marrow mesenchymal stromal cells (BM-MSCs) have been characterized and used in many clinical studies based on their immunomodulatory and regenerative properties. We have recently reported the benefit of autologous MSC systemic therapy in the treatment of type 1 diabetes mellitus (T1D). Compared with allogeneic cells, use of autologous products reduces the risk of eliciting undesired complications in the recipient, including rejection, immunization, and transmission of viruses and prions; however, comparable potency of autologous cells is required for this treatment approach to remain feasible. To date, no analysis has been reported that phenotypically and functionally characterizes MSCs derived from newly diagnosed and late-stage T1D donors in vitro with respect to their suitability for systemic immunotherapy. In this study, we used gene array in combination with functional in vitro assays to address these questions. MSCs from T1D donors and healthy controls were expanded from BM aspirates. BM mononuclear cell counts and growth kinetics were comparable between the groups, with equivalent colony-forming unit-fibroblast capacity. Gene microarrays demonstrated differential gene expression between healthy and late-stage T1D donors in relation to cytokine secretion, immunomodulatory activity, and wound healing potential. Despite transcriptional differences, T1D MSCs did not demonstrate a significant difference from healthy controls in immunosuppressive activity, migratory capacity, or hemocompatibility. We conclude that despite differential gene expression, expanded MSCs from T1D donors are phenotypically and functionally similar to healthy control MSCs with regard to their immunomodulatory and migratory potential, indicating their suitability for use in autologous systemic therapy. SIGNIFICANCE: The potential for mesenchymal stromal cells (MSCs) as a cell-based therapy in the treatment of immunologic disorders has been well established. Recent studies reported the clinical potential for autologous MSCs as a systemic therapy in the treatment of type I diabetes mellitus (T1D). The current study compared the genotypic and phenotypic profiles of bone marrow-derived MSCs from T1D and healthy donors as autologous (compared with allogeneic) therapy provides distinct advantages, such as reduced risk of immune reaction and transmission of infectious agents. The findings of the current study demonstrate that despite moderate differences in T1D MSCs at the gene level, these cells can be expanded in culture to an extent corresponding to that of MSCs derived from healthy donors. No functional difference in terms of immunosuppressive activity, blood compatibility, or migratory capacity was evident between the groups. The study findings also show that autologous MSC therapy holds promise as a T1D treatment and should be evaluated further in clinical trials.

Pre-transplant donor-specific anti-human leukocyte antigen antibodies are associated with high risk of delayed graft function after renal transplantation.

BACKGROUND: Sensitive screening methods have revealed that many patients have donor-specific human leucocyte antigen antibodies (DSAs) prior to transplantation, regardless of negative crossmatch results. The clinical significance of pre-transplant (pre-Tx) DSAs for early graft function has remained unclear. Our aim was to examine the association of DSAs with delayed graft function (DGF). METHODS: Pre-Tx sera of 771 patients who received kidney transplants in our single-centre study were retrospectively screened. All transplantations were performed after negative complement-dependent cytotoxicity (CDC) crossmatch. RESULTS: DSAs were detected in 13% of the patients. The overall DGF rate in our study was 29%. Patients with DSAs had a higher incidence of DGF when compared with non-sensitized patients (48 and 26%, respectively; P < 0.0001). Third-party antibodies had no effect for DGF incidence (28%; P = 0.6098). The relative risk (RR) of DGF for patients with DSAs in the multivariate analysis was 2.039 (95% CI 1.246-3.335; P = 0.0046). Analyses of the cumulative mean fluorescent intensity (MFI) value of the DSAs revealed a rate of DGF more than two times higher in patients with a cumulative value of 3000-5000 MFI compared with a cumulative value of 1000-3000 (65 versus 31%; P = 0.0351). DSAs against any loci showed an elevated DGF incidence of 44-69% when compared with patients without DSA (27%). CONCLUSIONS: The risk of DGF is twice as high in patients having pre-formed DSAs. Pre-Tx DSAs is a modifiable risk factor that can be obviated with careful organ allocation relying on careful pre-Tx analysis of non-accepted mismatches determined with sensitive solid phase methods.

