Serum HLA Research Articles

The possible protective role of HLA B27 and relevant immune markers in Juvenile Idiopathic Arthritis patients.

JIA is a disease with different immunological characteristics and a complicated genetic foundation. HLA B27 is a risk factor for the development of JIA, and its impact on immunopathogenesis of the disease is also an area of interest. To determine whether HLA B27 and immune markers varied between JIA patients and healthy population. This comparative cross-sectional study was conducted at Immunology Department of University of Health sciences (UHS), Lahore from February 2018 till August 2021. A total of (71) JIA patients and (34) healthy controls were enrolled. B cells were enumerated by flowcytometry, ELISA was used for serum cytokines estimation and HLA B27 allele was detected by SPSS polymerase chain reaction. The HLA B27 allele was significantly more in the control group than in the patient group, suggesting it is a protective allele to prevent JIA. Peripheral blood B cell counts and percentages were significantly lower in the HLA B27 positive group than in the HLA B27 negative group of control population. Serum cytokine levels were not significantly different between the HLA B27 positive and HLA B27 negative allele of the two study populations. In this study B cells are different between the two groups of control population however; serum cytokines are comparable between the study groups. Though, it was indicated that HLA B27 may be a preventive allele in the onset of JIA.

Post-COVID-19 and Post-COVID-19 Vaccine Arthritis, Polymyalgia Rheumatica and Horton's Arteritis: A Single-Center Assessment of Clinical, Serological, Genetic, and Ultrasonographic Biomarkers.

The potential role of the COVID-19 vaccine and infection to induce autoimmunity is currently underestimated despite the literature emphasizing arthralgia as a common adverse event. We aimed to study the impact of rheumatological complications post-COVID-19 (PC) and post-COVID-19 vaccine (PCV), comparing undifferentiated arthritis (UA) to Polymyalgia Rheumatica, Horton's Arteritis (PMR-HA) and isolated arthritis to UA with "connective-like" accompanying symptoms. We retrospectively included 109 patients with at least 6 months of follow-up, analyzing serum biomarkers, joint ultrasound (US), lung HRCT, DLCO, and HLA haplotypes. There were 87 UA patients showing increased gastrointestinal and lung involvement (p = 0.021 and p = 0.012), higher anti-spike protein IgG levels (p = 0.003), and anti-SARS-CoV-2 IgG positivity (p = 0.003). Among them, 66 cases progressed to ACR-EULAR 2010 early arthritis after 3 months, whereas PMR-HA patients were more commonly PCV (81.8%, p = 0.008), demonstrating higher CRP (p = 0.007) and ESR (p = 0.006) levels, a lower rate of ANA positivity (p = 0.005), and a higher remission rate after six months (p = 0.050). In UA patients, the prevalent HLA was DRB1*11 and C*07 (36.8% and 42.1%). Serum calprotectin, interleukin-6, and C*07 (p = 0.021, 0.041, 0.018) seemed more specific for isolated UA. Conversely, "connective-like" arthritis showed poorer DLCO (p = 0.041) and more frequent US synovitis (p = 0.041). In conclusion, UA is a frequent common PC and PCV complication and may persist over time when compared to PMR-HA.

Sarcolemmal Staining by Anti-C4d/C3d in Cardiac Transplant Endomyocardial Biopsies and Associated Serum HLA Antibodies and Pathologic Rejection

<h3>Purpose</h3> Immunologic evidence of complement fixation in endomyocardial biopsies (EMB) is a feature used to assess pathologic antibody-mediated rejection in cardiac transplants. Uncommonly, sarcolemmal staining (SS) of myocytes is observed. The significance of this is unclear and under-investigated. This study aims to temporally correlate SS with changes in HLA antibodies and pathologic rejection. <h3>Methods</h3> All EMBs that had SS by anti-C4d and/or C3d on immunofluorescence or immunohistochemistry (Figure 1) were identified via the laboratory information system between 10/1/2016 and 10/1/2019. The HLA antibodies and all other EMB results since time of transplant for each case were reviewed and temporally correlated to the biopsy that showed SS. <h3>Results</h3> 28 patients (mean: 31 yrs; range: 6-73 yrs) with 31 SS events were identified. Associated features are summarized in Table 1. Of note, 29/31 events of SS had HLA antibodies measured within a 2 week period before/after the time of biopsy. A temporally associated expansion of HLA Class I/II antibodies was noted in 12 (39%) cases. 21 (68%) cases had prior donor specific antibodies before EMB showing SS, with less immediate temporal association. <h3>Conclusion</h3> Sarcolemmal staining of complement split products in EMBs was associated with a concurrent expansion in strength and/or breadth of HLA antibodies in 39% of cases. This is a novel observation that may partially explain the appearance of SS by immunologic assays. Further investigation is required to fully elucidate the significance of SS in EMB.

