Pre- and post-transplant monitoring of granzyme B enzyme-linked immunosorbent spot assay in pediatric liver recipients

This study aims to investigate potential role of granzyme B enzyme-linked immunosorbent spot (GrB ELISPOT) for immunological monitoring in pediatric liver transplantation. PATIENTS AND METHODS: Peripheral blood mononuclear cells from 28 pediatric recipients were serially tested for GrB-producing donor-reactive cells at day 0 pre-transplantation (baseline) and days 7, 14, and 28 post-transplantation. RESULTS: At baseline, no difference of GrB value was found in acute rejection (14/28) compared to normal graft function patients (day 0: 4(3.9) spots versus 5(2.9) spots, respectively: p=0.65). At day 7 post-transplantation, acute rejection patients showed frequencies of GrB ELISPOT higher than those with normal graft function, but the differences observed were not statistically significant (day 7: 15(4.9) spots versus 10(4.0) spots, respectively: p=0.55). GrB increased significantly at day 7 from baseline in the rejection group (15(4.9) spots versus 4(3.9), respectively p=0.04), whereas corresponding changes were not significant in the group without rejection (10(4.0) versus 5(2.9), respectively: p=0.15). Conclusion: GrB ELISPOT pre-transplantation could not predict the occurrence of early post-transplant acute rejection; similarly frequencies at days 7, 14 and 28 could not be correlated with acute rejection in pediatric liver recipients. However, a kinetic study of GrB ELISPOT could be helpful to predict or confirm early rejection in the small group of liver allograft recipients analyzed in this study.     

Monitoring of cellular immunity by interferon-gamma enzyme-linked immunosorbent spot assay in kidney allograft recipients: preliminary results of a longitudinal study

Several efforts have been made in past years to identify markers for patients at heightened risk of acute and chronic immune-mediated allograft rejection. The ex vivo monitoring of cellular immunity by the enzyme-linked immunosorbent spot (ELISPOT) assay has recently emerged as a primary tool in predicting short- and long-term outcomes in kidney allograft recipients. Therefore, we started the systematic application of interferon-gamma (IFN-gamma) ELISPOT assay to measure the frequency of producing IFN-gamma in recipient peripheral blood lymphocytes (PBLs) stimulated with donor lymphocytes before and 7, 14, 21, 28, and 60 days after transplantation. Preliminary results in eight kidney transplant patients indicated that the number of HLA mismatches never correlated with the number of IFN-gamma spots. The frequencies of pretransplantation IFN-gamma spots were positively and significantly correlated with the number of posttransplantation IFN-gamma spots. Clinical outcomes were better among recipients with lower frequencies than those with higher frequencies of pre- and/or posttransplantation IFN-gamma spots. The highest pre- and posttransplantation number of IFN-gamma spots was observed in a patient who developed early acute rejection. Significant increases in the number of IFN-gamma spots preceded the onset of acute rejection events and were decreased by supplemental IV steroid administration. Considering the low number of observations, these preliminary results must be considered cautiously; nevertheless, we are encouraged to extend the systematic application of serial IFN-gamma ELISPOT assay measurements in a more consistent cohort of patients.     

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.     

Evaluation of an enzyme-linked immunosorbent assay in the serodiagnosis of amoebic liver abscess

An indirect enzyme-linked immunosorbent assay (ELISA) was evaluated for the detection of anti-amoebic IgG and IgM antibodies to assess its value in distinguishing past from current infection in invasive amoebiasis, particularly in amoebic liver abscess (ALA) patients. Using sera from 295 individuals, the ELISA was also compared with the amoebic gel diffusion (AGD) test. In 100 patients the IgG-ELISA at a single test dilution of 1/6,400 had a sensitivity of 99% for clinically diagnosed ALA. In these same patients the IgM-ELISA at a single dilution of 1/400, had a sensitivity of 64% and a specificity of 97.9%. No cross-reactions were observed in sera from patients with collagen vascular disease. In 121 patients without clinical invasive amoebiasis, 8 were AGD-positive and 12 were IgG-ELISA-positive, giving the latter assay a specificity of 91.7%. This is thought to be due to past infection with Entamoeba histolytica. In symptomless carriers of pathogenic zymodemes, 10/11 were seropositive by the IgG-ELISA and 11/11 by the AGD test. There was an excellent correlation between the IgG-ELISA and the AGD test (r = 0.99). The IgG-ELISA is a sensitive, specific, simple and rapid test. It has the clinical advantage that results are obtainable 2 1/2 hours after receipt of the specimen, compared with the 24-48 hours required for the AGD test. The prompt availability of IgG-ELISA results could prove advantageous for implementation of early therapy. The IgM-ELISA was not found to be sensitive enough to be used as an index of active amoebic infection.     

