Multiple Sera Research Articles

The fibrotic response in pericyte culture upon exposure to multiple sclerosis sera

The fibrotic response in pericyte culture upon exposure to multiple sclerosis sera

Comprehensive profiling of neutralizing polyclonal sera targeting coxsackievirus B3

Despite their fundamental role in resolving viral infections, our understanding of how polyclonal neutralizing antibody responses target non-enveloped viruses remains limited. To define these responses, we obtained the full antigenic profile of multiple human and mouse polyclonal sera targeting the capsid of a prototypical picornavirus, coxsackievirus B3. Our results uncover significant variation in the breadth and strength of neutralization sites targeted by individual human polyclonal responses, which contrasted with homogenous responses observed in experimentally infected mice. We further use these comprehensive antigenic profiles to define key structural and evolutionary parameters that are predictive of escape, assess epitope dominance at the population level, and reveal a need for at least two mutations to achieve significant escape from multiple sera. Overall, our data provide a comprehensive analysis of how polyclonal sera target a non-enveloped viral capsid and help define both immune dominance and escape at the population level.

494. Enteric bacteria are immune-reactive in patients with Crohn's disease with extraintestinal manifestations

Abstract Background Bacterial constituents of the intestinal microbiota can contribute to local and systemic inflammatory diseases. Crohn's disease (CD), a type of inflammatory bowel disease, can result in extraintestinal manifestations (EIM) including peripheral (CD-SpA) and axial spondyloarthritis (CD-AxSpA). Microbial contributors to CD pathogenesis can be identified by flow cytometric sorting and 16S sequencing of bacteria recognized by mucosal IgA. We expand this technique by incubating fecal samples with autologous sera, capturing bacterial species recognized by circulating IgG in a process called IgG-seq. We hypothesize that these systemically-recognized enteric bacteria are linked to development of CD-EIM. Methods Fecal samples from individuals with CD were sorted into IgG-positive and negative populations. DNA was extracted from each population, 16S sequencing performed, and sequences processed with QIIME2. Intestinal coating index (ICI) was calculated at the genus level. Serum cell-free DNA sequencing (cfDNA) was performed for a subset of samples. Results IgG-seq was conducted on 86 CD, 41 CD-SpA, and 16 CD-AxSpA samples. PCoA analysis demonstrates significant differences in microbiome composition in the three groups (P = 0.013, PERMANOVA). Relative abundances of Escherichia-Shigella and Ruminococcus are positively correlated with joint disease activity. Analysis of IgG ICI for genera present in > 10% of patients demonstrates overrepresentation of Ruminococcus, Escherichia-Shigella, and Bacteroides in IgG-recognized fractions. IgG recognition of bacteria does not cluster specifically by CD or EIM severity. cfDNA sequencing demonstrates no Ruminococcus DNA in serum, whereas E. coli DNA was detected in multiple patient sera. Conclusion IgG-seq is a robust method for identifying immune-reactive enteric bacteria; our results highlight the link between immune response to enteric bacterial genera, such as Escherichia-Shigella and Ruminococcus, and joint disease activity in CD-SpA and CD-AxSpA. While Escherichia-Shigella may induce immunity by breaching the mucosal barrier, the absence of Ruminococcus in serum cfDNA suggests immune activation at barrier sites. Further studies are needed to characterize the strain-specific features that underlie our findings. Disclosures Fardina Malik, MD, Pfizer: Advisor/Consultant Iwijn de Vlaminck, PhD, GenDX: Advisor/Consultant|GenDX: Board Member|Kanvas Biosciences: Board Member|Kanvas Biosciences: Ownership Interest|Karius Inc.: Board Member|Karius Inc.: Ownership Interest|Viracor Eurofins: Advisor/Consultant Randy S. Longman, MD, PhD, Pfizer: Advisor/Consultant.