Free Airway C4d after Lung Transplantation - A Quantitative Analysis of Bronchoalveolar Lavage Fluid

In recent years, the utility of vascular complement factor 4d (C4d) deposition as diagnostic tool for antibody mediated rejection (AMR) after lung transplantation, has become a controversial issue. We aimed to pinpoint the problematic nature of C4d as biomarker with a simple experiment.We quantified C4d in broncho-alveolar lavage (BAL) of lung transplant patients with diverse post-transplant complications in 3 different settings of clinically clear cases of: 1/ chronic lung allograft dysfunction (CLAD); 2/ acute complications acute rejection (AR), lymphocytic bronchiolitis (LB), antibody-mediated rejection (AMR) and respiratory infection (INF); 3/ patients with parallel C4d immunostaining and Anti-HLA. All groups were compared to BAL of stable patients. C4d was measured via standard ELISA.C4d was increased in CLAD, predominantly in RAS (p = 0.0026) but not in BOS (p = 0.89). C4d was increased in all acute events, AR (p = 0.0025), LB (p < 0.0001), AMR (p = 0.0034), infections (p < 0.0001). In patients with parallel C4d immunostaining and serum HLA antibodies, C4d was increased in C4d-/HLA- (p = 0.0011); C4d-/HLA+ (p = 0.013); HLA+/C4d + (p = 0.0081). A correlation of systemic C-reactive protein (CRP) with C4d was found in all patients (r = 0.49; p < 0.0001). We hypothesize that free C4d in BAL may only be representative of a general immune response in the transplanted lung.

Donor-specific B Cell Memory in Alloimmunized Kidney Transplant Recipients: First Clinical Application of a Novel Method.

HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell-derived HLA antibodies (DSA-M) in renal allograft recipients with pretransplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (AMR) using a recently developed method. Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells were collected at 3 timepoints (pretransplant, mo 6, mo 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated peripheral blood mononuclear cells using SAB assays and compared HLA antibody profiles with same day plasma results. Plasma SAB analysis revealed 35 DSA in 20 patients pretransplant. DSA-M were detected in 9 of 20 (45%) patients and for 10 of 35 specificities (29%). While median mean fluorescence intensity values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3 of 6 patients with AMR and low mean fluorescence intensity DSA (<3000) had DSA-M. Overall, pretransplant DSA/DSA-Mpos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical AMR (P = 0.032) and a higher extent (g≥1 + ptc≥1) of microvascular inflammation (67% vs 9%, P = 0.02). In 17 patients (28 DSA) with posttransplant analyses, persisting DSA posttransplant had more often DSA-M (6/12; 50%) than nonpersisting DSA (2/16; 13%). Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pretransplant risk stratification in patients with DSA.

Sarcolemmal Staining by Anti-C4d/C3d in Cardiac Transplant Endomyocardial Biopsies and Associated Serum HLA Antibodies and Pathologic Rejection

Immunologic evidence of complement fixation in endomyocardial biopsies (EMB) is a feature used to assess pathologic antibody-mediated rejection in cardiac transplants. Uncommonly, sarcolemmal staining (SS) of myocytes is observed. The significance of this is unclear and under-investigated. This study aims to temporally correlate SS with changes in HLA antibodies and pathologic rejection. All EMBs that had SS by anti-C4d and/or C3d on immunofluorescence or immunohistochemistry (Figure 1) were identified via the laboratory information system between 10/1/2016 and 10/1/2019. The HLA antibodies and all other EMB results since time of transplant for each case were reviewed and temporally correlated to the biopsy that showed SS. 28 patients (mean: 31 yrs; range: 6-73 yrs) with 31 SS events were identified. Associated features are summarized in Table 1. Of note, 29/31 events of SS had HLA antibodies measured within a 2 week period before/after the time of biopsy. A temporally associated expansion of HLA Class I/II antibodies was noted in 12 (39%) cases. 21 (68%) cases had prior donor specific antibodies before EMB showing SS, with less immediate temporal association. Sarcolemmal staining of complement split products in EMBs was associated with a concurrent expansion in strength and/or breadth of HLA antibodies in 39% of cases. This is a novel observation that may partially explain the appearance of SS by immunologic assays. Further investigation is required to fully elucidate the significance of SS in EMB.