Diagnosis of neurocysticercosis by enzyme-linked immunosorbent assay

The detection of neurocysticercosis by enzyme-linked immunosorbent assay, the criterion used for determining seropositivity and the method of interpreting results are discussed. Serum and cerebrospinal fluid (CSF) samples from each of 212 patients were analysed. The results showed that in some patients positive antibody titres may be found in only one specimen. If both specimens are analysed 87% of neurocysticercosis patients can be detected serologically. However, if only CSF is tested, the detection rate falls to 67%. It is recommended that clinicians submit both serum and CSF samples for analysis.     

Potential of real-time PCR assessment of granzyme B and perforin up-regulation for rejection monitoring in intestinal transplant recipients

Granzyme B (GrB) and perforin are promising markers to predict acute rejection episodes of transplanted organs. Having recently reported that immunohistochemical expression of GrB/perforin correlates with histologically assessed acute cellular rejection (ACR) episodes in intestinal transplantation recipients, herein we have additionally explored the potential of real-time polymerase chain reaction (PCR) assessment of GrB/perforin gene up-regulation in peripheral blood mononuclear cells. Both immunohistochemical evaluation of GrB/perforin expression and real-time PCR assessment of up-regulation, which was defined as a 2-fold increase with respect to "basal" levels during maintenance immunosuppressive protocols, were performed among a population of 23 intestinal transplant recipients under routine surveillance, in addition to histological analysis of ACR. The ACR scores showed direct relationships both with GrB/perforin immunohistochemistry (IHC) scores (P < .001) and with gene up-regulation by real-time PCR (P = .004). Furthermore, real-time PCR upregulation was associated with the IHC score (P < .001). A preliminary analysis of diagnostic accuracy-performed to gain information to plan future studies-indicated that when using histological assessment as the reference technique, our current definition of PCR up-regulation provided good specificity (84%) but insufficient sensitivity (44%) for a noninvasive prediction of ACR. The results of this pilot study suggested that real-time PCR analysis of GrB/perforin upregulation may help therapeutic decision making, and have the potential for detection of presymptomatic rejection. More extensive studies must investigate strategies to improve the sensitivity of the analyses of GrB/perforin up-regulation.     

The granzyme B and interferon-gamma enzyme-linked immunospot assay as alternatives for cytotoxic T-lymphocyte precursor frequency after renal transplantation

BACKGROUND: The interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay has gained increased popularity as a surrogate marker of cytotoxic T-lymphocyte (CTL) activity. However, the functional activity of CTL might be a more relevant surrogate marker of CTL. Therefore, the authors wondered whether the granzyme B (GrB) ELISPOT assay is a better marker for determining the number of CTL than the IFN-gamma ELISPOT assay. METHOD.: Peripheral blood mononuclear cells (PBMC) from 19 kidney transplant patients were stimulated with donor cells or third-party cells. The authors determined the CTL precursor frequency (CTLpf) and simultaneously measured the number of IFN-gamma- and GrB-producing cells (pc) by ELISPOT assay. RESULTS: In all three different assays, the reactivity to donor cells was significant lower than the reactivity to third-party cells: CTLpf, median: 9 versus 60/10(6) PBMC (P=0.0002); number of IFN-gamma pc: 10 versus 90/10(6) PBMC (P=0.0001); number of GrB pc: 60 versus 205/10(6) PBMC (P=0.05). When the authors compared the CTLpf after third-party stimulation with the corresponding ELISPOT results, they found a positive correlation between the CTLpf and the number of IFN-gamma pc (r(s)=0.47, P=0.05). No correlation was found between the CTLpf and the number of GrB pc (r(s)=0.23, P=0.36). However, when they compared the donor-specific CTLpf with the corresponding ELISPOT results, no correlation with the ELISPOT for IFN-gamma (r(s)=0.10, P=0.69) or GrB (r(s)=-0.24, P=0.34) was found. CONCLUSIONS: The authors feel that the CTLpf, as a measure of the actual endpoint of cytolytic activity and independent of the pathway of killing, remains the "gold standard" for determining donor-specific cytolytic activity after clinical organ transplantation.     

