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It is often difficult for students grasp the concept that some organisms can grow in the absence of oxygen and that oxygen can be toxic to organisms. However, as educators usually describe in the classroom, the importance of the anaerobic growth of microorganisms can be seen in many areas of industry, health, and the creation of several categories of food ranging from fermented alcohols to breads and cheeses. To demonstrate that microorganisms can grow and thrive in the absence of oxygen, we have developed an inexpensive and easy technique to culture facultative anaerobic bacteria from yogurt in high school or undergraduate biology laboratories. Using the method discussed, which uses readily available and low-cost equipment, students can culture and visually identify facultative anaerobic bacteria that they may consume on a regular basis, demonstrating the utility of organisms that thrive in an anaerobic environment.

BACKGROUND Panel-reactive antibody (PRA) testing provides assessment of the breadth of sensitization a patient might have against human leukocyte antigen (HLA) antigens. The evolution of calculated PRA (cPRA) reflects the commitment of the transplant community to increase accessibility and promote equity to all patients awaiting kidney transplantation. Recent data from our center and others, however, suggested that a significant diversity of HLA-DQ antigens is not captured, which may lead to inequity in allocating cPRA points. METHODS HLA-DRB1-DQA1-DQB1 typing of 2182 individuals was evaluated for this study using Luminex-based sequence-specific oligonucleotide typing. A total of 3182 haplotypes were confirmed to have the level of resolution required for this study. RESULTS The diversity of HLA-DQαβ alleles is greater than what is apparent using the serologic equivalents. The distribution of these alleles within a serologic group varies, with some alleles being more frequent than others; therefore, their representation within the current cPRA system is inaccurate. Three informative examples are given. Haplotypes of DR antigens with DQαβ alleles did not always follow the common published linkage disequilibrium, especially in populations where there is greater genetic diversity. CONCLUSIONS The current cPRA system does not take into account the distribution of molecular equivalents within DQ serologic specificities. This can result is inequitable allocation of sensitization points and disadvantaging the more sensitized patients. To ameliorate this situation, the United Network for Organ Sharing system should allow inputting HLA-DQαβ alleles both for donor typing and as antibody specificities, which will lead to better representation of unacceptable DQ alleles and improve organ allocation equity.

Endothelial cell antigens have been reported as potential targets for antibodies in the context of organ transplantation, leading to increased risk for graft failure. Serum samples from 142 consecutive living donor kidney recipients were tested for the presence of antibodies to angiotensin II - type 1 receptor (AT1R), donor endothelial cells, and donor HLA. Graft survival was monitored for five years post-transplant, and secondary outcomes, including biopsy-proven rejection, proteinuria, biopsy-proven vasculopathy, and renal function based on serum creatinine were also assessed for the first two to three years. AT1R antibody levels were positive (>17U/mL) in 11.3%, 18.8% and 8.1% of patients pre-transplant, post-transplant and at time of indication biopsy, respectively. XM-ONE assay was positive in 17.6% of patients pre-transplant (7 IgG+; 15 IgM+; 3 IgG+/IgM+). Overall, 4 patients experienced antibody-mediated rejection (AMR), 31 borderline cellular rejection (BCR), 19 cellular rejection (CR) and 3 mixed AMR and CR within the first 24months. While pre-existing and de novo donor-specific HLA antibodies were associated with graft failure and many secondary outcomes, no statistical association was found for either anti-endothelial or anti-AT1R antibodies, indicating that these tests may not be the best predictors of graft outcome in living donor renal transplantation.

Background. The United Network for Organ Sharing algorithm for deceased-donor kidney allocation considers only the human leukocyte antigen (HLA)-A, HLA-B, and HLA-DR loci. Although HLA-DQ serologic specificities can be entered as unacceptable antigens, they are assigned only by the identity of the DQA chain, disregarding the role of the similarly polymorphic > chain. DQ>/A combinations result in unique antigenic epitopes, which serve as targets to different antibodies. Therefore, the presence of HLA antibodies to one DQ>/A combination should not preclude negative crossmatch (XM) against another combination. In this retrospective analysis, patients were allowed XM against a particular donor if they had antibodies to some, but not all, DQ>/A allele combinations with the donor serologic HLA-DQ antigens. Methods. HLA antibody signature was obtained using solid-phase Luminex-based antibody analysis. Results were captured at the high-resolution level (as provided by the positive beads). Potential donors were typed to include information on both HLA-DQA and HLA-DQB alleles. Results. Of the 1130 flow XM assays performed, 147 patients had antibodies to donor serologic HLA-DQ antigens. Thirty-five of those patients had antibodies to an allelic DQ>/A combination within the donor serologic DQ specificity that were different from the donor’s DQ>/A, leading to negative flow XM results (24%). Virtual XM, accounting for donor DQ>/A combinations, successfully predicts more than 98% of XM outcomes. Conclusions. In patients with allelic DQ>/A antibodies, denying the opportunity for XM based on serologically defined unacceptable antigens can disadvantage the patient. Larger cohort studies are required to substantiate our observation. Introducing DQ>/A combination information may increase virtual XM accuracy and organ allocation equity.