Diagnostic Biomarkers to Diagnose Acute Allograft Rejection After Liver Transplantation: Systematic Review and Meta-Analysis of Diagnostic Accuracy Studies.

Objective: A systematic review and meta-analysis of diagnostic biomarkers for noninvasive diagnosis of acute allograft rejection following liver transplantation.Background: Noninvasive blood and urine markers have been widely explored in recent decades for diagnosing acute rejection after liver transplantation. However, none have been translated into routine clinical use so far due to uncertain diagnostic accuracy, and liver biopsy remains the gold standard.Methods: Systematic literature searches of Medline, Cochrane and Embase were conducted up to February 2019 to identify studies evaluating the use of noninvasive markers in diagnosing allograft rejection following liver transplantation. Meta-analysis was performed using a random effects model with DerSimonian–Laird weighting and the hierarchical summary receiver operating curve.Results: Of 560 identified studies, 15 studies (1,445 patients) met the inclusion criteria. The following markers were tested: acid labile nitroso-compounds (NOx), serum amyloid A protein, procalcitonin, peripheral blood eosinophil count, peripheral blood T-cell activation and interleukin 2 (IL-2) receptor, guanylate-binding protein-2 mRNA, graft-derived cell-free DNA, pi-glutathione S-transferase, alpha-glutathione S-transferase and serum HLA class I soluble antigens. Only eosinophil count was tested in multiple studies, and they demonstrated high heterogeneity (I2 = 72% [95% CI: 0.5–0.99]). IL-2 receptor demonstrated the highest sensitivity (89% [95% CI: 0.78–0.96]) and specificity (81% [95% CI: 0.69–0.89]).Conclusion: IL-2 receptor expression demonstrated the highest diagnostic accuracy, while the peripheral eosinophil count was the only marker tested in more than one study. Presently, liver biopsy remains superior to noninvasive diagnostic biomarkers as most studies exhibited inferior designs, hindering possible translation into clinical application.

Humoral Rejection in Lung Transplantation

Introduction The diagnosis of humoral rejection in lung transplant recipients is sometimes elusive, involving histopathologic (capillaritis), immunopathologic (C4d staining), and serologic (donor-specific anti-HLA antibodies, DSA) parameters. These criteria are not always present in cases and, moreover, the involvement of non-HLA antibodies in lung-allograft humoral rejection remains largely unclear. The aim of the study was to assess the involvement of HLA and non-HLA antibodies in humoral rejection diagnosis after lung transplantation. Materials and Methods A total of 60 consecutive lung transplant recipients were followed in our Institution from 2015. Protocol biopsy at day 21 post transplantation and screening for serum HLA and non-HLA antibodies by Luminex, LABScreen Autoantibody (One Lambda), before lung transplant and at the moment of the biopsy, were performed. Results and Discussion A total of 5 lung transplant recipients (8.33%) were C4d+ on biopsy but only one of them developed de novo DSA without evidence of non-HLA antibodies. The remaining 4 patients C4d+, were serum positive for non-HLA antibodies (2 against LG3 and other 2 against myosin) prior lung transplantation (6.67%) but negative at the moment of the biopsy. The incidence observed of preformed non-HLA antibodies in patients awaiting for lung transplantation was significant and potentially could drive C4d deposition on lung tissue with subsequent humoral rejection. Conclusion The screening of non-HLA and HLA antibodies could help to identify patient at risk of lung-allograft rejection.

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss.

Luminex multiplex immunoassays enable simultaneous monitoring of Abs against multiple Ags in autoimmune, inflammatory, and infectious diseases. The assays are used extensively to monitor anti-HLA Abs in transplant patients for donor organ selection, desensitization, and assessing the risk for graft rejection. To monitor IgG Abs, fluoresceinated IgG constant H chain-binding polyclonal F(ab')2 (IgHPolyFab) is used as the fluoresceinated secondary Ab (2nd-Ab), whereas IgG subclasses are monitored with Fc-specific monoclonal whole IgG (FcMonoIgG). The fluorescent signal from the 2nd-Ab is measured as mean florescence intensity (MFI). When IgHPolyFab is used, the signal is amplified as a result of the binding of multiple polyclonal Fabs to the C region of primary IgH. The reliability of such amplification for Ab measurements was not validated, nor were MFIs compared with 1:1 binding of FcMonoIgG to primary Abs. Comparing the MFIs of anti-HLA Abs obtained with IgHPolyFab and FcMonoIgG against normal human sera, IVIg, and allograft recipients' sera, it was observed that the number of HLA-Abs was notably higher with IgHPolyFab than with FcMonoIgG The MFIs of anti-HLA Abs also remained higher with IgHPolyFab in the normal sera and in IVIg, but the reverse was true when the autologous and allogeneic IgG concentrations were augmented in allograft recipients. Indeed, MFIs of the de novo allo-HLA Abs were markedly higher with FcMonoIgG than with IgHPolyFab. Serum titration established the superiority of FcMonoIgG for monitoring MFIs of de novo allo-HLA Abs in allograft recipients. Avoiding false amplifications of the number and MFIs of anti-HLA IgG with FcMonoIgG may minimize immunosuppressive therapies, maximize the number of donors for patients waiting for allografts, and enable better prediction of graft rejection.

