Accuracy of Serological Screening for the Diagnosis of Celiac Disease in Type 1 Diabetes Children.

Background and Objectives: Patients with type 1 diabetes (T1D) are considered at high-risk for developing celiac disease (CD). The purpose of our study was to determine the prevalence of CD among children who were followed in our unit for T1D using the latest ESPGHAN guidelines, and avoiding intestinal biopsies in some of the children. Materials and Methods: We performed a prospective monocentric study, which included 663 T1D children between June 2014 and June 2016. We considered CD according to serological (tissue transglutaminase (TGAs) and endomysium antibodies) results. Children were included either at the time of T1D diagnosis or during their follow up. We looked for clinical and biochemical signs of CD, and for T1D characteristics. Results: The children's ages ranged from 11 months to 18 years. CD was confirmed in 32 out of 663 patients with T1D, with a prevalence of 4.8%. CD was excluded in 619 children and remained uncertain for 12 children, who had positive TGAs without the required criteria. We found that 95% of T1D children express HLA-DQ2 and/or -DQ8, which was 2.4 times higher than in the general population. Conclusions: An intestinal biopsy could be avoided to confirm CD in the majority of T1D children. Silent forms of CD are frequent and screening is recommended for all patients. Importantly, repeated TGA assessment is required in HLA genetically predisposed T1D patients, while it is unnecessary in the 5% who are HLA-DQ2 and -DQ8 negative.

Glucocorticoid use as a cause of non-cellular immune response to SARS-Cov2 Spike in patients with immune system diseases.

Disease modifying therapies compromise immune response to SARS-Cov2 or its vaccine in patients with immune system diseases (ISD). Therefore, analysis of the humoral and cellular responses against Spike is of utmost importance to manage ISD patients. A single-center retrospective study was conducted to evaluate the impact of COVID-19 immunization in 87 ISD patients and 81 healthy controls. We performed a whole blood interferon gamma release assay using SARS-Cov2 Spike and Nucleocapsid recombinant proteins in order to evaluate T-cell memory response, and an IgG anti-Spike ELISA to evaluate humoral response. Cellular (26.4%) and humoral (44.8%) responses were negative against Spike in ISD patients following COVID-19 immunization. In univariate analysis, an anti-Spike T cell defective response was associated with the use of glucocorticoids (Odds ratio [OR] = 10.0; p < 10-4), serum albumin level ≤40 g/L (OR = 18.9; p < 10-4), age over 55 years old (OR = 3.9, p = 0.009) and ≤2 vaccine injections (OR = 4.9; p = 0.001). The impact of glucocorticoids persisted after adjustment for age and number of vaccine injections (OR = 8.38, p < 0.001). In contrast, the humoral response was impacted by the use of anti-CD20 mAb (OR = 24.8, p < 10-4), and an extended time since immunization (≥75 days; OR = 4.3, p = 0.002). Double defective cellular/humoral responses (6.9%) were typically encountered in glucocorticoids and/or anti-CD20 mAb treated ISD with a serum albumin level ≤40 g/L (OR = 17.5; p = 0.002). Glucocorticoid usage, B cell depleting therapies, and a low serum albumin level were the main factors associated with a non-response to COVID-19 immunization in ISD patients. These results need further confirmation in larger studies.

Impact of calcineurin inhibitor-free immunosuppression on de novo donor-specific antibody formation in liver transplant recipients.

BACKGROUND & AIMS: Low calcineurin inhibitor (CNI) levels expose liver transplant recipients to rejection episodes and potentially to antibody-mediated rejection. There are little data on the impact of CNI-free immunosuppression on de novo donor-specific HLA antibody (dnDSA) development. Here we evaluated the prevalence of dnDSA in liver transplant recipients on CNI-free maintenance regimens and their associations with histopathological abnormalities of allografts. METHODS: Seven hundred and twenty-seven liver transplant recipients underwent a first liver transplant between 2000 and 2018 in three French transplant centres and had protocolized follow-up with dnDSA screening and allograft biopsy 1, 5 and 10 years after transplantation. RESULTS: CNIs were withdrawn in 166 (22.8%) patients with or without conversion to mammalian target of rapamycin inhibitors and/or maintenance with mycophenolic acid. DSA were present after withdrawal in 30.1% (50/166) patients on CNI-free immunosuppression compared with 16% (90/561) on CNI maintenance therapy (p < 0.001). The cumulative incidence of dnDSA 10 years after transplant was 20% in the CNI group versus 28% in the CNI-free group (p < 0.01). dnDSAs were associated with histological graft abnormalities (significant allograft fibrosis or rejection) (HR 2.24, 95% CI 1.2-4.1; p = 0.01). In univariate Cox regression analysis, being on a CNI-free regimen did not impact graft histology. CONCLUSIONS: Patients on a CNI-free IS regimen have a higher prevalence of dnDSA than patients on a standard IS regimen. dnDSAs but not CNI-free immunosuppression were associated with abnormal allograft histology.