Serum neutrophil gelatinase-associated lipocalin and recovery of kidney graft function after transplantation.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a marker for acute kidney injury. We studied whether serum NGAL predicts delayed graft function (DGF) and recovery of kidney function after transplantation. METHODS: Serum NGAL was analyzed using commercial ELISA and point-of-care (POC) (Triage®, Biosite) methods. Serum samples were collected from 176 consecutive, deceased-donor kidney recipients just before transplant surgery and on day 1 and 14 after transplantation. The first 132 samples were analyzed with both methods and the remaining samples with the POC method. RESULTS: The correlation between the ELISA and POC methods was 0.89, p < 0.0001 and hence the POC method was used for the remaining analyses. DGF was seen in 66/176 patients. Day 1 sNGAL was significantly higher in DGF (588 ng/ml, SD 189.6) compared to early graft function (355 ng/ml, SD 166.2, p < 0.0001) and this difference persisted on day 14. Day 1 sNGAL predicted DGF with an area under the curve (AUC) of 0.853 (CI 0.792-0.914, p < 0.0001). At the optimal cutoff level of 423 ng/ml the sensitivity was 87% and the specificity 77%. In a multivariate analysis, day 1 sNGAL emerged as an independent predictor of DGF. The sNGAL also predicted DGF lasting longer than 14 days with an AUC of 0.825 (CI 0.751-0.899, p < 0.0001). At the optimal cutoff level of 486 ng/ml, the sensitivity was 80% and specificity 75%. CONCLUSION: Serum NGAL predicts clinically significant DGF and is useful in the care of kidney transplant recipients.

Deceased donor neutrophil gelatinase-associated lipocalin and delayed graft function after kidney transplantation: a prospective study.

INTRODUCTION: Expanding the criteria for deceased organ donors increases the risk of delayed graft function (DGF) and complicates kidney transplant outcome. We studied whether donor neutrophil gelatinase-associated lipocalin (NGAL), a novel biomarker for acute kidney injury, could predict DGF after transplantation. METHODS: We included 99 consecutive, deceased donors and their 176 kidney recipients. For NGAL detection, donor serum and urine samples were collected before the donor operation. The samples were analyzed using a commercial enzyme-linked immunosorbent assay kit (serum) and the ARCHITECT method (urine). RESULTS: Mean donor serum NGAL (S-NGAL) concentration was 218 ng/mL (range 27 to 658, standard deviation (SD) 145.1) and mean donor urine NGAL (U-NGAL) concentration was 18 ng/mL (range 0 to 177, SD 27.1). Donor S-NGAL and U-NGAL concentrations correlated directly with donor plasma creatinine levels and indirectly with estimated glomerular filtration rate (eGFR) calculated using the modification of diet in renal disease equation for glomerular filtration rate. In transplantations with high (greater than the mean) donor U-NGAL concentrations, prolonged DGF lasting longer than 14 days occurred more often than in transplantations with low (less than the mean) U-NGAL concentration (23% vs. 11%, P = 0.028), and 1-year graft survival was worse (90.3% vs. 97.4%, P = 0.048). High U-NGAL concentration was also associated with significantly more histological changes in the donor kidney biopsies than the low U-NGAL concentration. In a multivariate analysis, U-NGAL, expanded criteria donor status and eGFR emerged as independent risk factors for prolonged DGF. U-NGAL concentration failed to predict DGF on the basis of receiver operating characteristic curve analysis. CONCLUSIONS: This first report on S-NGAL and U-NGAL levels in deceased donors shows that donor U-NGAL, but not donor S-NGAL, measurements give added value when evaluating the suitability of a potential deceased kidney donor.

Posttransplant sCD30 as a predictor of kidney graft outcome.