A Memory B Cell Crossmatch Assay for Quantification of Donor-Specific Memory B Cells in the Peripheral Blood of HLA-Immunized Individuals.

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow-residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA-specific memory B cells are scarce and insufficient in quantifying the complete donor-specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor-specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme-linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II-specific memory B cells per million IgG-producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor-specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross-match assay to quantify donor-specific memory B cells in patients with a history of sensitizing events.

Should epitope-based HLA compatibility be used in the kidney allocation system?

The new kidney allocation system (KAS) still applies donor-recipient HLA compatibility mostly at the antigen level and although some four-digit alleles have been included. This system is used to record unacceptable mismatches for sensitized transplant candidates with serum HLA antibodies. Since the reactivities of such antibodies are specifically associated with epitopes rather than HLA antigens, a more scientifically accurate assessment of mismatch acceptability could be based on epitopes. HLA class I and class II epitope specificity analyses can now be readily performed with serum antibody assays with single allele panels. This report describes an epitope-based HLA compatibility system for KAS and involves recipient and donor HLA typing at the four-digit allele level. It focuses on sensitized patients who have serum antibodies specific for HLA epitopes that can be entered as unacceptable mismatches in the transplant candidate database. Newly developed software programs could readily identify compatible HLA types.

Detecting the humoral alloimmune response: we need more than serum antibody screening.

Whereas many techniques exist to detect HLA antibodies in the sera of immunized individuals, assays to detect and quantify HLA-specific B cells are only just emerging. The need for such assays is becoming clear, as in some patients, HLA-specific memory B cells have been shown to be present in the absence of the accompanying serum HLA antibodies. Because HLA-specific B cells in the peripheral blood of immunized individuals are present at only a very low frequency, assays with high sensitivity are required. In this review, we discuss the currently available methods to detect and/or quantify HLA-specific B cells, as well as their promises and limitations. We also discuss scenarios in which quantification of HLA-specific B cells may be of additional value, besides classical serum HLA antibody detection.

A single-centre, tertiary-care, Italian registry of celiac disease: Incidence and clinical presentation