Do anti-IL-6R blockers have a beneficial effect in the treatment of antibody-mediated rejection resistant to standard therapy after kidney transplantation?

Antibody-mediated rejection (AMR) that resists to standard of care (SOC) therapy remains a major challenge after kidney transplantation and leads to graft failure in a majority of cases. The use of anti-IL6 receptor antibodies was suggested to treat chronic antibody-mediated rejection (cAMR) after failure of classical treatments. We treated nine patients with AMR resistant to apheresis, rituximab, and intravenous immunoglobulins, with a monthly infusion of tocilizumab and compared them with a historical cohort of 37 patients with similar clinical, immunological, and histological characteristics. The 1-year graft survival and the decline in renal function did not differ between patients who received tocilizumab and those who did not. Histological follow-up showed that despite a decrease in inflammation and tubulitis scores after tocilizumab, the course of antibody-mediated lesions and chronic glomerulopathy were similar in both groups. In our study, the addition of monthly infusions of tocilizumab did not alter the course of AMR that resist to SOC therapy. Large randomized studies are urgently needed to assess the effect of tocilizumab in this context.

Reassessment of the clinical impact of preformed donor-specific anti-HLA-Cw antibodies in kidney transplantation.

Anti-denatured HLA-Cw antibodies are highly prevalent, whereas anti-native HLA-Cw antibodies seem to lead to random flow cytometry crossmatch results. We aimed to reassess crossmatch prediction for anti-HLA-Cw using 2 types of single antigen flow beads (classical beads and beads with diminished expression of denatured HLA), and to compare the pathogenicity of preformed anti-denatured and anti-native HLA-Cw antibodies in kidney transplantation. We performed 135 crossmatches with sera reacting against donor HLA-Cw (classical beads fluorescence ≥500); only 20.6% were positive. Forty-three (31.6%) were anti-denatured HLA antibodies (beads with diminished expression of denatured HLA fluorescence <300); all were crossmatch negative. The correlation between classical beads fluorescence and the crossmatch ratio was low (ρ = 0.178), and slightly higher with beads with diminished expression of denatured HLA (ρ = 0.289). We studied 52 kidney recipients with preformed anti-HLA-Cw donor-specific antibodies. Those with anti-native HLA antibodies experienced more acute and chronic antibody-mediated rejections (P = .006 and .03, respectively), and displayed a lower graft survival (P = .04). Patients with anti-native HLA-Cw antibodies more frequently had previous sensitizing events (P < .000001) or plausibility of their antibody profile according to known anti-native HLA-Cw eplets (P = .0001). Anti-native but not anti-denatured HLA-Cw antibodies are deleterious, which underscores the need for reagents with diminished expression of denatured HLA.

Outcome of Liver Transplant Patients With Preformed Donor-Specific Anti-Human Leukocyte Antigen Antibodies.