Anti-endothelial cell antibodies (AECAs) may play a role in allograft rejection. We prospectively tested 150 consecutive living donor kidney transplant recipients, with transplants performed at Northwestern Memorial Hospital between January and December 2010, using the donor-specific endothelial (XM-ONE) crossmatch. 88/150 Patients received standard of care (SOC) immunosuppression and analyzed separately, in addition to the complete study cohort. Patients were followed for one year and XM-ONE results were analyzed in relation to occurrence of acute rejection, proteinuria, serum creatinine levels, and biopsy proven fibrosis. No correlation was found between XM-ONE results and protocol or “for-cause” biopsy proven acute rejection or vasculopathy at 12 months. When IgG+ and IgM+ results of the XM-ONE assay were combined, a correlation with proteinuria at 12 months was observed ( p = 0.047). Although IgG + XM-ONE results were associated with significantly higher creatinine at 6 months ( p = 0.018), significance was lost at 12 months. Conversely, patients with an IgM + XM-ONE crossmatch had significantly lower creatinine at 1 month ( p = 0.019), 3 months ( p = 0.0045), and 6 months ( p = 0.038) post-transplant, but lost statistical significance at 12 months ( p = 0.67) post-transplant. In summary, the presence of AECAs as determined by a positive XM-ONE result was not predictive of overall poorer graft outcome after one year in our center.

Human CD8(+) regulatory T cells, particularly the CD8(+)CD28(-) T suppressor cells, have emerged as an important modulator of alloimmunity. Understanding the conditions under which these cells are induced and/or expanded would greatly facilitate their application in future clinical trials. In the current study, we develop a novel strategy that combines common gamma chain (γc) cytokines IL-2, IL-7 and IL-15 and donor antigen presenting cells (APCs) to stimulate full HLA-mismatched allogeneic human CD8(+) T cells which results in significant expansions of donor-specific CD8(+)CD28(-) T suppressor cells in vitro. The expanded CD8(+)CD28(-) T cells exhibit increased expressions of CTLA-4, FoxP3, and CD25, while down-regulate expressions of CD56, CD57, CD127, and perforin. Furthermore, these cells suppress proliferation of CD4(+) T cells in a contact-dependent and cytokine-independent manner. Interestingly, the specificity of suppression is restricted by the donor HLA class I antigens but promiscuous to HLA class II antigens, providing a potential mechanism for linked suppression. Taken together, our results demonstrate a novel role for common γc cytokines in combination with donor APCs in the expansion of donor-specific CD8(+)CD28(-) T suppressor cells, and represent a robust strategy for in vitro generation of such cells for adoptive cellular immunotherapy in transplantation.

Aim Single antigen reagent panels differ in the makeup of HLA alleles offered for testing. Additional alleles (2 HLADR, 7 HLA-DQ) were added for the newest lot of Gen-Probe MHC Class II reagents in an effort to improve diversity of the panel and increase the ability to interchange between commercial products. However, an analysis of the correlation between single antigen reagents is necessary before results may be compared. Methods Twenty-two sera samples were tested in parallel with Gen-Probe (GP; Lot 03022V) and One Lambda (OL; Lot 9) single antigen MHC Class II reagents. GP assay requires addition of 40 μl bead mix + 10 μl sera, and the OL assay requires 5 μl bead mix + 20 μl sera, while the absolute number of beads is similar between assays. Murine monoclonal antibodies against HLA-DR, -DQ, and -DP with a concentration capable of saturating reagents were used to compare antigen density of GP and OL reagents. GP Background Corrected Mean (BCM) value and OL Normalized value in mean fluorescence intensity (MFI) from antigen density experiments were analyzed by paired student’s t-test. Correlation analysis (Pearson r) was performed on GP BCM and OL Normalized values between beads with equivalent molecular antigens (50 total, 27 HLA-DR, 13 HLA-DQ, and 10 HLA-DP) as well as on titer analysis (neat, 1:16, 1:256, and 1:1024) of 5 sera samples. Results Antigen density was significantly greater on GP reagents versus OL reagents with mean strength of saturating murine monoclonal antibodies being 8,080 ± 2,257 MFI versus 2,150 ± 516 MFI for HLA-DR, 9,955 ± 2,866 MFI versus 5,175 ± 1,256 MFI for HLA- DQ, and 11,084 ± 1,615 MFI versus 3,291 ± 553 MMF for HLA-DP (p Conclusions Both the significantly higher density of HLA antigens on the GP beads and a dilution effect introduced by the difference in serum to bead-volume ratio may contribute to the lower correlation observed in this study. This is supported in part by the higher prozone effect observed with OL HLA-DQ specificities but not in GP, and the improved correlation observed with titers. Further investigation is warranted to determine correlation between GP and OL reagents.