s / Digestive and Liver Disease 46 (2014) e85–e127 e95 4.5±0.6pg/ml, mean FT4 1.2± .4 ng/dl. TSH levels were above cutoff laboratory range in 10% of our population and the 2 patients (0.2%) of the whole population presented TSH≥10 UI/ml. We found a significant positive correlation between TSH levels and BMI z-score (rho=0.121, p=0.001), even after adjustment for age and sex (r=0.117, p=0.001); a positive correlation was also observed between FT3 and BMI z-score (rho=0.161, p<0.001), but not for Ft4 (p=0.787). TSH and FT3 were also positively associated with Waist to Height ratio (WtHr, rho=0.114, p=0.002 and rho=0.095, p=0.011 respectively). No correlation was found between thyroid functionality and: Homa index, z-score of blood pressure and hepatic steatosis. Conclusions: Our data suggest a relationship between obesity and serum TSH and FT3 levels. In particular, WtHr, marker of visceral fat, is directly related to TSH levels. It is not clear whether thyroid alteration are cause or consequence of fat excess, but metabolic active visceral fat could have a role. http://dx.doi.org/10.1016/j.dld.2014.07.078 A SINGLE-CENTRE, TERTIARY-CARE, ITALIAN REGISTRY OF CELIAC DISEASE: INCIDENCE AND CLINICAL PRESENTATION Valentina Giorgio ∗, Maria Rita Sartorelli, Claudia Della Corte, Donatella Comparcola, Daniela Liccardo, Arianna Alterio, Valerio Nobili, Francesca Ferretti Malattie EpatoMetaboliche, Ospedale Pediatrico “Bambino Gesu”, Roma, Italy Objectives: The aim of this study was to assess clinical pattern and the incidence of celiac disease (CD) currently diagnosed in children living in Rome, describing a single-Italian-center experience. Methods: A prospective, single-center registry of new cases of CD in children under 18 years was conducted from 1 January 2011 to 1 June2013. The variables studiedwere: age at diagnosis, gender, breast feeding, clinical symptoms, associated diseases, IgA serum levels, CD serology, histological lesions, and HLA DQ2/QQ8. The crude incidence rate of CD was calculated as new cases per 1000 live births. Results: 800 new cases of CD were included (39% males). The median age at diagnosis was 8 years (range 0.9–18). 483/800 (60%) were breast fed≥2months, with amean breast feeding duration of 15.2±11.4months. Mean age at first gluten exposurewas 6.4±2.2 months. Regarding clinical presentation 68.9% showed classical symptoms, 27.1% were non-classical, and 4% were asymptomatic. Among atypical symptoms, celiac crises was present and seen in 3% of all children. 96.7% were positive for IgA endomysial antibodies, 97.3% were positive for IgA anti-transglutaminase type 2 antibodies. IgA selective deficiency was seen in 4% children. 85,6% performed duodenal biopsy. Villous atrophy was observed in 91% and increased intraepithelial lymphocytes with crypt hyperplasia in 10.3%. 3 children (0.37%) were diagnosed with CD after cutaneous biopsy for dermatitis herpetiformis. The HLA phenotype was performed in 200 children (25%) and was: DQ2: 84.5%, DQ2/DQ8: 5.5% and DQ8: 10%. The incidence rate was 7.08 cases of CD per 1000 live births. Conclusions: InRome, themost frequent clinicalpresentationof CD in the pediatric age is the classical form. The observed incidence of CD in children ismuchhigher than the current CD incidence rates observed in other countries. A multicenter study on larger series would be needed to verify this increasing incidence. http://dx.doi.org/10.1016/j.dld.2014.07.079 EVALUATION OF VITAMIN D STATUS IN A PEDIATRIC POPULATION OF SOUTHERN ITALY Donatella De Giovanni1,∗, Irene Rutigliano1, Clementina Calabrese1, Giovanna Nardella1, Maria Pia Falcone1, Angelo Guida1, Antonio Longo2, Massimo Pettoello-Mantovani1, Angelo Campanozzi1 1 Clinica Pediatrica, Universita’ di Foggia, Foggia, Italy 2 U.O. Pediatria, Ospedali Riuniti di Foggia, Foggia,

Sodium and potassium intake in children: Relationship with age, body mass and blood pressure

s / Digestive and Liver Disease 46 (2014) e85–e127 e95 4.5±0.6pg/ml, mean FT4 1.2± .4 ng/dl. TSH levels were above cutoff laboratory range in 10% of our population and the 2 patients (0.2%) of the whole population presented TSH≥10 UI/ml. We found a significant positive correlation between TSH levels and BMI z-score (rho=0.121, p=0.001), even after adjustment for age and sex (r=0.117, p=0.001); a positive correlation was also observed between FT3 and BMI z-score (rho=0.161, p<0.001), but not for Ft4 (p=0.787). TSH and FT3 were also positively associated with Waist to Height ratio (WtHr, rho=0.114, p=0.002 and rho=0.095, p=0.011 respectively). No correlation was found between thyroid functionality and: Homa index, z-score of blood pressure and hepatic steatosis. Conclusions: Our data suggest a relationship between obesity and serum TSH and FT3 levels. In particular, WtHr, marker of visceral fat, is directly related to TSH levels. It is not clear whether thyroid alteration are cause or consequence of fat excess, but metabolic active visceral fat could have a role. http://dx.doi.org/10.1016/j.dld.2014.07.078 A SINGLE-CENTRE, TERTIARY-CARE, ITALIAN REGISTRY OF CELIAC DISEASE: INCIDENCE AND CLINICAL PRESENTATION Valentina Giorgio ∗, Maria Rita Sartorelli, Claudia Della Corte, Donatella Comparcola, Daniela Liccardo, Arianna Alterio, Valerio Nobili, Francesca Ferretti Malattie EpatoMetaboliche, Ospedale Pediatrico “Bambino Gesu”, Roma, Italy Objectives: The aim of this study was to assess clinical pattern and the incidence of celiac disease (CD) currently diagnosed in children living in Rome, describing a single-Italian-center experience. Methods: A prospective, single-center registry of new cases of CD in children under 18 years was conducted from 1 January 2011 to 1 June2013. The variables studiedwere: age at diagnosis, gender, breast feeding, clinical symptoms, associated diseases, IgA serum levels, CD serology, histological lesions, and HLA DQ2/QQ8. The crude incidence rate of CD was calculated as new cases per 1000 live births. Results: 800 new cases of CD were included (39% males). The median age at diagnosis was 8 years (range 0.9–18). 483/800 (60%) were breast fed≥2months, with amean breast feeding duration of 15.2±11.4months. Mean age at first gluten exposurewas 6.4±2.2 months. Regarding clinical presentation 68.9% showed classical symptoms, 27.1% were non-classical, and 4% were asymptomatic. Among atypical symptoms, celiac crises was present and seen in 3% of all children. 96.7% were positive for IgA endomysial antibodies, 97.3% were positive for IgA anti-transglutaminase type 2 antibodies. IgA selective deficiency was seen in 4% children. 85,6% performed duodenal biopsy. Villous atrophy was observed in 91% and increased intraepithelial lymphocytes with crypt hyperplasia in 10.3%. 3 children (0.37%) were diagnosed with CD after cutaneous biopsy for dermatitis herpetiformis. The HLA phenotype was performed in 200 children (25%) and was: DQ2: 84.5%, DQ2/DQ8: 5.5% and DQ8: 10%. The incidence rate was 7.08 cases of CD per 1000 live births. Conclusions: InRome, themost frequent clinicalpresentationof CD in the pediatric age is the classical form. The observed incidence of CD in children ismuchhigher than the current CD incidence rates observed in other countries. A multicenter study on larger series would be needed to verify this increasing incidence. http://dx.doi.org/10.1016/j.dld.2014.07.079 EVALUATION OF VITAMIN D STATUS IN A PEDIATRIC POPULATION OF SOUTHERN ITALY Donatella De Giovanni1,∗, Irene Rutigliano1, Clementina Calabrese1, Giovanna Nardella1, Maria Pia Falcone1, Angelo Guida1, Antonio Longo2, Massimo Pettoello-Mantovani1, Angelo Campanozzi1 1 Clinica Pediatrica, Universita’ di Foggia, Foggia, Italy 2 U.O. Pediatria, Ospedali Riuniti di Foggia, Foggia,