BACKGROUND: Reliable markers for assessing the biological effect of immunosuppressive drugs and identification of transplant recipients at risk of developing rejection are not available. METHODS: In a prospective multicenter study, we investigated whether posttransplant measurement of the T-cell activation marker soluble CD30 (sCD30) can be used for estimating the risk of graft loss in kidney transplant recipients. Pre- and posttransplant sera of 2322 adult deceased-donor kidney recipients were tested for serum sCD30 content using a commercial enzyme-linked immunosorbent assay. RESULTS: sCD30 decreased posttransplant and reached a nadir on day 30. Patients with a high sCD30 of more than or equal to 40 U/mL on day 30 showed a subsequent graft survival rate after 3 years of 78.3±4.1%, significantly lower than the 90.3±1.0% rate in recipients with a low sCD30 on day 30 of less than 40 U/mL (log-rank P<0.001; Cox hazard ratio 2.02, P<0.001). Although an association was found between pre- and posttransplant sCD30 levels, patients with high sCD30 on posttransplant day 30 demonstrated significantly lower 3-year graft survival irrespective of the pretransplant level. CONCLUSIONS: Our data suggest that posttransplant measurement of sCD30 on day 30 is a predictor of subsequent graft loss in kidney transplant recipients and that sCD30 may potentially serve as an indicator for adjustment of immunosuppressive medication.

Urine neutrophil gelatinase-associated lipocalin is a marker of graft recovery after kidney transplantation.

Delayed graft function (DGF), especially long-lasting DGF, complicates kidney transplant outcome. Neutrophil gelatinase-associated lipocalin (NGAL) is an acute kidney injury marker; therefore, we tested whether urine NGAL could predict DGF, prolonged DGF (lasting over 14 days), or the quality of kidney function in transplant recipients without DGF (non-DGF). We collected urine samples from 176 recipients transplanted with deceased donor kidneys before and various days after transplantation. A total of 70 transplantations had DGF, of which 26 were prolonged. Patients who developed DGF had a significantly slower decrease in urinary NGAL compared with those without DGF, such that day 1 NGAL predicted DGF (area under the curve (AUC) 0.75) and predicted DGF in 15 of 112 cases with day 1 urine output over 1 l (AUC 0.70) and in 19 of 86 cases with a day 1 decrease in creatinine over 50 µmol/l (AUC 0.74). The urinary NGAL level on day 1 predicted prolonged DGF (AUC 0.75), which had significantly worse 1-year graft survival (73%), compared with shorter DGF (100%). In non-DGF, high day 3 NGAL (greater than the mean) was associated with significantly worse kidney function at 3 weeks compared with low NGAL, but not at 3 months and 1 year. NGAL did not correlate with long-term function in DGF. Hence, day 1 urinary NGAL predicted DGF even when it was not clinically expected early on, and importantly, it predicted prolonged DGF that led to worse graft survival.

Association of genetic variation in inducible costimulator gene with outcome of kidney transplantation.

BACKGROUND: The closely-linked genes of CD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS), and programmed cell death 1 on chromosome 2q encode costimulatory molecules, which are regulators of the T-cell activity. The T-cell mediated immune response has a major role in allograft rejection. Hence, the variation in these genes may have an effect on graft survival and the amount of immunosuppression needed, but so far the studies have restricted solely to the CTLA4 gene. METHODS: We determined 13 single nucleotide polymorphisms in CD28, CTLA4, ICOS, and PPCD1 genes in 678 adult patients who received a kidney from deceased donor. The effect of genetic variation on the outcome of renal transplantation was analyzed. RESULTS: Two markers on the ICOS gene, rs10183087 and rs4404254, were associated with delayed graft function (odds ratio=5.8; P=0.020 and odds ratio=5.8; P=0.019, respectively). Interestingly, the same ICOS variation has been shown to regulate the expression level of ICOS. We also demonstrated an association of the ICOS polymorphism rs10932037 with the graft survival (P=0.026). CONCLUSIONS: The present results indicate that potentially functional genetic variation in T-cell costimulatory molecule ICOS has an effect on the outcome of kidney transplantation.

Intragraft coagulation events and delayed graft function in clinical renal transplantation.

