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Viral infections represent significant morbidity and mortality factors in kidney transplant recipients, with CMV, EBV, and BKV infections being most common. Desensitization (DES) with IVIg and rituximab with/without plasma exchange followed by kidney transplantation with alemtuzumab induction increased successful transplant rates in HLA-sensitized patients but may represent an increased risk for viral infections due to severe lymphocyte depletion. Here, we report on the posttransplant viral infection status in 372 DES versus 538 non-DES patients. CMV and EBV viremia were significantly lower in DES patients, while BKV viremia was similar. This trend was observed primarily in CMV sero(−), EBV sero(+), and sero(−) patients. No patient developed PTLD. The incidence of BKAN, allograft, and patient survival was similar in both groups. These viral infections were not associated with subsequent allograft rejection which occurred within 6 months after the infection.Conclusions.The IVIg + rituximab desensitization combined with alemtuzumab induction with triple immunosuppression maintenance does not increase the risk for CMV, EBV, and BKV infections. Possible factors include, in addition to posttransplant antiviral prophylaxis and PCR monitoring, presence of memory T cells and antibodies specific to CMV and likely EBV, NK cell-mediated ADCC despite lymphocyte depletion, elimination of EBV and CMV reservoirs by rituximab and alemtuzumab, and use of IVIg with antiviral properties.

Background. Global immunosuppression can be measured by assessing adenosine triphospate (ATP) levels in mitogen-stimulated CD4+ T cells.Methods. We investigated the effect of storage time on ATP levels in 234 blood samples from 18 healthy individualsand 152 transplant patients. The difference between day 0 (G13 hours post-blood draw) and day 1 (24Y37 hours) mea-surements was analyzed and compared with various factors; a subset of samples was also analyzed in 6-hour intervals.Results. The ATP levels were significantly lower on day 1 compared with that on day 0 in healthy individuals(279T159 vs 414T159 ng/mL, PG0.001) and patients (356T209 vs 455T221 ng/mL, PG0.0001). Of the 18 healthyindividuals, 17 showed ATP reduction, whereas 192 (89%) of 216 patients did so on day 1 (24.8T24.1%). In the timecourse analysis, ATP levels decreased with the blood storage time in healthy and patient samples, and the reductionbegan as early as 7 hours post-blood draw. The reduction rate was significantly higher in patient samples with low day0 ATP levels compared with samples with moderate or high levels (44.7T31.3% vs 23.2T23.6% or 18.7T15.7%;PG0.001). The reduction rate in patients treated with alemtuzumab induction was slightly higher than that indaclizumab-treated patients (28.8T24.6% vs 21.3T21.3%, P=0.09). CD4+ cell number did not change within 24 hourspost-blood draw, but CD4 expression decreased 2.0T2.8% (PG0.05).Conclusions. The ATP levels are significantly lower in 1-day-old blood compared with fresh blood, suggesting thatfresh blood should be used for assessing the T cell immune function to obtain the most accurate results.Keywords: T cell immune function assay, Blood storage, ATP levels, Monitoring of transplant patients.(Transplantation 2012;94: 00Y00)

BACKGROUND: Studies show that alemtuzumab, a potent lymphocyte-depleting agent, is well tolerated in pediatric renal transplantation. We report on the use of alemtuzumab induction in highly HLA sensitized (HS) pediatric kidney transplant patients. METHODS: Fifty pediatric renal transplants were performed from 1/2009-12/2014. 15 HS patients received IVIG (2 g/kg ×2 doses)/rituximab (375 mg/m(2) ×1) for desensitization with alemtuzumab induction (15-30 mg, 1 dose, subcutaneous), whereas 35 nonsensitized patients received anti–IL-2R. Graft survival and infections were compared between 2 groups. RESULTS: All HS patients had received a prior transplant and were older with lower risk for viral infections due to serostatus. Patient survival was 100%, and graft outcomes were similar with mean 1-year creatinine of 1.03 ± 0.45 versus 0.99 ± 0.6 (P = 0.48). Although a higher incidence of acute cellular rejection was seen in HS patients receiving alemtuzumab (P = 0.001), there was a nonsignificant difference in antibody-mediated rejection. White blood cell and absolute lymphocyte count were significantly lower in alemtuzumab group at 30 days (P < 0.0001) and at 1 year (P = 0.026 and P = 0.001), respectively. There was no significant difference in bacterial, viral, or fungal infections after transplant. CONCLUSIONS: Alemtuzumab induction with desensitization led to nearly equivalent graft survival and functional outcomes in HS pediatric patients as nonsensitized patients receiving anti–IL-2R induction. With this small sample size, we observed significant reduction of white blood cell and absolute lymphocyte count up to 1 year posttransplant. The risk of infection was comparable between the 2 groups; however, patients who received alemtuzumab were older and at lower risk of viral infection due to serostatus.