57-P: HYPERTENSION AND HLA-A MISMATCHING ARE ASSOCIATED WITH RESTARTING DIALYSIS IN KIDNEY TRANSPLANTATION

Aim The interaction of the heart with other organs is well established. In particular, several donor and recipient factors involving the heart are known to be associated with graft loss in kidney transplantation. In this retrospective single-center study, we analyzed the effect of clinical, cardiological and immunological factors on kidney transplantation outcome. The study included 245 transplanted recipients from deceased donors between 2000 and 2006. Methods In donors, age, cause of death, history of hypertension, hypotension or cardiac arrest, length of intensive care unit stay, serum creatinine levels and HLA typing were evaluated; while age, waiting time, HLA typing, antibodies sensitization and allocation were evaluated in recipients. Age donor/recipient matching and HLA mismatches were also considered. Results Cox regression analysis shows that time spent in waiting list increases the risk of restarting dialysis (OR=1.01, 95% CI=1.00-1.03; p Conclusions Our study established that both hypertension and HLA-A mismatch can affect the time of restarting dialysis in patients undergoing kidney transplantation.

174-P: VIRTUAL CROSSMATCH IN A PATIENT WITH HLA ALLELE SPECIFIC ANTIBODY

Aim HLA typing at low/intermediate resolution levels is acceptable for the characterization of HLA genes of solid organ recipients and donors. However, the interpretation of virtual crossmatch may be problematic for transplant candidates with allele specific antibodies. We present here a case report and a possible strategy to alleviate this problem. Methods HLA typing was performed using SSO for low/intermediate resolution and SSP for high resolution. One Lambda Single Antigen Bead assay was used for the detection and identification of serum HLA antibodies. Crossmatch was performed using CDC and flow cytometry. Results The patient is a 52-year old Caucasian male, previously sensitized to HLA antigens, listed for heart transplantation. Due to the urgency of the transplant, an organ offer from a 28-year old donor was accepted even though the patient displayed an allele specific, potentially donor specific antibody (DSA) directed to DRB1∗13:03 antigen ( Table 1 ). [table1] The available donor HLA class II type was: DR13, DR15, DQ6. This donor was accepted based on the known frequency of HLA-DR13 alleles (13:03 allele is 5-fold less frequent than 13:01) and the presumed decreased risk of anti-HLA class II DSA compared to class I DSA in heart transplantation. CDC and flow cytometry crossmatches were performed at the time of transplant and yielded negative results. Extended typing of the donor revealed that the donor carried the HLA-DRB1∗13:01 gene. Thus, the allele specific DRB1∗13:03 antibody was not a DSA. Post-transplant outcome was favorable. C4d staining was negative in serial allograft biopsies. Conclusions In patients with allele specific antibodies, the frequency of related HLA alleles may guide the interpretation of the virtual crossmatch. Whenever possible, extended HLA typing of the donor should be performed to confirm the presence or absence of DSA.