After liver transplantation (LT), the role of preformed donor-specific anti-human leukocyte antigen antibodies (pDSAs) remains incompletely understood. We conducted a retrospective, case-control analysis to determine the impact of pDSAs after LT in 3 French transplant centers (Bordeaux, Lyon, and Toulouse). Among the 1788 LTs performed during the study period, 142 (7.9%) had at least 1 pDSA. The patient survival rate was not different between patients who received an LT with pDSAs and the matched-control group. A liver biopsy was performed 1 year after transplantation in 87 recipients. The metavir fibrosis score did not differ between both groups (1 ± 0.8 versus 0 ± 0.8; P = 0.80). However, undergoing a retransplantation (hazard ratio [HR] = 2.6, 95% confidence interval [CI], 1.02-6.77; P = 0.05) and receiving induction therapy with polyclonal antibodies (HR = 2.5; 95% CI, 1.33-4.74; P = 0.01) were associated with a higher risk of mortality. Nonetheless, high mean fluorescence intensity (MFI) donor-specific antibodies (ie, >10,000 with One Lambda assay or >5000 with Immucor assay) were associated with an increased risk of acute rejection (HR = 2.0; 95% CI, 1.12-3.49; P = 0.02). Acute antibody-mediated rejection was diagnosed in 10 patients: 8 recipients were alive 34 (1-125) months after rejection. The use of polyclonal antibodies or rituximab as an induction therapy did not reduce the risk of acute rejection, but it increased the risk of infectious complications. In conclusion, high MFI pDSAs increase the risk of graft rejection after LT, but they do not reduce medium-term and longterm patient survival. The use of a T or B cell-depleting agent did not reduce the risk of acute rejection.

Anti-IL-2R blockers comparing with polyclonal antibodies: Higher risk of rejection without negative mid-term outcomes after ABO-incompatible kidney transplantation.

There is no recommendation regarding the type of induction therapy to use in ABO-incompatible (ABOi) kidney transplantation. The aim of this retrospective study was to compare the outcome of ABOi living donor kidney transplant (LDKT) recipients who received either polyclonal antibodies or anti-interleukin-2 receptor (IL-2R) blockers as an induction agent. All ABOi HLA-compatible patients that received a LDKT between 03/11 and 03/18 in three French transplantation center (Paris Saint-Louis, Paris Necker, and Toulouse) were included in the study. Fifty-eight patients were given polyclonal antibodies and 39 patients received anti-IL-2R blockers. We identified by a Cox proportional hazard model the use of polyclonal antibodies as a protective factor against acute rejection (HR = 0.4, 95%CI [0-0.9], P < .05). However, pathological findings on protocol biopsies at 1 year were similar in both groups, as were patient and graft survivals, renal function, and complications. We conclude that the acute rejection rate was significantly higher in patients given anti-IL-2R blockers compared to polyclonal antibodies. However, in our series, there was no negative impact on mid-term outcome.

Distinct effect of age, sex, and CMV seropositivity on dendritic cells and monocytes in human blood.

We analyzed the impact of age, sex, and CMV on blood monocyte and dendritic cell (DC) subpopulations in 256 healthy individuals aged from 19 to 96 years. Flow cytometry was performed on whole blood within the 4 h following blood drawing. Myeloid (mDC) and plasmacytoid DC (pDC), classical, intermediate, and nonclassical monocytes were enumerated by means of TruCount tubes (BD Biosciences). We provided reference values for mDC, pDC and the three monocyte subpopulations. The numbers of classical, intermediate, and nonclassical monocytes slightly increased with age while the numbers of mDC and pDC did not vary significantly. The level of expression of CD64 and CD163 on monocytes significantly increased with age while HLA-DR expression did not vary significantly. More precisely, CD163 expression level on intermediate monocyte slightly increased with age in women only (Spearman P = 0.019) while CD64 expression increased on monocytes in CMV-positive individuals only. We observed that sex had almost no impact on the numbers of monocytes and DC and on their expression level of CD64 and HLA-DR. We observed a significant decrease in the numbers of pDC with age in CMV-positive individuals, but not in CMV negative individuals. This suggests that the lifelong subclinical infection by CMV could influence the number of circulating DC of lymphoid origin. In contrast, CMV serostatus had no significant impact on absolute numbers of mDC and monocytes.

Alternative complement pathway hemolytic assays reveal incomplete complement blockade in patients treated with eculizumab.

