Cutting through the weeds: Evaluation of a novel adsorption with crossmatch cells and elution protocol to sharpen HLA antibody identification by the single antigen bead assay.

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless, detection of spurious reactivities remains a recognized assay limitation. In addition, the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique, which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed, with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly, negligible reactivity was detected in the last wash control, confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly, antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis, AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity.

Reducing number of laboratories performing complement dependent cytotoxicity crossmatching: Reasons and conclusions.

Complement dependent cytotoxicity crossmatch (CDC-XM) has been the original standard crossmatch test, whereas, flow cytometry crossmatch (FCXM) is an enhanced and highly sensitive crossmatch assay performed to detect donor specific anti-HLA antibodies (DSA). We analyzed American Society for Histocompatibility and Immunogenetics (ASHI) proficiency testing data (2011-2020) and examined the number of laboratories performing CDC-XM vs. FCXM, the overall efficiency of laboratories in reporting ≥80% consensus CDC-XM vs. FCXM result, and reasons for non-consensus results in the two assays. Of 600 crossmatches in each crossmatch category, the percentage of laboratories reporting T cell CDC-XMs reduced from 40% in 2011 to 13% in 2020, T cell anti-human globulin (AHG) CDC-XM reduced from 56% in 2011 to 21% in 2020, and B cell CDC-XM reduced from 51% in 2011 to 20% in 2020. The percentage of laboratories performing T cell and B cell FCXM remained at approximately 80% throughout. CDC-XM performed on par with FCXM in providing a consensus negative result using negative DSA serum, but under-performed in comparison to FCXM in providing a consensus positive result using positive DSA serum. In addition, only minority of CDC-XMs was reported positive in presence of complement fixing DSA. This study shows that non-consensus CDC-XM was always in presence of HLA IgG DSA and that laboratories may be struggling to interpret the low sensitive CDC-XM results, where highly sensitive solid phase multi-antigen or single antigen assay shows the presence of HLA IgG DSA in serum.

Donor-Specific Human Leukocyte Antigen Antibody Formation After Allograft Glenoid Reconstruction Occurs But Does Not Impact Clinicoradiographic Outcomes.

BACKGROUND: Recurrent shoulder instability is a prevalent condition, with glenoid bone loss as a common cause. Arthroscopic repair using distal tibial allografts provides long-lasting treatment by restoring glenoid surface area and presumably avoids risks of sensitization against donor human leukocyte antigen (HLA). Two case studies have challenged this assumption, suggesting that small bone allografts are able to induce host adaptive immune responses to donor HLA. The incidence of small bone allograft HLA sensitization and its effects on resorption and patient outcomes are unclear. PURPOSE/HYPOTHESIS: The purpose was to assess the rate of sensitization against donor HLA after distal tibial allograft procedures for shoulder instability due to glenoid bone loss and to find whether HLA sensitization negatively affects patient-reported and radiographic outcomes. We hypothesized that sensitized patients would have worse radiographic and self-reported outcomes compared with nonsensitized patients. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: A total of 71 patients with a mean age of 28.85 years (range, 13.58-61.31 years) were enrolled, with 58 patients submitting sufficient pre- and postoperative blood samples for HLA antibody testing. In patients who developed HLA antibodies postoperatively, donor HLA typing was used to confirm donor-specific sensitization. Pre- and postoperative computerized tomography scans (0.9 ± 0.8 years follow-up) were used to grade resorption based on the modified Zhu resorption grade classification (ie, grade 0 = no resorption; grade 1 = less than 25% resorption; grade 2 = between 25% and 50% resorption; and grade 3 = larger than 50% resorption). The Western Ontario Shoulder Instability Index outcome scores were obtained preoperatively and at regular postoperative appointments. Resorption and outcome data were compared between sensitized and nonsensitized patients using the Fisher exact test, independent 2-tailed Student t tests, and the Wilcoxon rank-sum test to determine the effect of HLA sensitization on radiographic and patient-reported outcomes. RESULTS: A total of 7 (12.1%) patients with sufficient HLA samples were sensitized against donor HLA postoperatively. Sensitized patients did not have significantly higher rates of resorption (21.9% vs 14.3%, 21.9% vs 28.6%, 43.8% vs 28.6%, and 12.5% vs 28.6% for respective resorption grades 0-3; P = .67; α = .05). Self-reported outcomes were not statistically significant between sensitized and nonsensitized patients (24.9 ± 27.61 vs 40.16 ± 18.99; P = .37; α = .05) and did not differ significantly based on resorption grade (47.4 ± 0.0 vs 55.2 ± 18.8, 30.4 ± 15.8 vs 39.9 ± 20.9, 41.2 ± 0.0 vs 39.1 ± 13.1, and -24.9 ± 0 vs 24.4 ± 19.6 for resorption grades 0-3; P > .05; α = .05). CONCLUSION: Sensitization against donor HLA after small bone graft allografting was not previously considered but has been brought to light as a possibility. Aside from potential complications for future organ transplants, HLA sensitization does not introduce a risk for adverse outcomes or higher grades of resorption compared with nonsensitized patients after small bone allografting for shoulder instability.