Sa1289 Non Celiac Gluten Sensitivity: Lack of Response to In Vitro Gliadin Challenge and Basophils Activation Assay

In recent years it has become more frequent for physicians to come across types of gluten sensitivity other than celiac disease (CD). Regardless of the absence of any serological CD markers or intestinal damage, gluten sensitive (GS) patients often report intestinal and extraintestinal symptoms shortly after the ingestion of gluten and the disappearance of such symptoms on a gluten free diet (GFD). Regrettably, at present, there is no evidence of any mucosal or serological modifications in GS patients, or that gluten is really the trigger factor in gluten sensitivity. We aimed at evaluating the impact of gliadin challenge on two experimental settings, i.e. in vitro gliadin challenge of duodenal mucosa and peripheral blood basophils in GS patients compared to CD patients and healthy controls. We consecutively enrolled patients referred to tertiary centers for food intolerance and celiac disease. Participants underwent a complete clinical screening in order to rule out CD (serum a-tTG, EMA, total IgA and HLA status, where appropriate), an interview for evaluation of diet and symptoms, a skin prick test to exclude wheat allergy, upper endoscopy for duodenal biopsies used for both histological assessment and for the in vitro evaluation of the gliadin-induced mucosal expression of early and late inflammatory markers: PY99, epithelial HLA-DR, ICAM1, crypt HLA-DR, CD3, CD25 and CD69. In addition, a basophils activation assay, in which patients' basophils were challenged with a gliadin extract, was performed. Basophils were analyzed for the expression of hematopoietic membrane antigens CD203c, CD63 and CD45 by flow cytometry looking at differences in antigen expression. 119 subjects were screened for CD and GS and, according to the results, four groups were obtained: GS patients (no.16), CD on gluten free diet (no.34), CD on gluten containing diet (no.35) and healthy controls (no.34). As for the in vitro gliadin challenge, all CD patients, regardless of their dietetic status, showed a clear mucosal activation (increased immunofluorescence intensity both for early and delayed inflammation markers) when stimulated with gliadin, while only 3 controls and 3 GS patients showed an weak response for some, not all, inflammation markers. Also, there were no significant differences in the skin prick test results or in the level of CD63 and CD203c basophils expression, used as a marker of cell activation, among the groups. The present study shows that in GS patients, gliadin challenge test did not show any significant modifications in the expression of those mucosal inflammation markers found increased in CD patients. Therefore, this tool should not be used as a diagnostic instrument in case of GS. Moreover, the lack of differences in basophils activation and skin prick test seems to exclude this form of cellular sensitivity in our GS series also.

C4d by Immunofluorescence and Immunohistochemistry in Routine Lung Allograft Biopsies

Purpose The role of C4d in diagnosing antibody mediated rejection (AMR) in lung allografts is unclear. We compared C4d by immunofluorescence (IF) and immunohistochemistry (IHC) in routine allograft transbronchial bx (TBBx) and correlated the results with clinical findings. Methods and Materials Prospectively, consecutive paired frozen and paraffin-embedded samples from the same TBBx were stained for C4d by IF and IHC and scored as negative (neg), focal+ or diffuse 2+ or 3+. Serum HLA antibodies (ab) (within 6 mo of TBBx) were noted. Poisson regression (SAS version 9) was used to compare HLA ab between neg and focal+. Results 228 TBBx from 76 patients (pts) were reviewed. No pt was clinically suspected to have AMR. Pt 1 with C4d 3+ had pleural effusions; TBBx was unremarkable; apheresis was performed. The pt died 1 mo later (subdural hematoma). Pt 2 with C4d 3+ had pseudomonas bacteremia and progressive multifocal leukoencephalopathy (PML). TBBx showed intimitis. The pt died 4 mo later (PML). A pt (heart-lung transplant) had AMR on heart bx. A TBBx 8 mo later was C4d focal+ (IF); neg. by IHC; HLA ab 8% & 28% (class I & II, respectively) at time of TBBx. HLA ab were not significantly different between C4d (IHC) neg and C4d (IHC) focal+ TTBx. Conclusions IHC and IF are similar in detecting diffuse 3+C4d in routine TBBx. IHC appears superior in identifying focal C4d, likely because IHC provides better architecture that helps to neglect the high background of C4d stain in the lung. Routine C4d might not be a reliable marker of AMR in lung transplant. Results of C4d and HLA abs C4d by IHC (#pts) C4d by IF (#pts) HLA Abs (#pts with + abs), median (range) Neg (196) Neg (188) Screen (3/68), 2 (2-7) Class (Cl) I (50/76), 3 (0-76) Cl II (53/76), 5.5 (0-65) Diffuse 2+(3), focal + (4), equivocal (1) Screen (0/8) Cl I (4/8), 0.5 (0-9) Cl II (4/8), 3.5 (0-28) Focal+ (30) Neg (30) Screen, neg. Cl I (13/19), 3 (0-52) Cl II (13/19), 25 (0-65) Diffuse 3+ (2) Diffuse 3+ (2) Screen (1) neg. Cl I (2), 16.5 (3-30) Cl II (2), 1 (0-2)