Eculizumab is a monoclonal anti-C5 antibody used in the treatment of atypical hemolytic uremic syndrome (aHUS). We monitored complement inhibition in 16 eculizumab-treated patients suffering from HUS or transplant rejection (not aHUS patients). Blood samples were obtained one to four weeks after the last eculizumab injection. We observed that eculizumab efficiently blocked the terminal pathway (TP) through classical pathway (CP) activation measured by kinetic hemolytic assay (HA) (<10%) but incompletely blocked the TP through alternative pathway (AP) activation measured by rabbit (APH50>23%) or chicken erythrocytes HA (AP100>15%). Conversely, functional ELISA revealed a complete blockade of TP through AP activation in all patients (<10%). C5a and sC5b9 levels were not correlated with residual APH50 or AP100. Similar results were obtained after in vitro addition of increasing amounts of eculizumab to a control serum (in vitro APH50>60% and AP100>20%). We also showed that ELISA was less sensitive than HA.

Impact of transplant accessibility for sensitized patients by avoiding unacceptable antigens.

Recent data have confirmed the negative impact of preformed donor-specific antibodies (pDSAs) after liver transplantation (LT). In order to reduce the risk of developing lesions associated with acute and chronic antibody-mediated rejection in LT recipients, we evaluated the consequences in terms of transplant accessibility, associated with avoiding pDSAs according to several mean fluorescence intensity (MFI) titer thresholds that have been previously reported to be relevant in LT. Among the 484 included LT candidates, 99 (20.5%) presented with anti-human leukocyte antibodies (HLAs). The predictive factors for anti-HLA sensitization were a history of previous kidney transplantation (odds ratio [OR], 1.5; 95% confidence interval [CI], 1.30-1.9; P = 0.05), a history of previous LT (OR, 1.9; 95% CI, 1.6-2.1; P = 0.01), a history of blood transfusion (OR, 2.5; 95% CI, 2.2-4.1; P = 0.01), and a history of pregnancy (OR, 2.9; 95% CI, 2.4-3.3; P = 0.04). By applying a strategy of unacceptable mismatches for recipients with an antibody (Ab) MFI of > 5000, only 35 patients were affected (7% of the cohort), but 22 of these (63%) would have been considered incompatible with >50% of the donors. Using a MFI threshold of >10,000, only 16 patients were affected (1.4% of the cohort), but half of these would have been considered incompatible with >50% of the proposed donors. Considering only those with anti-class II Ab and a MFI >5000 and >10,000, respectively, 10/14 and 4/8 patients were considered incompatible with >50% of the donors. In conclusion, avoiding pDSAs affects a small but not negligible proportion of LT candidates. However, in these sensitive patients, avoiding pDSAs has the potential to significantly reduce the donor pool and, consequently, transplant accessibility. Liver Transplantation 23 880-886 2017 AASLD.

Histological long-term outcomes from acute antibody-mediated rejection following ABO-compatible liver transplantation.

BACKGROUND AND AIM: Acute antibody-mediated rejection (aAMR) is an unusual complication after orthotopic ABO-compatible liver transplantation. To date, the clinical and histological long-term outcomes after aAMR are not well known. METHOD: Herein, we describe nine cases of aAMR that occurred in our liver-transplant center between 2008 and 2016, with an initial and reevaluation liver biopsy available for reexamination. RESULTS: Two patients presented with aAMR at 10.5 (10, 11) days post-transplantation, caused by preformed donor-specific antibodies. Seven other recipients developed de novo donor-specific antibodies and aAMR at 11.2 (3-24) months post-transplantation. Eight of the nine patients received a B-cell targeting agent (rituximab, with or without plasma exchange), associated with polyclonal antibodies (three patients) or intravenous immunoglobulins (three patients). At the last follow up (i.e. 21 [4-90] months post-aAMR), seven patients were alive, including two patients with normal liver tests. Grafts' survival was 66%. A liver biopsy performed at 11.5 (5-48.5) months after the first biopsy showed no significant improvement in aAMR score (from 2 ± 1.3 to 1.6 ± 1.5, P = 0.6), a significant improvement in chronic AMR score (from 37 ± 9 to 25 ± 8, P = 0.003) and an increase in the Metavir score (1.2 ± 0.6 to 2.1 ± 0.9, P = 0.03). CONCLUSION: In this study, a B-cell-depleting agent seemed to improve the prognosis of aAMR in selected cases, but several patients kept active lesions antibody-mediated rejection.

Pilot conversion trial from mycophenolic acid to everolimus in ABO-incompatible kidney-transplant recipients with BK viruria and/or viremia.