Background. Vascular endothelial cells are primary targets for injury during both cellular and humoral allograft rejection (AR). In cardiac transplantation, the role of humoral immunity in mediating AR has not been extensively characterized. Methods. Antibodies against human vascular endothelial cells (AECA) were measured using a cellular ELISA developed from human umbilical vein endothelial cells in 80 consecutive patients after cardiac transplantation. The aim was to determine the incidence of AECA formation after transplantation and their association with different types of AR, graft survival, and development of cardiac allograft vasculopathy (CAV). At least eight serum samples obtained from each patient were examined for AECA and an endomyocardial biopsy was performed at regular intervals during the first year after transplantation. Results. Of the 80 patients examined, 31 were AECA (+) and 49 patients were AECA (-). There were no significant differences between the AECA (+) and (-) groups when examined for age, sex, and pretransplantation ischemia time. A significant correlation was found between the presence of AECA and humoral AR (P

Background. Cytomegalovirus (CMV) infection represents a significant morbidity factor for transplant recipients. A rapid, sensitive, specific, and reliable test is desirable for early detection of CMV infection and for monitoring the efficacy of antiviral therapy. Methods. We examined the incidence of CMV infection in 95 cardiac and 25 renal allograft recipients followed for up to 3 years using qualitative and quantitative polymerase chain reaction (PCR) techniques. Results were subsequently correlated with clinical findings. Of the 236 samples analyzed by the CMV PCR, 84 and 20 were also analyzed by blood buffy coat culture and anti-CMV antibody IgM assays, respectively. Results. The sensitivity of the CMV PCR was found to be superior to that of the other assays, although the specificity of the blood buffy coat culture is as good as that of the CMV PCR, which is higher than that of the anti-CMV antibody IgM assay. CMV infection was detected by the CMV PCR in 17 of 95 cardiac and 9 of 25 renal transplant recipients. Clinical symptoms were observed when

Intravenous immunoglobulin (IVIG) has the potential to regulate Ig production, but the mechanism(s) responsible for this effect is unknown. In experiments reported here, we examined the ability of IVIG to regulate Ig production in human peripheral blood mononuclear cells (PBMCs) stimulated with pokeweed mitogen (PWM). IVIG (2-10 mg/ml) showed a potent (80-85%) inhibition of PWM-stimulated IgG, IgM, and IgA production. To determine more precisely how IVIG mediated the inhibition of Ig production, we studied Ig promoting cytokine gene expression after PWM stimulation with or without IVIG (2 and 10 mg/ml) using dot-blot techniques. RNA was isolated from PBMCs at predetermined time points and probed with cDNAs specific for human cytokines (IL-1 beta, IL-2, IL-2R, IL-4, IL-5, IL-6, gamma-IFN, and TNF-alpha). IL-6 mRNA accumulation was maximal at 4.5 hr post-PWM stimulation and was inhibited 64-75% when IVIG (10 mg/ml) was present. gamma-IFN mRNA levels peaked at 72 hr poststimulation and were also 68-75% inhibited by IVIG. IL-2 mRNA levels peaked at 4.5 hr and were 23-46% inhibited by IVIG. The inhibitory effect of IVIG on production of these cytokines (IL-6 and gamma-IFN) was also observed at the protein level in sonicated PBMCs after incubation with PWM and IVIG. The mRNA levels for other cytokines were not or only minimally inhibited by IVIG. Addition of IL-6, gamma-IFN, or IL-2 partially restored Ig production in IVIG-treated PWM-stimulated cultures, suggesting that inhibition of other cytokines or another mechanism(s) independent of cytokine inhibition might also be involved, although inhibition of IL-6, gamma-IFN, and IL-2 may be one of the critical factors in the suppression of Ig production by IVIG.

Cytomegalovirus (CMV) infection represents a significant morbidity factor for transplant recipients. CMV infection has an association with the development of allograft rejection (AR) through graft endothelial cell (EC) damage, but the mechanisms are not yet clear. There are few reports addressing the role of humoral immunity in vascular EC injury mediated by CMV infection whereas many reports are available regarding the mechanism(s) of CMV-associated allograft EC injury mediated by cellular immunity. Here we examine the incidence of CMV infection in 40 cardiac and 25 renal allograft recipients using polymerase chain reaction (PCR) techniques. We also monitored sera for the development of anti-EC antibodies (AECA) using an ELISA with human umbilical vein ECs as targets, and IL-2 levels using an ELISA. AECA levels (immunoglobulin-G and immunoglobulin-M) were significantly elevated in allograft recipients who demonstrated CMV-PCR positivity when compared with the CMV-PCR negative group (IgG: 23.1 +/- 16.4 vs 4.7 +/- 4.5, p0.0001; IgM: 47.0 +/- 53.6 vs 7.0 +/- 11.2, p0.0001). Serum AECA (IgG and IgM) levels increased one to four weeks after CMV DNA was detected and elevated AECA levels persisted for at least one to two months, and sometimes for several months. Elevated AECA levels correlated well with serum IL-2 levels. These results suggest that CMV infection is associated with an increased humoral immune response to EC antigens, which may be a risk factor for vascular rejection, chronic rejection and decreased allograft survival.