A Novel ELISPOT Assay to Quantify HLA-Specific B Cells in HLA-Immunized Individuals

Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. Enriched peripheral B cells were pre-activated in a CD40L-driven fashion before being assayed by the HLA-specific B cell ELISPOT assay. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B cell hybridomas producing HLA antibodies of defined specificity. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non HLA-immunized individuals and transplant patients with serum HLA antibodies. B cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while non-immunized individuals had none (Figure 1). Specificities that can currently be tested in the HLA-specific ELISPOT include HLA-A1, HLA-A2, HLA-B7 and HLA-B44. The assay is highly reproducible, as determined by repeat testing of B cells from a single bleeding date.[Figure 1]: Figure 1. HLA-specific B cells are detected in HLA-immunized individuals, but not in non-immunized individuals. (A) Raw ELISPOT data of 2 HLA-A2 immunized and 2 non-immunized individuals. Frequency is the number of spots per million B cells. Enriched B cells were activated for 7 days and seeded into ELISPOT plates coated with monomeric HLA molecules (B). Frequencies of HLA-A2 specific antibody producing B cells of HLA-A2 immunized and non-immunized healthy individuals.Transplant recipients who were immunized by an HLA-mismatched graft had up to 143 HLA-specific B cells per million total B cells. Moreover, preliminary data suggest that immunosuppressive drugs do not interfere with the detection of HLA-specific B cells. In conclusion, we have developed and validated a highly specific, reproducible and sensitive HLA-specific B cell ELISPOT assay, can be used to determine the frequency of peripheral HLA-specific B cells in transplant recipients. This technique constitutes a new tool for quantifying humoral immune responses.

The Detection of Serum Hla Antibodies by Luminex Bead Assays Can Be Improved by the Addition of Edta or Dithiotreitol

The detection and specification of human leukocyte antibodies (HLA antibodies) has improved in the recent years through the introduction of solid phase techniques. The Luminex x-MAP technology with single HLA antigen coated beads allows a more precise and reproducible specification of HLA antibodies than cell based methods or ELISA using antigen extracts from cells. However, there are some drawbacks. The sensitivity is very high and the correlation of the results with clinical data and standard test systems like the complement dependent cytotoxicity (CDC) assay is not always satisfactory. Recently it has been shown, that Luminex-based HLA antibody assays might be disturbed by prozone phenomena resulting from binding of C1q and/or IgM, when sera with a high content of HLA antibodies are analyzed. Dilution, treatment of the sera with DTT or addition of EDTA seem to decrease these effects but may have marked influences on the results of these assays. We tested sera from five highly immunized patients for in CDC (for HLA-Class I antibodies), LABScreen Mixed and LABScreen single antigen tests (for HLA-Class I and II antibodies) using untreated, DTT-treated serum or serum supplemented with 8 mM EDTA. 4 CDC (-/+DTT) positive sera showed a high increase (DTT>EDTA) in reaction strengths for some HLA specificities, which was more pronounced for the antibody specificities reactive in CDC. DTT or EDTA treatment changed the number of HLA antibody specificities detected to a variable extent in all five sera. The CDC negative serum showed minor changes in reaction strength. The signal strenghs of the different specificities mainly remained in the order of the untreated serum. In summary Luminex bead assay systems are sensitive to prozone phenomena which might be diminished by DTT treatment or addition of EDTA. By using these methods a further increase of sensitivity and a change of detected specificities with results differing from the results with untreated sera have to be expected. However, the use of DTT or EDTA might improve the results. In our cases we found, that specificities confirmed by data from the immunizer and/or found in the CDC-assay are better represented in the results. Further efforts should be taken to verify, if one of these methods could replace the regular Luminex bead assay with untreated serum.