Neutralizing Antibodies Responses against SARS-CoV-2 in a Sardinian Cohort Group Up to 9 Months after BNT162b2 Vaccination.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, has caused over 460 million cases of infection and over 6 million deaths worldwide. The pandemic has called for science, technology, and innovation to provide solutions and, due to an incredible scientific and financial global effort, several prophylactic and therapeutic apparatuses such as monoclonal antibodies and vaccines were developed in less than one year to address this emergency. After SARS-CoV-2 infection, serum neutralizing antibodies are produced by B cells and studies on virus-neutralizing antibodies’ kinetics are pivotal. The process of protective immunity and the duration of this kind of protection against COVID-19 remain to be clarified. We tested 136 sera from 3 groups of individuals, some of them providing multiple sequential sera (1—healthy, no previous CoV2-infected, vaccinated; 2—healthy, previous CoV2 infected, vaccinated; 3—healed, previous CoV2-infected, not vaccinated) to assess the kinetics of antibodies (Abs) neutralizing activity. We found that SARS-CoV-2 infection elicits moderate neutralizing antibody activity in most individuals; neither age nor gender appear to have any influence on Abs responses. The BNT162b2 vaccine, when administered in two doses, induces high antibodies titre endowed with potent neutralizing activity against bare SARS-CoV-2 in in vitro neutralizing assay. The residual neutralization capability and the kinetic of waning immunity were also evaluated over 9 months after the second dose in a reference group of subjects. Neutralization titre showed a decline in all subjects and the median level of S-protein IgG, over 270 days after the second vaccination dose, was below 10 AU/mL in 53% of serum tested.

Menthol Flavor in E-Cigarette Vapor Modulates Social Behavior Correlated With Central and Peripheral Changes of Immunometabolic Signalings.

The use of electronic cigarette (e-cigarette) has been increasing dramatically worldwide. More than 8,000 flavors of e-cigarettes are currently marketed and menthol is one of the most popular flavor additives in the electronic nicotine delivery systems (ENDS). There is a controversy over the roles of e-cigarettes in social behavior, and little is known about the potential impacts of flavorings in the ENDS. In our study, we aimed to investigate the effects of menthol flavor in ENDS on the social behavior of long-term vapor-exposed mice with a daily intake limit, and the underlying immunometabolic changes in the central and peripheral systems. We found that the addition of menthol flavor in nicotine vapor enhanced the social activity compared with the nicotine alone. The dramatically reduced activation of cellular energy measured by adenosine 5′ monophosphate-activated protein kinase (AMPK) signaling in the hippocampus were observed after the chronic exposure of menthol-flavored ENDS. Multiple sera cytokines including C5, TIMP-1, and CXCL13 were decreased accordingly as per their peripheral immunometabolic responses to menthol flavor in the nicotine vapor. The serum level of C5 was positively correlated with the alteration activity of the AMPK-ERK signaling in the hippocampus. Our current findings provide evidence for the enhancement of menthol flavor in ENDS on social functioning, which is correlated with the central and peripheral immunometabolic disruptions; this raises the vigilance of the cautious addition of various flavorings in e-cigarettes and the urgency of further investigations on the complex interplay and health effects of flavoring additives with nicotine in e-cigarettes.

Specificity of Anti-Citrullinated Protein Antibodies to Citrullinated α-Enolase Peptides as a Function of Epitope Structure and Composition.

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1–2% of the world population. In addition to the first discovered serologic markers for RA, the rheumatoid factors (RFs), anti-citrullinated protein antibodies (ACPAs) are even more specific for the disease compared to RFs and are found in 70–80% of RA patient sera. RA etiopathogenesis still needs to be elucidated, as different factors are proposed to be involved, such as Epstein–Barr virus infection. Hence, understanding the interaction between ACPAs and their citrullinated peptide targets is relevant for a better knowledge of RA pathophysiology and for diagnostic purposes. In this study, a cohort of RA sera, healthy control sera and multiple sclerosis sera were screened for reactivity to a variety of citrullinated peptides originating from α-enolase, pro-filaggrin, proteoglycan and Epstein–Barr nuclear antigen-2 by enzyme-linked immunosorbent assay. ACPA reactivity to citrullinated α-enolase peptides was found to depend on peptide length and peptide conformation, favouring cyclic (disulfide bond) conformations for long peptides and linear peptides for truncated ones. Additional investigations about the optimal peptide conformation for ACPA detection, employing pro-filaggrin and EBNA-2 peptides, confirmed these findings, indicating a positive effect of cyclization of longer peptides of approximately 20 amino acids. Moreover, screening of the citrullinated peptides confirmed that ACPAs can be divided into two groups based on their reactivity. Approximately 90% of RA sera recognize several peptide targets, being defined as cross-reactive or overlapping reactivities, and whose reactivity to the citrullinated peptide is considered primarily to be backbone-dependent. In contrast, approximately 10% recognize a single target and are defined as nonoverlapping, primarily depending on the specific amino acid side-chains in the epitope for a stable interaction. Collectively, this study contributed to characterize epitope composition and structure for optimal ACPA reactivity and to obtain further knowledge about the cross-reactive nature of ACPAs.