BACKGROUND: Cold preservation, reperfusion damage, immunosuppressive drugs, and uremia-induced acquired thrombophilias increase the risk of thrombotic complications in renal transplantation. Intragraft fibrin deposition may be associated with delayed graft function. METHODS: We studied coagulation and fibrinolysis in 45 patients of a larger trial in renal transplantation: perioperative antithymocyte globulin (group A, n=15), perioperative basiliximab (group B, n=16), and conventional triple therapy (group C, n=14). Blood samples for prothrombin fragment F1+2, plasminogen activator inhibitor (PAI)-1, d-dimer, tPA antigen, tPA activity, and platelet counts were obtained simultaneously at 1 and 5 min after reperfusion from iliac artery and graft vein for calculation of transrenal changes. Because antithymocyte globulin activates coagulation and fibrinolysis, group A was analyzed separately. Groups B and C were pooled (group BC). RESULTS: In group BC, transrenal D-dimer release occurred at 1 min, tPA-antigen release at 1 and 5 min, and transrenal PAI-1 uptake at 5 min postreperfusion. tPA activity increased marginally only at 1 min. High graft tPA-antigen release at 5 min and D-dimer release at 1 min were associated with delayed graft function. In group A, transrenal tPA-antigen release occurred at 1 and 5 min and D-dimer release at 1 min. There were no transrenal F1+2 changes in either group. CONCLUSION: Although graft PAI-1 uptake inhibits tPA activity, graft releases D-dimer at early reperfusion without concomitant F1+2 release. Data suggest thrombin and fibrin formation already before cold preservation during donor care and organ retrieval. This fibrin deposition increases risk of delayed graft function.

Lack of association between thrombosis-associated and cytokine candidate gene polymorphisms and acute rejection or vascular complications after kidney transplantation.

BACKGROUND: Acute rejection episodes and vascular complications are common after renal transplantation and have negative impact on the long-term patient and graft survival. We investigated whether the risks of acute rejection, thrombosis, infarction and graft loss could be predicted based on the presence of functional polymorphisms in the genes of the coagulation and endothelial inflammation cascade. METHODS: The study consisted of 772 consecutive cadaver kidney transplantations from a single centre. The effects of gene polymorphisms FVL, F5R2, FII G20210A, MTHFR C677T, F13A1 V34L, TFPI P151L, PROC W380G, TNF G(-308)A, IL10 A(-592)C, IL10 A(-1082)G and IL6 C(-174)G of recipients and donors were investigated. RESULTS: We were unable to find statistically significant associations between any of the studied polymorphisms and clinical outcomes. CONCLUSIONS: Our results indicate that high-risk renal transplant candidates cannot be identified through the routine analysis of the polymorphisms.

Single bolus antithymocyte globulin versus basiliximab induction in kidney transplantation with cyclosporine triple immunosuppression: efficacy and safety.

BACKGROUND: The aim of this prospective randomized study was to examine the effect of induction immunosuppression and low initial cyclosporine (CsA) on the onset of graft function and its long-term consequences. METHODS: During 1999-2001, 155 patients were randomized to single 9 mg/kg dose antithymocyte globulin (ATG)-Fresenius (group A) or two 20-mg doses of basiliximab (group B) with reduced dose CsA or conventional CsA triple therapy without induction (group C). RESULTS: Delayed function (DGF) was lower in group A than in groups B or C (5.7% vs. 24.1% and 15.9%, P<0.025) and need of dialysis was less in groups A and B compared to C (10.3 and 10.4 vs. 20.0 days, P<0.05). Acute rejections occurred in 11.3%, 12.1% and 20.5%, and the mean (median) time to rejection was 16 (13), 97 (46) and 101 (35) days in groups A, B, and C, respectively (P<0.005). One-and 5-year graft survivals (GS) were 98.1% and 90.6% (group A), 96.6% and 96.6% (group B), and 93.2% and 84.1% (group C). Five-year GS was significantly better in group B than in group C (P<0.05). The death censored 5-year GS in groups A, B, and C were 94.3%, 96.6%, and 90.0% (P=NS). Single high-dose ATG induction was associated with hemodynamic and pulmonary disturbances without, however, serious or long-term consequences. CONCLUSIONS: ATG induction significantly reduced DGF. Both induction regimens together with low initial CsA led to significantly less posttransplant dialysis and excellent survival. The high dose ATG was associated with significant hemodynamic and pulmonary side effects during drug infusion.

Diagnosis of acute renal allograft rejection by analyzing whole blood mRNA expression of lymphocyte marker molecules.