Immunosuppression using everolimus (EVR) plus low-dose tacrolimus (Tac) is commonly used in organ transplantation. EVR has potential antiviral effects. Herein, the long-term outcomes and impacts of Tac-EVR on the BK virus are reported in ABO-incompatible kidney-transplant recipients. The initial immunosuppressive regimen combined steroids, Tac, and mycophenolic acid (MPA). At a median of 141 (34-529) days post-transplantation, seven stable ABO-incompatible kidney-transplant recipients were converted from MPA to EVR because of active BK replication, and compared with a reference group of fourteen ABO-incompatible patients receiving classical Tac plus MPA. At 1 month before conversion, at 1, 3 months after, and at last follow-up, clinical and biological parameters were monitored. The median time from conversion to the last follow-up was 784 (398-866) days. Conversion to EVR caused no change to rejection episodes or immunological status (isoagglutinin titers, anti-HLA antibodies). At last follow-up, median eGFR was similar in the Tac-MPA versus Tac-EVR group (40 [range: 14-56] vs. 54.5 ml/min/1.73 m(2) [range: 0-128], P = 0.07). The major adverse event was dyslipidemia. Interestingly, conversion from MPA to EVR decreased BK viral load in five patients. ABO-incompatible kidney-transplant recipients with an active BK virus infection may benefit from conversion to EVR.

ADAM17-mediated shedding of FcγRIIIA on human NK cells: identification of the cleavage site and relationship with activation.

FcγRIIIA/CD16A, the low-affinity receptor for the IgG Fc portion expressed on human CD56(dim) NK cells and involved in Ab-dependent cell cytotoxicity, is shed upon NK cell activation. We found that recombinant a disintegrin and metalloprotease (ADAM) 17 cleaved the ectodomain of FcγRIIIA/CD16A and a peptide for which the sequence encompasses aa 191-201 of the FcγRIIIA/CD16A stalk region but not ADAM10. MALDI-TOF analysis revealed that the peptide was cleaved between Ala(195) and Val(196) (i.e., 1 aa upstream of the expected position). This location of the cleavage site was confirmed by the finding that ADAM17 failed to cleave a peptide in which Ala and Val were reversed. ADAM17 was found to be expressed on NK cells, and stimulation with PMA or N-ethyl-maleimide resulted in the shedding of FcγRIIIA/CD16A and CD62L, a specific substrate of ADAM17. Selective inhibition of ADAM17 prevented the shedding of both molecules. Moreover, the shedding of FcγRIIIA/CD16A was strongly correlated with degranulation when a wide range of CD56(dim) NK cell activating receptors were stimulated, whereas both ADAM17-dependent shedding and internalization were involved in FcγRIIIA/CD16A downmodulation when the latter was engaged. Finally, the shedding of FcγRIIIA/CD16A was restricted to activated cells, suggesting that ADAM17 acts mainly, if not exclusively, in cis. Taken together, our results demonstrated for the first time, to our knowledge, at the molecular level that ADAM17 cleaves the stalk region of FcγRIIIA/CD16A and identified its cleavage site. The shedding of FcγRIIIA/CD16A was at least partially ADAM17 dependent, and it may be considered as a marker of FcγRIIIA/CD16A-independent NK cell activation highly correlated with degranulation.

De novo donor-specific anti-HLA antibodies mediated rejection in liver-transplant patients.

The incidence and consequences of de novo donor-specific anti-HLA antibodies (DSAs) after liver transplantation (LT) are not well known. We investigated the incidence, risk factors, and complications associated with de novo DSAs in this setting. A total of 152 de novo liver-transplant patients, without preformed anti-HLA DSAs, were tested for anti-HLA antibodies, with single-antigen bead technology, before, at transplantation, at 1, 3, 6 and 12 months after transplantation, and thereafter annually and at each time they presented with increased liver-enzyme levels until the last follow-up, that is, 34 (1.5-77) months. Twenty-one patients (14%) developed de novo DSAs. Of these, five patients had C1q-binding DSAs (24%). Younger age, low exposure to calcineurin inhibitors, and noncompliance were predictive factors for de novo DSA formation. Nine of the 21 patients (43%) with de novo DSAs experienced an acute antibody-mediated rejection (AMR). Positive C4d staining was more frequently observed in liver biopsies of patients with AMR (9/9 vs. 1/12, P < 0.0001). Eight patients received a B-cell targeting therapy, and one patient received polyclonal antibodies. Only one patient required retransplantation. Patient- and graft-survival rates did not differ between patients with and without DSAs. In conclusion, liver-transplant patients with liver abnormalities should be screened for DSAs and AMR.

