CD4+ lymphocyte adenosine triphosphate determination in sepsis: a cohort study.

INTRODUCTION: Patients suffering from sepsis are currently classified on a clinical basis (i.e., sepsis, severe sepsis, septic shock); however, this clinical classification may not accurately reflect the overall immune status of an individual patient. Our objective was to describe a cohort of patients with sepsis in terms of their measured immune status. METHODS: Fifty-two patients with sepsis (n = 13), severe sepsis (n = 21), or septic shock (n = 18) were studied. The immune status was determined by measuring the CD4+ lymphocyte adenosine triphosphate (ATP) content after mitogen stimulation in whole blood. RESULTS: The measured CD4+ lymphocyte ATP content at the time of ICU admission did not differ among the various groups defined by the sepsis classification system (sepsis = 454 +/- 79 ng/ml; severe sepsis = 359 +/- 54 ng/ml; septic shock = 371 +/- 53 ng/ml; P = 0.44). Furthermore, survivors of sepsis had a significantly higher CD4+ lymphocyte ATP content at the time of ICU admission than did nonsurvivors of sepsis (431 +/- 41 ng/mL vs. 266 +/- 53 ng/mL, respectively; P = 0.04). CONCLUSIONS: The sepsis classification system that is currently used is not representative of the individual immune status as determined by measuring the CD4+ lymphocyte ATP content. Moreover, a lower CD4+ ATP content at the time of ICU admission is associated with a worse clinical outcome in those suffering from sepsis.

Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6.

OBJECTIVES: To investigate immunological mechanisms underlying accelerated antibody-mediated rejection (AMR) of a living-related renal allograft in a patient with no detectable antibodies to donor human leukocyte antigens (HLA) in pre-transplant sera. METHODS: Pre- and post-transplant HLA antibody specificities were determined by single-antigen bead assay, and crossmatching was performed by flow cytometry- and complement-dependent cytotoxicity-based methods. Intermediate- and high-resolution HLA typing were performed by molecular methods. RESULTS: Pre-transplant patient serum reacted weakly against Bw6-positive beads; cytotoxicity and flow crossmatches were negative. The patient was mismatched for the donor antigens B62 and C10 (Bw6-positive). Following transplantation, strong antibody responses against B62, C10, and all Bw6-positive beads were detected. This reactivity was initially masked by complement interference, but became apparent at 1:20 dilution. High-resolution typing suggested that the anti-C16 antibody reactivity detected was an allele-specific response to donor C∗16:01 (Bw6-positive) but not recipient C∗16:02 (Bw6-negative). Alloimmunization likely occurred during pregnancy, during which HLA-C14 (Bw6-positive) was the only mismatched paternal HLA Class I allele. CONCLUSIONS: Sensitization to HLA-Bw6 via exposure to paternal HLA-C14 during pregnancy likely predisposed this patient to AMR. The case demonstrates the immunogenicity of HLA-C14-associated Bw6 epitopes in vivo and the clinical significance of low-level antibodies to HLA-Bw6.

A significant role for histocompatibility in human islet transplantation.

BACKGROUND: In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipient's immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation. METHODS: We retrospectively analyzed seven patients that were transplanted with islets under the auspices of the Juvenile Diabetes Research Foundation and Islet Cell Resource Center/National Institutes of Health. Humoral sensitization towards donor antigens both prior to and following islet transplantation was detected by FLOW panel reactive antibodies (PRA) and donor-specific cellular sensitization was detected by performing enzyme-linked immunospot assay analysis for cytokines interferon-gamma and interleukin-2. RESULTS: Our analysis demonstrates that humoral and cellular sensitization to histocompatibility antigens prior to and after islet transplantation are associated with the failure of transplanted islets CONCLUSION: Patient selection based on sensitization to donor HLA may be one of the factors crucial for the success of islet transplant. Further, in some patients, rejection of islets can be associated with sensitization to mismatched donor histocompatibility antigens.

HLA class I antibody mediated accommodation of endothelial cells via the activation of PI3K/cAMP dependent PKA pathway.