Recurrent Emergence and Transmission of a SARS-CoV-2 Spike Deletion H69/V70

SARS-CoV-2 Spike amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six-nucleotide out of frame deletion in the S gene, which results in loss of two amino acids: H69 and V70. We show that 𝚫H69/V70 and other common NTD deletions occur at loop structures in RNA where polymerase activity is often compromised. In addition, the same two amino acid deletion is also observed in a set of closely related pangolin sarbecoviruses, but not in the bat sarbecovirus RaTG13. We report that in human infections 𝚫H69/V70 often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F, and in the latter two cases has followed the RBD mutation suggesting an epistatic interaction. One of the +.-|.-|.-| N501Y lineages, B.1.1.7, has undergone rapid expansion and includes eight other S gene mutations: RBD (N501Y and A570D), S1 (𝚫H69/V70 and 𝚫144/145) and S2 (P681H, T716I, S982A and D1118H). In vitro, we show that 𝚫H69/V70 does not impact serum neutralisation across multiple convalescent sera. However, the 𝚫H69/V70 does increase infectivity of Spike and is able to compensate for infectivity defects induced by RBD mutations N501Y, N439K and Y453F. In addition replacement of H69 and V70 in B.1.1.7 Spike substantially reduces its infectivity. Mechanistically, we demonstrate that 𝚫H69/V70 is associated with two fold increased Spike incorporation into virions, though it does not increase cell-cell fusion. Based on our data 𝚫H69/V70 increases Spike infectivity, likely acting as a permissive mutation that allows acquisition of otherwise deleterious immune escape mutations. Enhanced surveillance for the 𝚫H69/V70 deletion with and without RBD mutations should be considered as a global priority not only as a marker for the B.1.1.7 variant, but potentially also for other emerging variants of concern.Funding Statement: RKG is supported by a Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA). COG-UK is supported by funding from the Medical Research Council (MRC) & UK&ch & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute. SAK is supported by the Bill and Melinda Gates Foundation via PANGEA grant: OPP1175094. DLR is funded by the MRC (MC UU 1201412). WH is funded by the MRC (MR/R024758/1).We thank Dr James Voss for the kind gift of HeLa cells stably expressing ACE2. SL is funded by Medical Research Council MC_UU_12014/12. This study was also partly funded by Rosetrees Trust. Work in the AML lab is supported by NIHR Biomedical Research Centre Grant RCAG 817.Declaration of Interests: RKG has received consulting fees from UMOVIS lab, Gilead Sciences and ViiV Healthcare, and a research grant from InvisiSmart Technologies. On behalf of COG-UK, there are no other interests to declare.Ethics Approval Statement: Ethical approval for use of serum samples. Controls with COVID-19 were enrolled to the NIHR BioResource Centre Cambridge under ethics review board (17/EE/0025).

Clinical evaluation of SARS-CoV-2 point-of-care antibody tests

The aim of this study was to assess the analytic and clinical performance of four rapid lateral flow point-of-care tests (POCTs) for identifying SARS-CoV-2-specific antibodies. A retrospective study was conducted between 22 January and 30 March 2020 on 132 serum samples for SARS-CoV-2-specific antibody detection referred to a tertiary referral hospital laboratory in New South Wales. Multiple sera were tested from 20 confirmed or suspected COVID-19 patients with SARS-CoV-2-specific antibodies detected by immunofluorescence (IFA) or neutralisation, and 71 SARS-CoV-2 uninfected individuals. We measured the sensitivity and specificity for detection of SARS-CoV-2 IgM and IgG antibodies for each POCT in comparison to positive SARS-CoV-2-specific IFA and viral neutralisation, our current laboratory benchmark tests.All POCTs were found to have a low analytic sensitivity for SARS-CoV-2 antibodies, ranging from 27.3% to 58.2%, with a specificity between 88.3% and 100%, and a low clinical sensitivity from 45% to 65%, with a clinical specificity between 87.3% and 100%. All POCTs had an increased sensitivity when specimens were collected more than 14 days from onset of symptoms. The detection using point-of-care testing of SARS-CoV-2-specific antibodies after disease onset lagged behind IFA by a range of 0–9 days.POCTs promise the benefit of providing quick easy testing for SARS-CoV-2-specific antibodies. However, their poor sensitivity and delayed antibody detection make them unsuitable as a diagnostic or screening tool alone.