Deleterious impact of feto-maternal MHC compatibility on the success of pregnancy in a macaque model.

The impact of feto-maternal histocompatibility on reproduction has inspired long-lasting debates. However, after the review of numerous articles, the impact of HLA allele sharing within couples on fecundity remains questionable. We decided to explore the impact of major histocompatibility complex (MHC) feto-maternal compatibility on reproduction in a cynomolgus macaque facility composed of animals of Mauritian descent. The Mauritian-derived macaque population presents a very restricted MHC polymorphism (only seven founding haplotypes) due to a strong founding bottleneck effect. The MHC polymorphism was investigated in 237 trios (male, female and offspring) using 17 microsatellite markers distributed across the MHC. Haplotypes were confirmed by segregation analysis. We evaluated the relative frequencies of MHC-compatible and MHC-semi-compatible offspring with the mothers. Among the 237 trios, we selected 42 trios for which the identity of the father is certain and for which the theoretical probabilities of fully compatible and semi-compatible offspring were equal. We found 11 offspring fully compatible and 31 offspring semi-compatible with their respective mother. The observed proportions were clearly outside the interval of confidence of 99 % and therefore most probably resulted from a selection of the semi-compatible offspring during pregnancy. We concluded that MHC fully compatible cynomolgus macaque offspring have a selective survival disadvantage in comparison with offspring inheriting a paternal MHC haplotype differing from maternal haplotypes.

Pregnancy after kidney transplantation: outcome and anti-human leucocyte antigen alloimmunization risk.

BACKGROUND: Kidney transplantation increases the chances for pregnancy and live birth for women with end-stage kidney disease. The aims of this study were to describe the outcomes of pregnancies in women with a kidney transplant and to evaluate the impact on anti-human leucocyte antigen (HLA) alloimmunization. METHODS: We analysed 61 pregnancies that occurred in 46 patients after having excluded 10 miscarriages during the first trimester and 10 other pregnancies from which important data were missing. Anti-HLA antibodies were screened using the Luminex assay. RESULTS: Overall, the live birth rate was 83% (94% after exclusion of miscarriages during the first trimester). Pre-eclampsia and gestational diabetes occurred in 26 and 21% of cases, respectively. The use of tacrolimus was an independent predictive factor for gestational diabetes. Twenty-four newborns (42%) were premature (<37 weeks). The median birth weight was 2720 (1040-3730) g. Nine newborns (15%) had low birth weights (<2.5 kg). At least one severe complication occurred in 56% of pregnancies. A high glomerular-filtration rate (GFR) before pregnancy was the sole independent protective factor that avoided a severe complication. Death-censored kidney-allograft survival was 80.4% at 6 years. De novo donor-specific anti-HLA antibodies were detected after only 5.9% of pregnancies: for two women, the father had the same HLA antigens as those from the deceased organ donor. The determination of the HLA of the father before pregnancy can better inform the woman about the possible impact of pregnancy on her kidney-allograft function. CONCLUSIONS: Despite many complications, the outcomes for pregnancy and kidney allografts are good. The risk of anti-HLA alloimmunization was low.

Identification of the novel HLA-DQB1*03:512 allele by next-generation sequencing.

HLA-DQB1*03:512 differs from DQB1*03:02:01 by one nucleotide substitution in codon 89 in exon 2.

Identification of the novel HLA-DRB1*14:253 allele by next-generation sequencing.

HLA-DRB1*14:253 differs from DRB1*14:54:01 by one nucleotide substitution in codon 233 in exon 5.

Identification of the novel HLA-DQA1*02:32 allele by next-generation sequencing.

HLA-DQA1*02:32 differs from DQA1*02:01:01 by one nucleotide substitution in codon 210 in exon 4.