Impact of the donor body mass index on the survival of pediatric liver transplant recipients and post-transplant obesity

In adult liver transplant recipients, the donor body mass index (dBMI) is associated with posttransplant obesity but not with graft or patient survival. Because of the obesity epidemic in the United States and the already limited supply of liver donors, clarifying whether the dBMI affects pediatric outcomes is important. United Network for Organ Sharing data for pediatric liver transplants in the United States (1990-2010) were evaluated. Data on transplants performed between 2004 and 2010 (n = 3788) were used for survival analyses with Kaplan-Meier and Cox proportional hazards models and for posttransplant obesity analyses with generalized estimating equations. For children receiving adult donor livers, a dBMI of 25 to <35 kg/m(2) was not associated with graft or patient survival in univariate or multivariate analyses. A dBMI ≥ 35 kg/m(2) increased the risk of graft loss [hazard ratio (HR) = 2.54, 95% confidence interval (CI) = 1.29-5.01, P = 0.007] and death (HR = 3.56, 95% CI = 1.64-7.72, P = 0.001). For pediatric donors, the dBMI was not associated with graft loss or mortality in a univariate or multivariate analysis. An overweight or obese donor was not a risk factor for posttransplant obesity. Overweight and obesity are common among liver transplant donors. This analysis suggests that for adult donors, a body mass index (BMI) of 25 to <35 kg/m(2) should not by itself be a contraindication to liver donation. Severe obesity (BMI ≥ 35 kg/m(2)) in adult donors increased the risk of graft loss and mortality, even after adjustments for recipient, donor, and transplant risk factors. Posttransplant obesity was not associated with the dBMI in this analysis. Further research is needed to clarify the impact of donor obesity on pediatric liver transplant recipients.     

Diagnostic sensitivity of enzyme-linked immunosorbent assay for IgG in hydatid disease

Context  

Hydatid disease in humans is most commonly caused by Echinococcus granulosus and results in development of cysts in various organs of body. The diagnosis is made by serology i.e. by estimation of antibody levels or on imaging studies. Enzyme-linked immunosorbent assay (ELISA) is widely used for serological confirmation of the disease.     

Pre-transplant risk factors predicting post-transplant cytomegalovirus infection in liver transplant recipients

Cytomegalovirus (CMV) infection causes significant morbidity and mortality among transplant recipients. Although it is still not clear if a preemptive strategy is superior to a prophylactic strategy, many transplant programs elect for preemptive treatment for post-transplant CMV infection. In order to improve the preemptive strategy, we analyzed a series of liver recipients by means of quantitative real-time polymerase chain reaction (PCR). Ninety-one liver transplant recipients were monitored by real-time PCR for CMV, and the results were analyzed in terms of preoperative conditions. Multivariate analysis revealed fulminant hepatic failure as an underlying disease (odds ratio, 6.8; 95% CI, 1.2-39.2), while an ABO-incompatible graft (odds ratio, 5.0; 95% CI, 1.3-19.1), and a serological combination of the donor (D) being positive with the recipient (R) being negative for CMV (D+/R-) (odds ratio, 5.8; 95% CI, 1.3-26.0) were independently associated with the development of significant CMV infection. Patients with risk factors had higher peak CMV DNA concentrations than those without, and developed CMV infections faster (P = 0.0002). Screening of recipients according to risk factors and PCR monitoring may result in an optimization of the preemptive strategy.     

Detection of fibronectin on vascular flow surfaces by enzyme-linked immunosorbent assay

Numerous variables are involved in the attachment of endothelial cells to a substrate. Quantifying these factors both on native vessels and on synthetic substrates is important in determining the success of endothelial cell attachment, retention, and growth on these substrates. Fibronectin is an important cell attachment molecule and is likely to be key to the successful attachment of endothelial cells to any substrate. For this reason we have developed an enzyme-linked immunosorbent assay for interrogation of the luminal surface of native and synthetic vessels for the presence of fibronectin. A plexiglass chamber was designed with two blocks, an upper block with wells and a lower supporting block. The chamber was then assembled with a vessel between the two blocks, forming the bottom of the well. This luminal surface was then interrogated by conventional enzyme-linked immunosorbent assay. Native vessels, collagenase-digested vessels, acellular matrices and PTFE preclotted with whole blood were assayed to determine the quantity of fibronectin present. These results were correlated with a bioassay developed to determine the quantity of fibronectin necessary for cell attachment. It was concluded that all of the samples assayed had ample fibronectin for cell attachment and that other factors must be responsible for successful maintenance of a cell monolayer.     