A Novel Pseudomonas aeruginosa Virulence Factor Predicts CLAD and Death after Lung Transplantation

Gram -ve bacteria have recently been found to induce O-antigen-specific IgG2 and IgA 'inhibitory' antibodies (iAb) in the host which are not only impotent but block complement-mediated killing. Our group has demonstrated the clinical benefit of plasmapheresis in patients infected with Pseudomonas aeruginosa (Psa) strains which have deployed this virulence factor. Here we assessed the prevalence and clinical impact of iAb in a large lung transplant cohort. Our centre maintains a biobank of sera from the majority of recipients. Recipients with at least one archival serum sample from the first post-transplant year were included. Presence of iAb in sera was determined by measuring titre of IgG2 and IgA against the three most prevalent (∼80% coverage) local Psa serotypes. Presence of iAb was confirmed by serum-bactericidal assays against serotype matched Psa. 123 recipients (54(44%) female; aged 54yrs (range 19-70); 38(31%) cystic fibrosis (CF), 89(72%) Psa colonised; median follow-up 33 (19.3-70.6) months) with 425 archived sera were studied. 25% of the whole cohort and 47% of CF subjects had iAb. iAb were persistent in subjects with multiple sera. The demographic factors independently associated with iAb were CF (OR 4.52, 95% CI 1.83-11.23, p 0.001) and younger age (OR 0.95, 95% CI 0.92-0.98, p 0.002); however, Psa-colonisation, gender, transplant type, CMV status, induction agent use and follow-up time were unassociated. In those with iAb, CLAD-free and overall survival was inferior in all (overall, CF, Psa-colonised) cohorts (Figure 1). iAb and CMV mismatch status but not Psa-colonisation status, were independently associated with inferior CLAD free survival and overall survival. iAb are prevalent after lung transplantation, in part explain the known association between Psa-colonisation and CLAD, and independently predict poor outcomes. Development of strategies to counter iAb could significantly improve outcomes in patients with Gram -ve infection.

Epstein Barr virus nuclear antigen 1 (EBNA-1) peptides recognized by adult multiple sclerosis patient sera induce neurologic symptoms in a murine model

Multiple sclerosis (MS) is an autoimmune demyelinating disease with progressive neurodegeneration and complex etiology likely involving genetic and environmental factors. MS has been associated with Epstein Barr virus (EBV) infection, with patients often showing enhanced responses to EBV antigens. To determine whether abnormal EBV nuclear antigen-1 (EBNA-1) humoral immunity can serve as an initiator of autoimmune responses in MS, we investigated the fine specificities of the humoral immune response against EBNA-1 in MS patients using solid phase epitope mapping. Antibodies from MS patients recognized an EBNA-1 epitope spanning amino acids 411–426, previously unknown to be recognized specifically by untreated MS patients. Antibodies against this epitope cross-reacted to myelin basic protein (MBP). Furthermore, animals immunized with this EBNA-1 polypeptide mounted a response against MBP and developed signs of experimental autoimmune encephalitis (EAE). These data support a link between MS and EBV through antibodies that cross-react between EBV proteins and the MBP autoantigen.

The role of serum and urinary biomarkers in the diagnosis of early diabetic nephropathy in patients with type 2 diabetes.

BackgroundPrevious studies have shown that a variety of biomarkers are closely related to the occurrence and development of early-stage diabetic nephropathy (DN) in patients. The aim of this study was to evaluate the role of multiple sera and urinary biomarkers in the diagnosis of early-stage DN in patients with type 2 diabetes.MethodsWe enrolled 287 patients with type 2 diabetes, who were classified into normoalbuminuria (n = 144), microalbuminuria (n = 94), or macroalbuminuria (n = 49) groups based on their urine albumin to creatinine ratios (UACR), along with 42 healthy controls. We assessed 13 biomarkers, including transferrin (Tf), immunoglobulin G (IgG), podocalyxin, neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl-beta-glucosaminidase, α-1-microglobulin, 8-hydroxy-deoxyguanosine, tumor necrosis factor-alpha (TNF-α), and interleukin-18 in urine samples, along with cystatin C, total bilirubin, and uric acid in sera samples, to evaluate their diagnostic roles. From the measurements, the blood neutrophil to lymphocyte ratio was also calculated.ResultsUrinary Tf, IgG, NGAL, and TNF-α were significantly related to the UACR. We calculated the area under the receiver operating characteristic curves (area under the curve) and found that urinary IgG (0.894), NGAL (0.875), Tf (0.861), TNF-α (0.763), and the combination of urinary Tf + IgG + TNF-α + NGAL (0.922) showed good diagnostic value for early-stage DN.ConclusionsUrinary Tf, IgG, NGAL, TNF-α, and the combination of all four biomarkers demonstrated excellent diagnostic value for early-stage DN in patients with type 2 diabetes.