Aim To evaluate and validate utilization of the more sensitive engraftment monitoring method, real-time quantitative polymerase chain reaction (qPCR), in detecting chimerism levels during early stages of relapse or rejection. Methods Twenty-four samples from 22 stem cell recipients (14 male; 8 female) were used for this study. The average age of recipients was 54.9 ± 12.6 years. Samples used for analysis were obtained from peripheral blood (n=14) or bone marrow (n=10) and were 274.2 ± 302.7 days post-transplant. Sixteen received allogeneic hematopoietic stem cell transplants and 6 received double cord transplants. Samples were tested for inter- and intra-assay variation. Short tandem repeat (STR) analysis was performed using the AmpFLSTR® Identifiler® PCR Amplification Kit (Life Technologies). qPCR was performed using the AlleleSEQR® Chimerism Assay (Celera Corporation). Results Initial studies with contrived mixtures confirmed the sensitivity of the qPCR to be lower than 0.5%. Based on these studies, we chose to run 2 informative qPCR markers, unique for the minor population, in our clinical assays. These markers should demonstrate the lowest delta Ct values and should not be located on the same chromosome or on chromosomes with known disease-related abnormalities. qPCR results were comparable with STR results for 22 of the 24 samples. Concordance was poor when the pre-determined “minor population” tested at greater than 50%. Reproducibility of the screen and quantitative qPCR assays using informative markers chosen by each method was also demonstrated. Conclusions Validation studies were successful. The sensitivity of the qPCR assay is excellent in the lower ranges of chimerism (below 30-40%), but the accuracy can be reduced when the “minor population” levels increase. The increased sensitivity of the qPCR assay can be useful in evaluating double cord blood recipients, particularly in the early times post-transplant.

Aim Donor specific HLA antibodies (DSA) in liver transplant recipients have been associated with allograft dysfunction. This analysis aims to describe the clinical outcomes in simultaneous liver-kidney (SLK) recipients with and without HLA antibodies. Methods A retrospective review of 50 SLK recipients transplanted from 03/2010 to 03/2013 identified 31 patients with pre- and post-transplant HLA testing and donor HLA typing. HLA antibodies were monitored by Flow PRA and Luminex single antigen assays. Results The 31 SLK recipients evaluated were 58 ± 10 years old at transplant, had a MELD score of 38 ± 5, were primarily male (52%), Caucasian (68%), on dialysis (81%), and had liver disease due to hepatitis C and/or alcoholic cirrhosis (65%). Mean follow-up was 11 ± 8 mos. Sensitization and clinical outcomes are described in the figure. Flow crossmatch (FXM) was performed on pre-transplant sera in 14 patients. Two sensitized patients without a DSA evaluation had a negative FXM. In DSA+ patients, 11 of 12 FXM were positive. DSA became negative post-transplant in 54% of pre-transplant DSA+ patients. De novo DSA developed in 17% of unsensitized patients. Acute cellular (ACR) or antibody mediated rejection of the kidney were most common in sensitized patients, with the majority seen in DSA+ patients. Liver ACR was seen in 1 unsensitized and 1 DSA+ patient. Three deaths occurred in DSA+ patients.[Fig. 1] Conclusions DSA levels may decrease post-transplant in some patients; however, persistent DSA post-transplant increases likelihood of rejection. Further prospective analysis of DSA in SLK recipients will be important to determine the role of DSA in allograft survival. Download full-size image