Determination of unacceptable antigens by summation of anti-HLA eplet antibody strength (MFI) based on single antigen bead assays: Excellent correlation with negative cell based cross matches.

The reliability of single antigen bead (SAB) assays and their use in predicting a negative cell based cross match (CBXM) is essential in the era of expanded organ sharing. A wide range of accuracy (80-95%) in predicting negative CBXM has been reported. We hypothesized that in SAB assays an antibody against an HLA eplet that was common among a number of different HLA alleles would be distributed among all of the shared eplet positive SABs. This would reduce binding to the donor specific SAB resulting in an under-estimate of antibody strength. We tested this proposal in adsorption studies using, instead of lymphocytes, a novel reagent, single-SAB (sSAB). Properties of SAB assays were examined that provided a basis for conducting adsorption - elution experiments with the sSABs. We found that incubation of sera with sA*02:01 or sB*42:01 not only depleted reactivity to these alleles but also depleted reactivity to beads that shared the reactive eplet. Anti-eplet strength from SAB data (sum of the MFI of eplet positive SABs (MFI-s) was compared with CBXM out comes in two case studies and with 99 proficiency testing sera. In these validation studies, an MFI-s above 11,000 was associated with a positive FCXM. This approach was placed into clinical practice for listing unacceptable antigens that shared a common eplet. CDCXMs (n = 3261) and FCXMs (n = 1012) were performed on patients listed in UNOS for deceased donor kidneys. All CDCXMs were negative and all FCXMs except one were negative. We conclude that summation of eplet strength provides a highly reliable method of predicting prospective negative CBXMs resulting in substantial savings of time and effort. Based on shared eplet summation data, CMS/NYSDOH has accepted our bead based XM (BBXM) method (aka, virtual XM) performed prior to transplant as fulfilling the regulation that XM results be available before kidney transplantation.

Poor kidney allograft survival associated with positive B cell - Only flow cytometry cross matches: a ten year single center study.

The presence of donor specific antibody (DSA) to class 1 or class 2 HLA as detected respectively in T cell or B cell - only flow cytometry cross matches (FCXMs) are risk factors for renal allograft survival, though the comparative risk of these XMs has not been definitively established. Allograft survival and FCXM data in 624 microcytotoxicity (CDC) XM negative kidney transplants were evaluated. Short and long term allograft survival was significantly less in recipients with T(-) B(+) FCXMs (1 year, 74%, 10 year, 58%) compared to T(+) B(+) FCXMs (1 year, 84%, 10 year, 68%) and to T(-) B(-) FCXM (1 year, 90%, 10 year, 85%). Risk factors were positive FCXM, deceased donor (DD) transplantation and donor age, but not race, gender, recipient age or previous transplant. Recipients with T(-) B(+) and T(+) B(+) FCXMs were at 4.5 and 2.5 fold greater risk, respectively, of DD allograft failure compared to patients with T(-) B(-) FCXMs. The quantitative value of FCXM did not correlate with the duration of graft survival. We conclude that patients with DSA to class 2 HLA have a greater risk of early and late allograft failure compared to patients with DSA to class 1 HLA.

Anti-inflammatory effects of phospholipid transfer protein (PLTP) deficiency in mice.

We previously reported that phospholipid transfer protein-deficient (PLTP KO) mice exhibit a lower rate of atherosclerosis. We proposed two possible mechanisms: a reduction in hepatic apoB secretion (Nat Med 7 (2001) 847) and induction of lipoprotein anti-oxidation activity (J Biol Chem 277 (2002) 31850). We now hypothesized that PLTP KO mice may exhibit an anti-inflammatory state per se. First, we found that PLTP KO mice have significantly lower IL-6 levels than wild type (WT) mice. Secondly, we found that IL-6 treatment increased plasma TNFalpha levels in WT mice, but not in PLTP KO mice. Thirdly, we used flow cytometric analyses to measure the mean fluorescence intensity of I-A(b), a MHC-class II molecule, on peripheral monocytes and found that IL-6 treatment significantly increased the I-A(b)-positive cell levels in WT mice, whereas no changes were observed in the cell levels in PLTP KO mice. The results of our experiments demonstrated an anti-inflammatory effect of PLTP deficiency as a further aspect of its proatherogenic potency.