Ginsenoside Rg1 Prevents Chemotherapy-Induced Cognitive Impairment: Associations with Microglia-Mediated Cytokines, Neuroinflammation, and Neuroplasticity.

Chemotherapy-induced cognitive impairment, also known as "chemobrain," is a common side effect. The purpose of this study was to examine whether ginsenoside Rg1, a ginseng-derived compound, could prevent chemobrain and its underlying mechanisms. A mouse model of chemobrain was developed with three injections of docetaxel, adriamycin, and cyclophosphamide (DAC) in combination at a 2-day interval. Rg1 (5 and 10mg/kg daily) was given 1week prior to DAC regimen for 3weeks. An amount of 10mg/kg Rg1 significantly improved chemobrain-like behavior in water maze test. In vivo neuroimaging revealed that Rg1 co-treatment reversed DAC-induced decreases in prefrontal and hippocampal neuronal activity and ameliorated cortical neuronal dendritic spine elimination. It normalized DAC-caused abnormalities in the expression of multiple neuroplasticity biomarkers in the two brain regions. Rg1 suppressed DAC-induced elevation of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but increased levels of the anti-inflammatory cytokines IL-4 and IL-10 in multiple sera and brain tissues. Rg1 also modulated cytokine mediators and inhibited DAC-induced microglial polarization from M2 to M1 phenotypes. In in vitro experiments, while impaired viability of PC12 neuroblastic cells and hyperactivation of BV-2 microglial cells, a model of neuroinflammation, were observed in the presence of DAC, Rg1 co-treatment strikingly reduced DAC's neurotoxic effects and neuroinflammatory response. These results indicate that Rg1 exerts its anti-chemobrain effect in an association with the inhibition of neuroinflammation by modulating microglia-mediated cytokines and the related upstream mediators, protecting neuronal activity and promoting neuroplasticity in particular brain regions associated with cognition processing.

Optimization of a magnetic bead-based assay (MAGPIX\xae-Luminex) for immune surveillance of exposure to malaria using multiple Plasmodium antigens and sera from different endemic settings

BackgroundSerological markers are potentially useful tools for monitoring the progress of malaria control programs, but a better understanding of antibody response dynamics is necessary. The use of a magnetic bead-based immunoassay (MBA) is advantageous compared to ELISA, due to its multiplexing capacity, but limited information is available on the standardization and validation of this assay.MethodsSeveral parameters for multiplex testing of antibodies to Plasmodium antigens were analysed using a set of 4 antigens and 98 sera from Senegalese rural asymptomatic and urban symptomatic individuals. The 4 antigens included Plasmodium falciparum CSP and PfAMA1 peptides, recombinant P. falciparum MSP4p20 and a Plasmodium malariae CSP (PmCSP) peptide. Comparisons with ELISA were done using MSP4p20 and whole schizont extract (SE) antigens.ResultsThe use of fewer beads (1000 beads per well instead of 2000) and 5 µg of antigen per 106 bead were validated as lower amounts. The use of a carrier protein (BSA) was shown to be critical when using peptides and the effect of a 24 h delayed measures was evaluated (5–25% signal decrease). Analysis of Ab responses showed almost equally high levels and prevalence in all transmission settings. Clear distinctions between rural and urban malaria were noted using PmCSP and SE antigens.ConclusionsThis study underlines the importance of further optimization of the MBA technique and highlights the interest of using multistage/multispecies antigens for surveillance of malaria in endemic settings.

P054 Unexpected quiescence of expected memory response against prior kidney allografts – Novel lessons learned from an 11 year old recipient of 3 kidney transplants