The Acute Phase Protein Hepcidin Is Cytotoxic to Human and Mouse Myeloma Cells.

BACKGROUND/AIM: Hepcidin is a cationic acute phase reactant synthesized by the liver. It has bactericidal properties and is a major regulator of iron homeostasis. Cationic antimicrobial peptides represent an innate antimicrobial defense system. We hypothesized that, like other cationic antimicrobial peptides, hepcidin is cytotoxic to cancer cells. MATERIALS AND METHODS: The cytotoxicity of human hepcidin against myeloma cells was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and DNA fragmentation assays. Plasma membrane damage was quantified by propidium iodide (PI) staining. Cell membrane changes were visualized by scanning electron microscopy. RESULTS: Hepcidin impaired myeloma cell survival and induced DNA fragmentation. PI staining and scanning electron microscopy revealed hepcidin-induced disruption of the plasma membrane. CONCLUSION: Human hepcidin is an anti-cancer peptide that induces myeloma cell lysis, and therefore may play a role in innate anticancer immunity. To our knowledge, this is the first biological function ascribed to human hepcidin that is not related to its antimicrobial and iron-regulatory properties.

Low Hydrophobic Mismatch Scores Calculated for HLA-A/B/DR/DQ Loci Improve Kidney Allograft Survival.

We evaluated the impact of human leukocyte antigen (HLA) disparity (immunogenicity; IM) on long-term kidney allograft survival. The IM was quantified based on physicochemical properties of the polymorphic linear donor/recipient HLA amino acids (the Cambridge algorithm) as a hydrophobic, electrostatic, amino acid mismatch scores (HMS\AMS\EMS) or eplet mismatch (EpMM) load. High-resolution HLA-A/B/DRB1/DQB1 types were imputed to calculate HMS for primary/re-transplant recipients of deceased donor transplants. The multiple Cox regression showed the association of HMS with graft survival and other confounders. The HMS integer 0-10 scale showed the most survival benefit between HMS 0 and 3. The Kaplan-Meier analysis showed that: the HMS=0 group had 18.1-year median graft survival, a 5-year benefit over HMS>0 group; HMS ≤ 3.0 had 16.7-year graft survival, a 3.8-year better than HMS>3.0 group; and, HMS ≤ 7.8 had 14.3-year grafts survival, a 1.8-year improvement over HMS>7.8 group. Stratification based on EMS, AMS or EpMM produced similar results. Additionally, the importance of HLA-DR with/without -DQ IM for graft survival was shown. In our simulation of 1,000 random donor/recipient pairs, 75% with HMS>3.0 were re-matched into HMS ≤ 3.0 and the remaining 25% into HMS≥7.8: after re-matching, the 13.5 years graft survival would increase to 16.3 years. This approach matches donors to recipients with low/medium IM donors thus preventing transplants with high IM donors.

Donor-Specific Human Leukocyte Antigen Antibody Formation After Distal Tibia Allograft and Subsequent Graft Resorption.

The association between donor-specific human leukocyte antigen (HLA) antibody formation and small bone allograft resorption has not been studied. We present the case of a patient treated for glenoid bone loss using a distal tibial allograft with Bankart repair who formed donor-specific HLA antibodies against the allograft and had subsequent graft resorption. X-ray and computed tomography (CT) scans were performed before and after surgery at standard checkpoints. Patient blood and serum samples were collected before and after surgery for HLA typing and HLA antibody testing. Human leukocyte antigen antibodies against the donor-specific HLA-A2 antigens were identified 6 weeks after surgery and were still detected at 5 months after surgery. At 6 months after surgery, a CT arthrogram revealed significant graft resorption. This case shows a temporal correlation between HLA antibody formation and clinical findings, potentially suggesting an association between HLA antibody formation and graft resorption. Further study is required to confirm this.