BACKGROUND: Currently, the diagnosis of acute rejection after kidney transplantation is based on a kidney biopsy taken after clinical rejection suspicion. A robust, noninvasive diagnostic method would allow easier and more frequent monitoring of the patient and the graft. Potentially, a straightforward method would be the analysis of lymphocyte marker molecule expression from whole blood samples. METHODS: Whole blood samples were collected prospectively in a single kidney transplantation center from 50 adult kidney recipients transplanted between 2001 and 2005. The mRNA expression of granzyme B, perforin, FasL, granulysin, CD154, ICOS, CTLA4 and PD-1 were analyzed with real-time quantitative polymerase chain reaction. RESULTS: The expression of ICOS and CD154 were significantly lower in rejection patients than in control patients (P<0.001). Both genes gave statistically significant area under receiver operating characteristic curve (AUC; 0.87, 0.88) with 84% sensitivity and 100% specificity for CD154 and 76% and 86% for ICOS, respectively. In paired rejection and postrejection therapy samples, the expression of both genes significantly increased during rejection therapy (P<0.001). When rejection patients were compared to patients biopsied because of other reasons of graft dysfunction, both CD154 and ICOS were lower in rejection patients but only CD154 was statistically significant (P=0.028, AUC=0.740, sensitivity 52%, specificity 90%). The other studied genes gave no consistent statistically significant results. CONCLUSIONS: The whole blood gene expression quantities of costimulatory molecules CD154 and ICOS reasonably robustly differentiated rejection patients from control patients. The clinical use of the analysis is limited by poor capability to differentiate patients with rejection from patients with other causes of graft dysfunction.

The impact of donor cytokine gene polymorphisms on the incidence of cytomegalovirus infection after kidney transplantation.

Cytomegalovirus (CMV) seronegative recipients of kidneys from CMV seropositive donors are at a high risk of CMV infection after transplantation since viruses in the allograft may reactivate in patients without prior immunity. We hypothesized that the genetic background of the graft has an influence on the incidence of infection. Effects of IL10, IL6 and IFNG gene polymorphisms, known to affect CMV infectivity, were investigated in 71 CMV seronegative recipients of grafts from CMV seropositive cadaver donors. Donor IL10(-1082 AA) genotype reduced the incidence of CMV infection (p=0.031) and CMV episodes in these patients tended to occur later (AA: median 83 days, AG/GG: median 45 days, p=0.072). In multivariate analysis, other explaining factors than the donor IL10(-1082 AA) genotype alone did not improve Cox hazard model (HR=0.3, 95% CI=0.09-0.96, p=0.043). Recipient polymorphisms did not reduce the incidence of CMV infection. We conclude that donor IL10 gene polymorphisms may influence the likelihood of CMV infection in the high risk patients investigated.

Early cyclosporine C0 and C2 monitoring in de novo kidney transplant patients: a prospective randomized single-center pilot study.

BACKGROUND: C2 monitoring of cyclosporine (CsA) has been promoted as improving the results of organ transplantation. No randomized, controlled studies in de novo kidney transplant recipients are available. METHODS: Between June 2003 and August 2004, 160 consecutive cadaveric kidney recipients allocated to CsA, mycophenolate and steroids were randomized to either C0 or C2 monitoring of CsA for the first 3 weeks posttransplant. Both levels were measured, keeping the other level blinded until 3 weeks. Altogether, 1451 double measurements were done. The target C0 was 200-300 microg/L and C2 1500-2000 microg/L. From the fourth week on, only C0 monitoring was used. Median follow up time was 505 days. RESULTS: The overall 3-month rejection rate was 7.5% in Group C0 vs. 10.8% in Group C2 and the one-year graft survival rates were 92.5% vs. 94.6% (NS). Rate of delayed graft function was similar in the groups. Plasma creatinine tended to be higher in group C2 at 3 weeks, but not thereafter. During the first three weeks posttransplant, the mean CsA dose was 57%, mean C2 levels were 55%, and mean C0 levels were 98% higher in group C2 than in group C0 (P < 0.00001). CONCLUSION: This pilot study showed no advantages of C2 monitoring but led to significantly higher CsA doses and blood levels than C0 monitoring.

Serum sCD30 in monitoring of alloresponse in well HLA-matched cadaveric kidney transplantations.