We present an 11 yr old girl who received 3 kidney transplants (KTx). She received 1st-KTx from a living donor at age 4 due to the end-stage renal disease. She had no HLA antibodies (Ab) at pre-1st-KTx, and thus pre-tx crossmatch (XM) and donor-specific HLA antibodies (DSA) were −ve. Six weeks later, the allograft was removed due to torsion (twisting of blood vessels) and thrombosis (formation of a blood clot). The C4d staining of Tx-biopsy was −ve indicating no antibody-mediated rejection (ABMR). She was relisted for the 2nd-KTx. Two-mo post tx-nephrectomy sample revealed Abs to all 10 mismatched (MM) donor’s HLA types (and associated CREG) resulting in 97% CPRA. After a 3 yr wait, she received 2nd-KTx from a deceased donor (DD) with −ve XM and DQ8 DSA (MFI = 1961). Shortly, she developed complications by BK viremia/nephropathy and immunosuppression was reduced, which potentially increased alloimmune activation. At 2-mo post-2nd-KTx, she developed de novo DSAs directed against 4 mismatched HLAs of 2nd-KTx (1600–14600 MFI). She was treated with multiple rounds of eculizumab, plasmapheresis and IVIG until Dec. 2015, when she presented with an acute increase in serum creatinine to 8.7 mg/dL. KTx biopsy showed acute ABMR. She was relisted for 3rd-KTx on Feb. 2016 with a cPRA 100%. After 2 yr wait, she received 3rd-KTx on Feb. 2018 from a DD with −ve XM and no DSA. The 3rd donor was mismatched by 8 HLA antigens - 1 of them was a repeat-MM with 1st donor and another was repeat-MM with both 1st and 2nd donors. However, Abs to 5 mismatched HLAs of the 3rd-KTx (B8, B37, Cw6, DP2, DP4) found in the historical sera (MFIs 1000–16,000) were completely −ve in multiple sera tested during 2 mo post-3rd-KTx (Fig.). No rejection was noted. Lessons learned from this case were: A 4-yr old human immune system can recognize and respond to alloantigens, and can trigger HLA-Abs production efficiently. However, acquired memory response, an integral component of adaptive immunity is not fully developed until 11 yr of age. These findings have vital implications in pediatric KTxs. Download : Download high-res image (508KB) Download : Download full-size image

OR5 Donor specific antibody (DSA) assessments using HLA P groups: The devil is in the details

Aim Use of HLA P groups for reporting purposes and DSA assessments for potential donors has been steadily increasing. P groups for HLA class II encompass exon2 sequences and capture the polymorphic rich peptide binding domain that serves as the primary target for alloantibody. Nevertheless, we have observed multiple sera exhibiting HLA-DQA1 or HLA-DPB1 reactivity to polymorphic sites outside of exon 2 and confirmed this reactivity using cell adsorptions. Methods HLA antibodies were assessed using Lifecodes single antigen; Immucor and LabScreen Single Antigen; One Lambda. Sera were pretreated to remove interference using Melon Columns™ (ThermoFisher). HLA typing was performed using rSSO LabType, One Lambda and confirmed using NGS TruSight HLA, Illumina. 10th international workshop B cell lines were used for adsorptions. Results We present a representative example in Fig. 1. Patient #1 typed as a DQA1∗05:05/DQB1∗03:19 yet her sera reacted with the DQA1∗05:03/DQB1∗03:01 bead with a median fluorescence intensity of 11,000. Sequence alignments illustrate an amino acid difference between DQA1∗05:03 and DQA1∗05:05 at position 159. There is also one amino acid difference between DQB1∗03:01 and DQB1∗03:19 but the DQA1∗05:05/DQB∗03:01 bead reactivity was negative, negating the possibility that the antibody was directed at HLA-DQB1. Serum adsorptions using a DQA1∗05:03/DQB1∗03:01 cell line showed an 84% reduction in the DQA1∗05:03/DQB1∗03:01 bead reactivity and a concomitant 3% reduction in reactivity with the DQA1∗03:01/DQB1∗03:01 bead. These data show that the serum reactivity with the DQA1∗05:03/DQB1∗03:01 bead was directed toward the HLA-DQA1 protein, which differed from the recipient by a single polymorphic difference outside of exon 2. Conclusions We show evidence of sensitization to a polymorphism outside of exon 2 implying the potential danger of using P group designations when performing virtual crossmatch assessments or unrelated stem cell donor searches for patients with complex HLA sensitization. A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; Thermo Fisher. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; ThermoFisher.

Calorimetric manifestation of IgA monoclonal immunoglobulins in multiple myeloma sera

Multiple myeloma (MM) with secretion of monoclonal immunoglobulin A (IgA) is among the common myeloma types. The diagnosis of IgA MM is based on a panel of clinical and paraclinical markers, the primary one being the IgA paraprotein level. One of the drawbacks of IgA MM diagnostics and monitoring, especially at low IgA levels, is the migration of monoclonal IgA in the β-globulins region of the serum protein electrophoresis profile where it overlaps with “healthy” β-globulin proteins and is thus not clearly resolved.The present study explores the manifestation of IgA monoclonal immunoglobulins in the thermograms of multiple myeloma sera. We show that the electrophoretic mobility of IgA paraproteins is related to altered intermolecular interactions, plausibly the formation of IgA oligomers and/or albumin-IgA complexes. We demonstrate that high IgA levels exhibit specific calorimetric features that discriminate IgA MM from other MM subtypes.