Getting on target: Development of the novel, prozone-resistant, dual antibody rapid test (DART) for the LABScreen single antigen bead (SAB) assay.

A major limitation of the single antigen bead (SAB) assay is the so called prozone effect, whereby the detection of high titer complement fixing HLA antibodies is compromised due to complement split product (from C3 and C4 components) deposition and interference with the reporter anti-IgG-PE antibody binding. Strategies to minimize prozone include serum titration or treatment with heat, dithiotreitol (DTT), or ethylenediaminetetraacetic acid (EDTA). While effective, these treatments may compromise HLA antibody binding and detection. Here we describe the Dual Antibody Rapid Test (DART), a modified version of the rapid optimized SAB (ROB) protocol, in which we use an IgG-PE/C3d-PE antibody cocktail to simultaneously detect bead bound IgG and C3d, which allows for detection of HLA antibodies independent of the prozone effect. Twenty prozone positive sera (10 class I and 10 class II), identified by titration, were tested by the ROB protocol, with or without EDTA pre-treatment, using three reporter antibody cocktails: (1) IgG-PE, (2) C3d-PE, or (3) IgG-PE/C3d-PE (DART). Mean fluorescence intensity (MFI) values were then compared. IgG negative (n = 735) vs IgG positive (n = 1185) reactions were identified using a 1000 MFI IgG EDTA cutoff. IgG positive reactions were classified based on ΔMFI (IgG EDTA - IgG) as follows: (1) prozone negative (ΔMFI < 3000; n = 737), (2) slight prozone (ΔMFI 3001-5000; n = 49), (3) moderate prozone (ΔMFI 5001-10,000; n = 93), and (4) marked prozone (ΔMFI > 10,001; n = 306). No C3d deposition was present on IgG negative beads, and the majority of prozone positive specificities (438/448; 98%) fixed complement and were detected with the C3d-PE reporter. Interestingly, C3d-PE MFI was directly proportional to the degree of prozone (mean C3d-PE MFI = 4419.5 ±â€¯1606.3 for slight, 5991.0 ±â€¯2302.7 for moderate, and 12,417.4 ±â€¯2969.9 for marked prozone specificities). Interestingly, EDTA treatment was found to have a negative impact on MFI of up to 15% of prozone negative specificities. Importantly, the DART protocol detected all prozone positive specificities while MFI for prozone negative specificities correlated well with those seen with the IgG-PE reporter alone (R2 = 0.97). In conclusion, the DART protocol accurately detects HLA antibodies independent of the prozone effect. Implementation of DART is an easy way to overcome the prozone effect without compromising HLA antibody detection.

The ABCs (DRDQDPs) of the prozone effect in single antigen bead HLA antibody testing: Lessons from our highly sensitized patients.

Accurate identification of HLA antibodies using the single antigen bead (SAB) assay is critical for assessment of pre/post-transplant immunological risk and successful virtual crossmatching. Unfortunately, high titer HLA antibodies can be missed or underestimated in the SAB assay as a result of interference with the detection of IgG. This so called prozone effect has been attributed to both complement- and IgM-dependent mechanisms and can be minimized with serum dilution or treatment with heat, EDTA, or DTT. In this study we describe the frequency, nature, and degree of prozone in a cohort of highly sensitized patients (cPRA ≥ 95%), in whom accurate detection of HLA antibodies and virtual crossmatching is of paramount importance. Sera were tested by the SAB assay ±â€¯EDTA treatment, ±1:10 dilution to identify the prozone effect. The relative contribution of complement vs IgM to prozone was assessed using anti-C3d and anti-IgM reporter antibodies, respectively. We found that prozone was very frequent in highly sensitized patients (80%), especially those with a history of previous transplantation (87%). Class I HLA specificities were more commonly affected than class II and the susceptibility to prozone was locus dependent with HLA-A(31%), -B(29%) and -DQ(26%) being affected more frequently than HLA-DP(17%), -C(16%) and -DR(5%) antigens. Interestingly, the presence of prozone could be predicted by C3d positivity (MFI ≥ 4000; sensitivity = 95.2%, specificity = 97.2%) and the degree of prozone correlated directly with the extent of C3d deposition. The role of IgM was less clear. However, serum dilution studies suggested that IgM may contribute to interference in a small subset of prozone positive specificities. Our study underscores the importance of serum treatment to inhibit complement activation and minimize prozone in the SAB assay, especially in highly sensitized patients.