Allografts transplanted across ABO incompatibility or human leucocyte antigen (HLA)-sensitization undergoes antibody (Ab) mediated hyperacute rejection. Depleting anti-graft Ab from the recipient by plasmapheresis prior to transplantation can prevent this Ab-mediated rejection. Under these conditions, allografts have been shown to function even when the Ab rebound in the recipients. We have developed an in vitro model using human aortic endothelial cells (EC) and elucidated the ability of W6/32 HLA class I monoclonal Ab to provide signals following binding to MHC class I molecules. Using this model, we show that ECs undergo caspase 3-dependent cell death by apoptosis upon exposure to saturating concentrations of W6/32 and complement. In contrast, exposure of ECs to sub-saturating concentrations of W6/32 conferred resistance towards Ab/complement-mediated lysis that has been termed accommodation. Accommodated ECs exhibited a significant increase in the expression of anti-apoptotic genes Bcl-xL, Bcl-2 and Heme Oxygenase-1 and the induction of Phosphatidylinositol 3 kinase (PI3K) and cyclic adenosine monophosphate (cAMP) dependent protein kinase A activities that facilitate the phosphorylation of Bad at positions Ser(136) and Ser(112). In conclusion, exposure of sub-saturating concentrations of HLA class I Ab results in the induction of signals downstream that confers resistance to endothelial cells against Ab-complement mediated cell death. Together, the observations made in this study will provide the basis for delineating the molecular mechanisms involved in mediating accommodation and developing strategies to induce accommodation in grafts prior to transplantation in highly sensitized patients.

Association of the HLA-DR15/HLA-DQ6 haplotype with development of choroidal neovascular lesions in presumed ocular histoplasmosis syndrome.

Associations of human leukocyte antigen DR2 (HLA-DR2) and HLA-B7 with presumed ocular histoplasmosis syndrome (POHS) in the United States has been previously described. However, these associations were determined by means of low-resolution, complement-dependent cytotoxicity assays for HLA-A, HLA-B, and HLA-DR molecules. To determine whether POHS is associated with other HLA alleles within the HLA-A, HLA-B, HLA-DR, and HLA-DQ loci, we performed a case control study of 34 patients diagnosed with macular choroidal neovascular membrane secondary to POHS and 45 healthy control individuals. Peripheral blood-derived DNA from the study patients was typed for HLA genes by means of sequence-specific primers that gave low-medium allele resolution. Significant associations were observed between HLA-B7 (X2 = 14.30, pc = 0.004, relative risk = 8.23), HLA-DR15 (X2 = 29.08, pc = 0.000001, relative risk = 27.50), and HLA-DQ6 (X2 = 23.09, pc = 0.00001, relative risk = 27.43) and POHS. Because there are strong linkage disequilibria between HLA-DR15 (a subtype of HLA-DR2) and HLA-B7 as well as HLA-DQ6, the significantly higher association of HLA-DR15 and HLA-DQ6 with POHS as compared to HLA-B7 suggests that the former alleles mediate susceptibility to the disease. In conclusion, there is a significant association between the HLA-DR15/HLA-DQ6 haplotype and development of choroidal neovascular lesions in POHS.

Cutoff values and data handling for solid-phase testing for antibodies to HLA: effects on listing unacceptable antigens for thoracic organ transplantation.

Application of single-antigen solid-phase immunoassay (SPI) in virtual crossmatch-based organ allocation has been hindered by continued debate over the biologic relevance of detected antibodies and the relationship between cutoff mean fluorescence intensity (MFI) values with crossmatch testing results. To define SPI parameters accurately predicting crossmatch testing, we analyzed a series of anti-HLA antibodies from highly-sensitized patients awaiting lung or heart transplantation. Serial dilution of serum for SPI and cytotoxic crossmatch (CXM) enabled comparison over a wide spectrum of antibody "strengths". Receiver operating characteristic (ROC) analysis identified predictive cutoff values for HLA Class I and DR-specific antibodies. However, antibodies to HLA-DQ antigens demonstrated a significantly different characteristic, highlighting difficulties in interpretation of clinical significance. We also quantitatively characterized two data handling methods, MFI ratio (MR) and relative ratio (RR), to examine their potential impact on identifying unacceptable antigens. In combination with user defined cutoff values, MFI, MR and RR lead to discordant identification of antibodies. Establishment of cutoff values for MR and RR that are comparable to MFI demonstrated increased consistency in antibody identification. This single laboratory experience is an example of establishing statistically robust cutoff values and validation across different data handling methods for use of SPI in virtual crossmatch.

Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: a 30-month analysis in living donor kidney transplantation.