In kidney transplantation, pretransplant serum sCD30 testing has been proposed in immunological risk estimation together with anti-HLA antibodies. We evaluated the risks associated with high pretransplant serum sCD30 in well HLA-matched cadaveric kidney recipients recruited in a clinical study comparing different immunosuppressive regimens. Rejection rate was similar in 37 recipients with high pretransplant serum sCD30 compared to 117 recipients with low serum sCD30 (16% vs. 15%, P=NS). Compared to pretransplant levels, the posttransplant sCD30 levels generally decreased, also in patients with rejection, although on day 21 posttransplant, rejecting patients had significantly higher relative sCD30 than nonrejecting patients (P<0.01). However, steroid-resistant rejection was associated with increasing posttransplant sCD30 levels. High pretransplant sCD30 values were associated with tubulointerstitial rejection. There was no correlation of sCD30 with delayed graft function. Good HLA matching seems to be effective in neutralizing the negative effect of a high pretransplant serum sCD30.

Activated protein C reduces graft neutrophil activation in clinical renal transplantation.

We studied the role of endogenous activated protein C (APC), the major physiological anti-coagulant with concomitant anti-inflammatory properties, on ischemia/reperfusion (I/R) in 45 patients participating in a larger trial comparing three immunosuppressive protocols in cadaveric renal transplantation: perioperative anti-thymocyte globulin (ATG, Fresenius AG, Bad Homburg, Germany), perioperative basiliximab and conventional triple therapy. Blood samples for assessing plasma APC, protein C, and lactoferrin concentrations, neutrophil CD11b and L-selectin expressions and blood leukocyte differential counts were obtained preoperatively and before reperfusion from central venous cannula, complemented with simultaneous samples from iliac artery and graft vein for calculation of transrenal differences (Delta) of study parameters at 1 and 5 min after reperfusion. Unlike basiliximab or conventional therapy groups, ATG infusion induced a substantial increase in plasma APC concentration (119 [88-144]% before infusion vs. 232 [85-1246]% after infusion, p<0.001), resulting in renal graft sequestration of APC at 1 min after reperfusion (Delta=-72 [-567 to 12]%, p<0.001). Graft APC consumption was associated with transrenal reduction of neutrophil activation markers (L-selectin r=0.7, p=0.01; lactoferrin r=-0.6, p=0.02; CD11b r=-0.8, p=0.001), and with both warm (r=0.6, p=0.01) and cold ischemia time (r=0.6, p=0.02) and donor age (r=0.6, p=0.01). These findings suggest that APC has an anti-inflammatory role in I/R injury in clinical renal transplantation.

Cytokine gene polymorphisms and risks of acute rejection and delayed graft function after kidney transplantation.

BACKGROUND: Pretransplantation identification of patients at an increased risk for adverse events would allow more individualized treatment strategies possibly improving long-term outcome. We studied cytokine gene polymorphisms of kidney allograft recipients and their donors to identify factors predisposing for acute rejection (AR) and delayed graft function (DGF). METHODS: A total of 291 adult cadaver kidney recipients transplanted at a single transplantation centre between 1999 and 2002 were investigated. Recipients and donors were typed for TNF-alpha(-308G/A), TGF-beta1(codon 10T/C, codon 25C/G), IL-10(-1082G/A, -819C/T, -592C/A), IL-6(-174C/G), and IFN-gamma(+874T/A) polymorphisms using a SSP-PCR kit. An AR episode was defined based on clinical and histological findings (Banff criteria). RESULTS.: The incidence of AR was 17%. In univariate statistical analyses recipients with TNF-alpha -308AA-genotype were found to be at a significantly increased risk for rejection (odds ratio [OR] 5.0, 95% CI 3.0-8.3, P = 0.003). The association was independent from the patient-donor HLA-mismatch status. In addition, patients with IL-10 ACCACC, ATAATA, GCCATA (-1082A/G, -819C/T, -592C/A, respectively) haplotypes were predisposed to rejection (OR 1.9, 95% CI 1.1-3.1, P = 0.016). Further, the combination of recipient TGF-beta1 25GG-genotype and donor IL-10 -819T-allele was associated with rejection (OR 1.8, 95% CI 1.1-3.0, P = 0.027). These variables remained significant risk factors also in a multivariate logistic regression analysis. The incidence of DGF was 22%. The risk was increased by a donor TNF-alpha -308GA-genotype (OR 1.6, 95% CI 1.1-2.6, P = 0.040). CONCLUSIONS: Our results confirm that cytokine gene polymorphisms influence the outcome of kidney transplantation. Our data especially identify the TNF-alpha -308AA-genotype as a factor predisposing for AR episodes.