(F)Utility of the physical crossmatch for living donor evaluations in the age of the virtual crossmatch.

Flow cytometric crossmatches (FCXM) are routinely performed to support living-donor renal transplantation. While long a laboratory mainstay, a physical crossmatch is costly, time consuming, and frequently poses interpretative conundrums with both false-positive and false- negative results. Given the increased utilization of the virtual crossmatch (vXM) in the deceased donor setting, our aim was to assess its utility in living donor evaluations. We reviewed 100 living donor FCXMs and retrospectively performed a vXM for each pair. Seventy-five (75) cases were concordant, (i.e., FCXM-/vXM- or FCXM+/vXM+) while 25 cases were discordant; Five were vXM+/FCXM- and 20 were FCXM+/vXM-. Since donor-specific antibodies (DSA) were not detected in the 20 FCXM+/vXM- cases, these were interpreted as false-positive, i.e., due to non-HLA antibodies. Importantly, none of these patients, when transplanted across a positive FCXM, experienced early antibody mediated rejection or subsequently developed HLA DSA. These data reveal that, for the vast majority of living donor evaluations, a vXM is an acceptable vetting procedure.

Ticagrelor inhibits platelet-tumor cell interactions and metastasis in human and murine breast cancer.

Activated platelets promote the proliferation and metastatic potential of cancer cells. Platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets. We examined the potential of the reversible P2Y12 inhibitor ticagrelor, an agent used clinically to prevent cardiovascular and cerebrovascular events, to reduce tumor growth and metastasis. In vitro, MCF-7, MDA-MB-468, and MDA-MB-231 human mammary carcinoma cells exhibited decreased interaction with platelets treated with ticagrelor compared to untreated platelets. Prevention of tumor cell-platelet interactions through pretreatment of platelets with ticagrelor did not improve natural killer cell-mediated tumor cell killing of K562 myelogenous leukemia target cells. Additionally, ticagrelor had no effect on proliferation of 4T1 mouse mammary carcinoma cells co-cultured with platelets, or on primary 4T1 tumor growth. In an orthotopic 4T1 breast cancer model, ticagrelor (10 mg/kg), but not clopidogrel (10 mg/kg) or saline, resulted in reduced metastasis and improved survival. Ticagrelor treatment was associated with a marked reduction in tumor cell-platelet aggregates in the lungs at 10, 30 and 60 min post-intravenous inoculation. These findings suggest a role for P2Y12-mediated platelet activation in promoting metastasis, and provide support for the use of ticagrelor in the prevention of breast cancer spread.

Of Cells and Microparticles: Assets and Liabilities of HLA Antibody Detection.

The evolution of antibody detection from cell- to bead-based technology has positively impacted the ability to allocate organs in a safe and timely manner. The devil, of course, is in the details that delineate how these assays are performed and applied and to recognize that while there have been some truly amazing technological advances (assets), they are still imperfect and subject to error (liabilities). This review identifies the strengths of HLA antibody assays, highlights their weaknesses and offers approaches for standardization.

Rapid optimized flow cytometric crossmatch (FCXM) assays: The Halifax and Halifaster protocols.