P060 Next generation virtual crossmatch: A program that captures data and provides rapid and accurate virtual crossmatch assessments

Aim To test the benefits of a software tool that captures HLA antibody analyses, highlights DSA and other risk factors, and provides a comprehensive report to allow fast and accurate Virtual Crossmatches (VXMs). Methods The VXM program used embedded, tiered reviewed HLA antibody data obtained from phenotype and single antigen bead panels. HLA antibody assignments in the VXM program required 2 independent reviewers and were not based on a MFI threshold; rather, assessments included HLA reactivity patterns, confirmation on multiple assays or in multiple sera, and previous HLA exposures (pregnancies and transplants). HLA typing for each potential donor was entered and DSA specificities, if present, were highlighted with individual strength assessments. Embedded HLA typing results for each candidate and all previous allografts allowed reporting of repeated HLA mismatches. Additional alerts included recent HLA sensitization changes, dates for peak and last serum tested, and HLA allele-specific antibodies that cannot be listed as avoids. Results A comparison was performed using results from the VXM program and 251 manual VXMs performed by experienced laboratory personnel during the first quarter of 2017. The majority (82%) of these VXMs involved sensitized candidates and >50% involved 99–100% CPRA candidates. One hundred of 251 VXMs examined were limited to one assessment per patient, but for the majority of cases VXM HLA analyses were performed repeatedly for the same patient. In 7.5% of manual VXMs cases, a secondary review of HLA data improved final DSA assignments. The number of VXMs per donor ranged from 1 to 6 with an average of 1.5 patients per donor. The average time for a manual VXM per patient was 30 min, while the VXM program and embedded data required 5 min per patient. In addition, the VXM program stored all previous donor assessments to assist in the decision to accept or decline the current offer. Conclusions For centers that do not list all HLA antibodies as unacceptable for sensitized waitlist candidates, VXMs can present a complex challenge given that potential donors must be reviewed and accepted/declined within one hour of initial offer. A VXM program that uses tiered reviewed HLA data increases efficiency and reduces possible errors in assessing risk for highly sensitized candidates.

P007 Transfusion-induced HLA antibodies are transient and do not rebound following heart transplantation

Aim To determine the impact of blood transfusions (BTf) given at the time of ventricular assist device (VAD) implantation on HLA antibody (Ab) production, and the clinical relevance of BTf-induced HLA Abs in heart transplantation (HTx). Methods This study includes 54 patients that received a VAD implant between 03/2012 and 05/2016. The amount and type of BTf given during VAD procedure were obtained from patient charts. Multiple sera collected during pre-VAD, post-VAD, and post-HTx periods were analyzed for HLA Abs using One Lambda single antigen beads. Results Of the 54 VAD-implanted patients, 19 (35%) showed a significant increase in HLA Ab specificities and/or strength in post-VAD samples compared to pre-VAD samples; 26 (48%) did not show any significant change in HLA Ab profile; 9 (17%) remind negative for all HLA Abs. Patients who showed significant changes in HLA Abs received more BTf (across all blood components) compared to those displayed little/no change in HLA Abs.BTf-induced HLA Abs are transient and disappear in 5–6 months, while pregnancy-induced HLA Abs are persistent (see figure). Majority of these VAD patients ( n = 37; 69%) received HTx, and 29 HTx recipients had no detectable donor-specific HLA antibodies (DSA) in pre- and post-VAD sera, and remained to be DSA negative following HTx. Three recipients had no DSA in pre-VAD samples, but developed DSA in post-VAD samples, then completely negative following HTx; 5 recipients had DSAs (∼2000 MFI) in both pre- and post-VAD samples, and persistent in post-HTx sera with similar MFIs. The average wait time for a HTx differs among the 3 groups: patients with no HLA Abs waited for ∼5 months, patients with little/no change waited for ∼9 months, and those showed significant change waited for a year. Conclusions HLA Ab production after VAD implantation is common and correlates with amount of BTf a patient receives. This sensitization does not affect HTx as well as post-tx DSA development. Therefore, the BTf-induced HLA Abs appear to be transient and clinically irrelevant in HTx. Download high-res image (446KB) Download full-size image

Abstract B05: Differential autoimmune response to glycolysis and plasminogen antigens from aggressive prostate cancer cells in African American and European American men with prostate cancer