Weak humoral posttransplant alloresponse after a well-HLA-matched cadaveric kidney transplantation.

BACKGROUND: Screening of donor-specific antibodies or alloantibodies after kidney transplantation has not been performed routinely. The aim of this study was to evaluate the humoral antidonor and alloresponse of immunologically low-risk recipients of cadaveric renal allografts during the first posttransplant year. METHODS: Alloresponse against the donor was analyzed by means of T-cell immunoglobulin (Ig)G and IgM and B cell IgG flow cytometric crossmatch (FCXM) tests with sera from days 0, 21, 90, and 365 posttransplant. In addition, panel reactive anti-human leukocyte antigen (HLA) class I and class II antibodies (PRA I and PRA II) were analyzed using flow cytometric methods. The recipients were treated either with a low initial cyclosporine regimen with single-bolus antithymocyte globulin (ATG) or basiliximab induction or conventional cyclosporine triple therapy. RESULTS: No significant posttransplant anti-HLA class I or class II sensitization was found in the recipients as a whole. Recipients receiving a single-bolus ATG showed significantly higher proportion of PRA I positivity in the day 21 sample compared with the other groups. Flow cytometric donor-specific T- and B-cell IgG alloresponses remained low, but the proportion of T-cell IgM crossmatch-positive recipients increased during the study. Positive T-cell IgM FCXM was found to be associated with acute rejection episodes and cytomegalovirus (CMV) infections. CONCLUSIONS: In immunologically low-risk kidney-graft recipients, positive T-cell IgM FCXM at transplantation was found to be a risk factor for rejection episodes. Conversion of T-cell IgM FCXM to positive was found to be associated with CMV infections.

Association of graft neutrophil sequestration with delayed graft function in clinical renal transplantation.

BACKGROUND: The authors studied the impact of neutrophil activation, detected in experimental models, on reperfusion injury in clinical renal transplantation. METHODS: Forty-five patients from a larger trial comparing three immunosuppressive protocols were recruited: perioperative antithymocyte globulin (ATG) with low initial cyclosporine A (CsA) triple therapy (group A, n=15); two-dose basiliximab with low initial CsA triple therapy (group B, n=16); and conventional triple therapy (group C, n=14). Blood samples were obtained preoperatively, before reperfusion, and at 1 and 5 min after reperfusion. During reperfusion, samples were collected from the iliac artery and the graft vein for calculation of transrenal differences (Delta) of study parameters. Leukocyte differential counts, plasma lactoferrin concentration, and neutrophil CD11b and L-selectin expressions were assessed. Graft blood flow was measured at 2 and 30 min after reperfusion. RESULTS: ATG induced neutrophil activation already before reperfusion. Thus, group A was excluded, but groups B and C were pooled for analysis of reperfusion-induced neutrophil activation. At 1 min after reperfusion, lactoferrin concentration was higher in graft vein than iliac artery, yielding Delta=15 microg/L (P<0.05). Concomitantly, Delta neutrophil count correlated with both Delta L-selectin expression (R=0.49, P=0.012) and graft blood flow at 2 min (R=0.51, P=0.007). At 5 min after reperfusion, 0.17 (-1.0-0.24)x10 cells/L neutrophils were sequestered in the graft (P<0.001). This sequestration correlated with graft blood flow at 30 min (R=0.53, P=0.005) and was stronger in patients with delayed graft function (DGF) (Delta = -0.38 [-1.45 to -0.2]) than those without (Delta = -0.12 [-0.41-0.24], P<0.001). In multiple regression analysis, sequestration was the most important parameter associated with DGF. CONCLUSIONS: Neutrophils are activated and sequestered in the reperfused graft during clinical renal transplantation. Neutrophil sequestration is a powerful independent factor explaining the incidence of DGF.