The flow cytometric crossmatch (FCXM) assay, which detects the presence of donor specific HLA antibodies in patient sera, is a cornerstone of HLA compatibility testing. Since relatively long FCXM assay turnaround times may contribute to transplant delays and increased graft ischemia time, we developed and validated two modified crossmatch procedures, namely the Halifax and Halifaster FCXM protocols. These protocols reduce FCXM assay time >60% and simplify their set-up without compromising quality or sensitivity. Optimization of the FCXM (the Halifax protocol) includes a 96-well tray platform, reduced wash times, increased serum to cell suspension volume ratio, shortened incubations and higher incubation temperature. The Halifaster protocol is a further modification, employing methods that improve lymphocyte purity compared to density gradient centrifugation (96 ±â€¯2.63% vs 69 ±â€¯19.06%), reduce cell isolation time (by ∼40%) and conserve FCXM assay reagents. Importantly, linear regression analysis of the median channel fluorescence shift (MCFS) values revealed excellent concordance (R2 of 0.98-0.99) among all three FCXM protocols (standard vs Halifax vs Halifaster). Finally, a retrospective review of 2013 crossmatches performed using the Halifax protocol demonstrated excellent correlation with the virtual crossmatch (95.7% and 96.8% specificity and sensitivity, respectively) regarding the identification of donor specific antibodies (HLA-A/B/DR) assigned based on the single antigen bead (SAB) assay testing with a 2000 mean fluorescence intensity (MFI) cutoff. Implementation of the Halifax or Halifaster protocols will expedite pre-transplantation work-up and improve patient care.

Unrelated donor choices for allogeneic hematopoietic cell transplantation in Canada: an evaluation of factors influencing donor selection.

BACKGROUND: Utilization of unrelated donors and cord blood units (CBUs) for allogeneic hematopoietic cell transplantation continues to increase. Understanding the practices of donor selection by transplant centers is critical for unrelated donor registries and cord blood banks to optimize registry composition and inventory to meet patient need. STUDY DESIGN AND METHODS: Unrelated donor and CBU selection practices of Canadian transplant centers served by Canadian Blood Services' OneMatch Stem Cell & Marrow Network (OM) were reviewed, including HLA match level, locus of disparity, age, sex, and product choice (donor vs. CBU). RESULTS: HLA-matched donors within OM and/or international (INT) registries were preferentially investigated, underscoring the primary importance of HLA matching. In the case of HLA-mismatched donors, HLA-A disparities were most common while DRB1 mismatches were least common. Advanced age, sex, and lack of donor availability were the most frequent reasons that high-probability OM donors were overlooked in favor of INT donors. High-probability 10 of 10 HLA-matched female donors from OM were often avoided in favor of INT male donors. Use of female donors, however, increased in cases restricted to more HLA-disparate donor options. Caucasian patients were more likely to find 10 of 10 matched donors, whereas use of mismatched donors and CBUs were more prevalent among non-Caucasian patients. CONCLUSIONS: Recruitment and retention of young, male donors from diverse ethnic backgrounds may increase the usage of histocompatible OM donors for patients in need.

Combination of poly I:C and Pam3CSK4 enhances activation of B cells in vitro and boosts antibody responses to protein vaccines in vivo.

Vaccines that can rapidly induce strong and robust antibody-mediated immunity could improve protection from certain infectious diseases for which current vaccine formulations are inefficient. For indications such as anthrax and influenza, antibody production in vivo is a correlate of efficacy. Toll-like receptor (TLR) agonists are frequently studied for their role as vaccine adjuvants, largely because of their ability to enhance initiation of immune responses to antigens by activating dendritic cells. However, TLRs are also expressed on B cells and may contribute to effective B cell activation and promote differentiation into antigen-specific antibody producing plasma cells in vivo. We sought to discover an adjuvant system that could be used to augment antibody responses to influenza and anthrax vaccines. We first characterized an adjuvant system in vitro which consisted of two TLR ligands, poly I:C (TLR3) and Pam3CSK4 (TLR2), by evaluating its effects on B cell activation. Each agonist enhanced B cell activation through increased expression of surface receptors, cytokine secretion and proliferation. However, when B cells were stimulated with poly I:C and Pam3CSK4 in combination, further enhancement to cell activation was observed. Using B cells isolated from knockout mice we confirmed that poly I:C and Pam3CSK4 were signaling through TLR3 and TLR2, respectively. B cells activated with Poly I:C and Pam3CSK4 displayed enhanced capacity to stimulate allogeneic CD4+ T cell activation and differentiate into antibody-producing plasma cells in vitro. Mice vaccinated with influenza or anthrax antigens formulated with poly I:C and Pam3CSK4 in DepoVax™ vaccine platform developed a rapid and strong antigen-specific serum antibody titer that persisted for at least 12 weeks after a single immunization. These results demonstrate that combinations of TLR adjuvants promote more effective B cell activation in vitro and can be used to augment antibody responses to vaccines in vivo.