双抗体夹心一步法检测前列腺特异抗原及应用的评价

用聚乙二醇、葡聚糖凝胶（ＰＥＧ、Ｓｅｐｈａｄｅｘ）两步法提取精浆前列腺特异抗原（ＰＳＡ），建立了一步检测血清ＰＳＡ的ＥＬＩＳＡ法及正常参考值范围。经５８例临床标本及９５例正常男性血清ＰＳＡ测定，对前列腺癌诊断的敏感性和特异性分别为１００％和９４．２％；６０岁以下和６０岁以上的男性血清ＰＳＡ临界值分别为３．４μｇ／Ｌ和４．６μｇ／Ｌ。表明年龄因素对ＰＳＡ测定有较大影响，在判断测定结果时必须加以考虑。     

Pre- and post-transplant monitoring of soluble CD30 levels as predictor of acute renal allograft rejection

Identification of renal graft candidates at high risk of impending acute rejection (AR) and graft loss may be helpful for patient-tailored immunosuppressive regimens and renal graft survival. To investigate the feasibility with soluble CD30 (sCD30) as predictor of AR, sCD30 levels of 70 patients were detected on day 0 pre-transplant and day 1, 3, 5, 7, 10, 14, 21, and 30 post-transplant. AR episodes in 6 months were recorded and then patients were divided into Group AR (n=11) and Group UC (n=59). Results showed that the patients had higher pre-transplant sCD30 levels than healthy people. A significant decrease of sCD30 was observed on the first day post-transplant and continued until day 14 post-transplant. Soluble CD30 presented a stable level from day 14 to 30 post-transplant. Pre-transplant sCD30 levels of Group AR were much higher than those of Group UC (P<0.001). Patients of Group AR also had higher sCD30 levels than those of Group UC on day 1, 3, 5, 7, 10 and 14 (P<0.001). The sCD30 level presented a significantly delayed decrease in the patients of Group AR. Statistical results showed that the highest value of area under ROC curve (0.95) was obtained on day 5 post-transplant, suggesting that sCD30 levels on day 5 are of high predictive value. Therefore, sCD30 level may be a good marker of increased alloreactivity and of significant predictive value. It's necessary to monitor the variation of sCD30 in the early period post-transplant.     

Enzyme-linked immunosorbent spot assay for donor-reactive interferon-gamma-producing cells identifies T-cell presensitization and correlates with graft function at 6 and 12 months in renal-transplant recipients

BACKGROUND: A major goal in clinical transplantation is the individualization of immunosuppression. This requires a definition of markers that identify patients at heightened risk of acute rejection and immune-mediated chronic allograft nephropathy. METHODS: Frequencies of interferon-gamma-producing donor-reactive cells were serially determined in unselected renal-transplant patients in an enzyme-linked immunosorbent spot assay (ELISPOT) before transplantation (n = 42) and up to 10 (mean 5.0) times during the first 6 months posttransplantation (n = 48) to determine detailed kinetics and analyze for correlation with acute rejection and graft function at 6 and 12 months posttransplantation. RESULTS: Pretransplant ELISPOT frequencies were significantly higher in patients with acute rejection (16/42) versus nonrejecters (26/42). Highly elevated pretransplant frequencies (>200 spots/300,000 peripheral blood mononuclear cells [PBMCs], n = 5/42) were associated with a risk of severe acute rejection episodes but were independent of risk factors such as high panel reactive antibodies. Early graft failure exclusively occurred in this group. Importantly, mean ELISPOT frequencies at weeks 2 and 3 but not at month 6 posttransplant correlated inversely with 6 and 12 months glomerular filtration rate. The correlation between ELISPOT frequencies and renal function showed the highest significance in patients without acute rejection. CONCLUSIONS: The pretransplant ELISPOT assay might be useful to identify T-cell presensitized patients, who are at heightened risk for severe early acute rejection. An analysis of ELISPOT donor-reactive cells during the early posttransplant period might allow an identification of patients at risk for immune-mediated graft deterioration.