Selection of donors for kidney transplantation depends on accurate prediction of risk factors for immunologic rejection. Historically, cytotoxicity crossmatch (CXM) examining lysis of donor cells by preformed anti-human leukocyte antigen (HLA) antibodies (Abs) has been considered the best predictor of immunologic rejection. However, there is much interest in defining anti-HLA Ab specificity in recipient sera by immunoassay to predict crossmatch results and aid in donor selection. Current immunoassays for anti-HLA Abs are highly sensitive, though correlation between Abs detected by immunoassay and their functional relevance in CXM and subsequent transplantation is not well defined. In this study, we retrospectively examined the predictive value of detection of donor-specific anti-HLA Abs (DSA) by Luminex Single Antigen assay from 149 consecutive living donor kidney transplant recipients. We demonstrate that detection of DSA by immunoassay accurately predicted negative crossmatch and graft survival. However, this approach had limited sensitivity for predicting positive crossmatch, attributable to either limited typing of donor HLA-DQ and -DP alleles or due to non-HLA Abs. False-positive prediction of CXM correlated with detection of "weak" Abs with low mean fluorescence intensity (MFI < 2000). Furthermore, we found that a ratio of the MFI of the DSA bead to the MFI of the positive control bead was a better method for identifying weak DSA that did not result in CXM-positive reactions. Interestingly, patients with weak DSA and negative CXM had equivalent graft survival over an 18 month follow-up period, suggesting that weak DSA may not preclude transplantation.

Mortality and morbidity in pre-sensitized pediatric heart transplant recipients with a positive donor crossmatch utilizing peri-operative plasmapheresis and cytolytic therapy.

BACKGROUND: The difficulty in obtaining a prospective negative donor/recipient crossmatch limits the ability to successfully transplant pediatric heart transplant candidates who show evidence of antibodies to multiple human leukocyte antigens (pre-sensitized patients). METHODS: We utilized a protocol that included peri-operative plasmapheresis, thymoglobulin and cyclophosphamide in 17 pre-sensitized (panel-reactive antibodies [PRA] >10%) pediatric patients to accept donors for these patients without a prospective crossmatch between 1995 and 2005. A retrospective review of survival, rejection and infection was performed, comparing the frequency of rejection and infection in our patients who were transplanted with a complement-dependent cytotoxic (CDC)-positive donor/recipient crossmatch to those patients transplanted with a negative crossmatch. RESULTS: Thirteen of 17 patients were found to have a CDC-positive crossmatch. Actuarial survival after transplantation was 85% at 1 year and 73% at 3 years. Twelve of 13 (92%) of these patients experienced rejection, and 5 of 13 (38%) had recurrent rejection, generally in the first 2 months after transplantation. Rejection was associated with hemodynamic compromise in 58% of first rejection episodes and 67% of episodes of recurrent rejection. The frequency of rejection in these patients was significantly greater than the frequency in patients with a negative crossmatch in the first 6 months after transplantation, but not afterward. The frequency of infection episodes was not significantly different between the groups. CONCLUSIONS: Heart transplantation in pre-sensitized pediatric recipients with a CDC-positive donor/recipient crossmatch may result in reasonable short-term survival, but with a high frequency of early rejection, often with hemodynamic compromise.

Pre-exposure to sub-saturating concentrations of HLA class I antibodies confers resistance to endothelial cells against antibody complement-mediated lysis by regulating Bad through the phosphatidylinositol 3-kinase/Akt pathway.

Allografts transplanted across HLA-sensitization results in an antibody-mediated rejection known as hyperacute rejection. Depleting anti-graft antibodies from the recipient by plasmapheresis prior to transplantation can prevent this rejection. We developed an in vitro model using polyclonal HLA class I antibodies obtained from highly sensitized patients awaiting transplantation,and analyzed their ability to provide signals following binding to human aortic endothelial cells (EC). Using this model, we show that EC undergo caspase 3-dependent cell death by apoptosis upon exposure to saturating concentrations of HLA class I antibodies and complement accompanied by loss of Akt activation and phosphorylation of Bad. In contrast, exposure of EC to sub-saturating concentrations of HLA class I antibodies conferred resistance towards antibody/complement-mediated lysis termed accommodation. Accommodated EC exhibited reduction in the expression of the adhesion molecules ICAM-1 and VCAM-1 and a significant increase in the expression of anti-apoptotic genes Bcl-xL, Bcl-2 and heme oxygenase-1. Further, induction of phosphatidylinositol 3-kinase (PI3K) and Akt activities that facilitate the phosphorylation of Bad were also noted. In conclusion, exposure of sub-saturating concentrations of HLA class I antibodies results in the induction of PI3K/Akt pathway that confers resistance to endothelial cells against antibody/complement-mediated cell death.