Becoming a chef in the human leukocyte antigen kitchen: interpretation and modification of human leukocyte antigen-antibody assays.

PURPOSE OF REVIEW: Fluorescence-based human leukocyte antigen (HLA) antibody detection methods, including flow cytometric crossmatch and single antigen bead assays revolutionized HLA antibody identification and assessment of immunological risk in transplant candidates and patients. Nevertheless, these assays are not flawless and their interpretation can be complex. This review highlights the limitations of the single antigen bead and flow cytometric crossmatch assays and discusses protocol modifications and interpretive approaches to address these issues. RECENT FINDINGS: Several limitations of HLA antibody detection methods have been identified in recent years. Protocol variability, denatured epitopes, and interfering factors can all significantly impact the identification of clinically relevant HLA antibodies. A number of solutions to address these challenges have been developed. These include pretreatment of sera, method standardization, and protocol modifications. In addition, HLA epitope-based analysis approaches to improve interpretation of antibody test results have been introduced. SUMMARY: In the 50 years, since Patel and Terasaki first developed the crossmatch assay there have been remarkable advances in HLA antibody testing methodology. However, with these advances, new problems emerged and solutions had to be developed. As the technology continues to evolve, our methods and ability to interpret results must keep pace to provide transplant patients with the best possible care.

It's about time: The development and validation of a rapid optimized single antigen bead (ROB) assay protocol for LABScreen.

The LABScreen single antigen bead assay (SAB) is a method widely used for the identification and monitoring of human leukocyte antigen (HLA) antibodies in patients pre-and post-transplant. While accurate testing of patient samples is key for optimal patient care, time can also be important, especially during deceased donor workups or post-transplant assessments. Here we describe the development and validation of the Rapid Optimized SAB (ROB) protocol, a modified version of the One Lambda LABScreen SAB (OLSAB) procedure, which reduces assay time from 85 to 25min (>70% reduction) without impacting assay quality or sensitivity. Optimization steps included shortened centrifugation cycles and reduced serum and secondary antibody incubation times in combination with increased secondary antibody concentration. Linear regression analysis of baseline median fluorescence intensity (MFI) values showed excellent correlation between the ROB and OLSAB protocols (r2>0.98) for both class I and class II antibodies in 58 sera tested in two HLA laboratories. Importantly, the ROB protocol demonstrated a trend towards improved inter-laboratory MFI concordance when compared to the OLSAB procedure (r2=0.9816 vs 0.9451), especially for HLA antibody specificities in the 500-2000 MFI range (r2=0.7824 vs 0.6313). Implementation of the ROB protocol will expedite HLA antibody testing and may improve reproducibility of the SAB assay.

Piperine, a Pungent Alkaloid from Black Pepper, Inhibits B Lymphocyte Activation and Effector Functions.

Piperine has several well-documented anti-inflammatory properties; however, little is known regarding its effect on humoral immunity. In this study, we describe the immunosuppressive effect of piperine on B lymphocytes, which are integral to the humoral immune response. Mouse B cells were cultured in the absence or presence of non-cytotoxic concentrations (25, 50, and 100 µM) of piperine during T-dependent or T-independent stimulation. Piperine inhibited B cell proliferation by causing G0/G1 phase cell cycle arrest in association with reduced expression of cyclin D2 and D3. The inhibitory effect of piperine was not mediated through transient receptor potential vanilloid-1 ion channel (TRPV1) because piperine also inhibited the proliferation of B cells from TRPV1-deficient mice. Expression of class II major histocompatibility complex molecules and costimulatory CD40 and CD86 on B lymphocytes was reduced in the presence of piperine, as was B cell-mediated antigen presentation to syngeneic T cells. In addition, piperine inhibited B cell synthesis of interleukin (IL)-6 and IL-10 cytokines, as well as IgM, IgG2b, and IgG3 immunoglobulins. The inhibitory effect of piperine on B lymphocyte activation and effector function warrants further investigation for possible application in the treatment of pathologies related to inappropriate humoral immune responses. Copyright © 2017 John Wiley & Sons, Ltd.

T Cell Immune Deficiency in zap70 Mutant Zebrafish.

ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP + thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency.

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia.

Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.