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related male deaths in the U.S. This cancer is more aggressive in African American (AA) men, who are twice as likely to die from the disease compared to other racial groups. Despite racial disparities in PCa mortality, immunoproteomic studies profiling sera from thousands of men for the presence of autoantibodies to tumor-associated antigens (TAA) have mostly used sera from European or Asian ancestry. These autoantibodies can serve as both cancer biomarkers and potential tools in anti-tumor immunotherapy. In this study we profiled sera of AA and European American (EA) men with (N=157) and without (N=183) PCa against protein lysates from aggressive PCa cell lines using one and two-dimensional Western blotting. Sera from AA-PCa patients showed stronger immunoreactivity to proteins in PC3 cell lysates compared to EA-PCa sera. Common bands of immunoreactivity were observed around 37, 40, 50, 55, 60, and 70 kD from the sera of multiple patients. Several AA-PCa sera showed common immunoreactivity against a 50 kD protein band (16 out of 24, 67%) which was identified by serological proteomic analysis (SERPA) as alpha-enolase (ENO1), an enzyme that participates in both the glycolysis and plasminogen pathways. ELISA using purified ENO1 was conducted on 360 sera and showed significantly elevated frequency in men with PCa compared to non-PCa controls. Interestingly, SERPA analysis of other strongly immunoreactive spots outside the 50 kD region recognized of the other highly reactive PCa sera revealed additional candidate TAAs involved in the glycolysis and plasminogen pathways. These included key glycolytic enzymes GAPDH, fructose bisphosphate aldolase, phosphoglycerate kinase and lactate dehydrogenase, and other members of the plasminogen system such as GRP78 and annexin A2. Autoantibodies to stress survival proteins HSP60 and STIP1 were also identified in multiple PCa sera. We then probed by immunoblotting representative sera recognizing some of these candidate TAAs against a panel of 12 PCa and non-PCa cell lines. Anti-STIP1 sera showed elevated immunoreactivity predominantly in docetaxel-resistant cell lines. In the anti-ENO1 positive sera, we observed a race-related differential immune response, with anti-ENO1 sera from AA-PCa men showing elevated immunoreactivity mostly in metastatic cell lines, and anti-ENO1 sera from EA-PCa men showing uniform immunoreactivity across the entire panel of prostate cell lines. Interestingly, while several anti-ENO1 positive sera from AA-PCa men had increased immunoreactivity to this protein in the metastatic PCa cell lines LNCaP, MDA-PC2b, PC3 and DU145, they lacked reactivity against ENO1 in the docetaxel-resistant PC3-DR and DU145-DR cells, or purified ENO1 used in the ELISA. Anti-ENO1 EA-PCa sera did not display this differential pattern of immunoreactivity. To better understand these differences in anti-ENO1 reactivity, we interrogated the pattern of ENO1 post-translational modifications (PTMs) from PC3, PC3-DR and purified ENO1. Several different acetylated, methylated, phosphorylated, citrullinated, and glycosylated ENO1 PTMs were identified in the PC3 cells that were not present in the PC3-DR cells nor purified ENO1 which might explain the differential reactivity of AA-PCa sera against these three sources of ENO1. Taken together, these data suggest the anti-ENO1 autoantibodies from AA-PCa men may target a modified form of ENO1 that is different from that recognized by EA-PCa men. These discrepancies point to racial differences in immune response to TAAs in prostate tumors, and may have implications for identifying immunobiological factors contributing to PCa health disparities. Citation Format: Tino W. Sanchez, Guangyu Zhang, Jitian Li, Liping Dai, Rowaid Kellow, Saied Mirshahidi, Nathan R. Wall, Clayton Yates, Colwick Wilson, Susanne Montgomery, Jian-Ying Zhang, Carlos Casiano. Differential autoimmune response to glycolysis and plasminogen antigens from aggressive prostate cancer cells in African American and European American men with prostate cancer. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B05.

MicroRNAs in Cancer: challenges and opportunities in early detection, disease monitoring, and therapeutic agents.

The goals of this review are to examine the usefulness of miRNAs as diagnostic and prognostic biomarkers for cancer and to evaluate the applicability of miRNAs as cancer therapeutics. Examination of miRNA milieu from body fluids offers a new alternative for quick, affordable and easy analysis of disease status in patients. Blood-based exosomal miRNAs have increased stability and are an excellent choice for clinical cancer diagnostics and prognostics. Currently, there are many miRNA signatures associated with cancer and progression but there is no consensus among multiple sera and tumor sample studies. Off-target and immunological effects remains an obstacle for use of miRNAs as novel chemotherapeutics in the clinic. Recent developments in nanotechnology and drug delivery systems which target the tumor microenvironment may provide an alternative therapeutic approach with decreased toxicity. This review critically evaluates the literature investigating the use of miRNAs as biomarkers and their future as potential therapeutics.