Abdulgabar Salama

Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells

In recent years, human dendritic cells (DCs) could be subdivided into CD304+ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c+, CD16+, and CD141+ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141+ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca2+ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141+ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8+ T cells when directly compared with CD1c+ DCs, CD16+ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141+ DCs correspond to mouse CD8+ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141+ DCs as professional antigen cross-presenting DCs in the human.

Effect of eltrombopag on platelet counts and bleeding during treatment of chronic idiopathic thrombocytopenic purpura : a randomised, double-blind, placebo-controlled trial

BACKGROUND Eltrombopag is an oral, non-peptide, thrombopoietin-receptor agonist that stimulates thrombopoiesis, leading to increased platelet production. This study assessed the efficacy, safety, and tolerability of once daily eltrombopag 50 mg, and explored the efficacy of a dose increase to 75 mg. METHODS In this phase III, randomised, double-blind, placebo-controlled study, adults from 63 sites in 23 countries with chronic idiopathic thrombocytopenic purpura (ITP), platelet counts less than 30 000 per muL of blood, and one or more previous ITP treatment received standard care plus once-daily eltrombopag 50 mg (n=76) or placebo (n=38) for up to 6 weeks. Patients were randomly assigned in a 2:1 ratio of eltrombopag:placebo by a validated randomisation system. After 3 weeks, patients with platelet counts less than 50 000 per microL could increase study drug to 75 mg. The primary endpoint was the proportion of patients achieving platelet counts 50 000 per microL or more at day 43. All participants who received at least one dose of their allocated treatment were included in the analysis. This study is registered with ClinicalTrials.gov, number NCT00102739. FINDINGS 73 patients in the eltrombopag group and 37 in the placebo group were included in the efficacy population and were evaluable for day-43 analyses. 43 (59%) eltrombopag patients and six (16%) placebo patients responded (ie, achieved platelet counts >/=50 000 per microL; odds ratio [OR] 9.61 [95% CI 3.31-27.86]; p<0.0001). Response to eltrombopag compared with placebo was not affected by predefined study stratification variables (baseline platelet counts, concomitant ITP drugs, and splenectomy status) or by the number of previous ITP treatments. Of the 34 patients in the efficacy analysis who increased their dose of eltrombopag, ten (29%) responded. Platelet counts generally returned to baseline values within 2 weeks after the end of treatment. Patients receiving eltrombopag had less bleeding at any time during the study than did those receiving placebo (OR 0.49 [95% CI 0.26-0.89]; p=0.021). The frequency of grade 3-4 adverse events during treatment (eltrombopag, two [3%]; placebo, one [3%]) and adverse events leading to study discontinuation (eltrombopag, three [4%]; placebo, two [5%]), were similar in both groups. INTERPRETATION Eltrombopag is an effective treatment for managment of thrombocytopenia in chronic ITP.

Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection of renal allografts.

Background. Although the incidence of early acute rejection could have been diminished in the past, the long-term renal allograft survival could not benefit from the introduction of more effective immunosuppressive regimens mainly aiming at cellular rejection mechanisms. The cause of chronic rejection is still discussed controversially. Here, we demonstrate to what extent human leukocyte antigen (HLA) antibodies (HLAab) posttransplant contribute to late graft outcome. Methods. A total of 1014 deceased kidney transplant recipients transplanted at the Charité hospital were monitored in a cross-sectional manner for the development of HLAab using Luminex Single Antigen beads. Patients with stable kidney function at a median of 5-years posttransplant were tested once for HLAab and monitored for 5.5 years after testing. Results. Thirty percent of recipients showed HLAab. Donor-specific antibodies (DSA) were found in 31% of antibody positive patients. The presence of DSA was associated with a significantly lower graft survival of 49% vs. 83% in the HLAab negative group (P≤0.0001). Non-DSAs also had an adverse effect on graft survival (70% vs. 83%; P=0.0001). In a prospective analysis of 195 patients with repeatedly no detectable HLAab, the survival probability was 94% as opposed to 79% survival among patients who developed HLAab de novo after the first testing (P=0.05). Conclusions. We confirmed that HLAab produced even late after transplantation are detrimental to graft outcome. DSA were proven to have a strong adverse impact on graft survival. The results indicate that a posttransplant HLAab monitoring routine could be appropriate to improve long-term results.

Deposition of Terminal C5b–9 Complement Complexes on Erythrocytes and Leukocytes during Cardiopulmonary Bypass

Hemolysis, leukopenia, a hemostatic deficit, and nonspecific systemic reactions collectively known as the postperfusion syndrome develop in patients who undergo cardiopulmonary bypass. We now report that terminal C5b-9 complement complexes are deposited on erythrocytes and polymorphonuclear neutrophilic leukocytes during cardiopulmonary bypass. Plasma samples taken from 48 unselected patients during and at the end of cardiopulmonary bypass contained raised levels of fluid-phase SC5b-9 complement complexes, indicating that the complement sequence had been activated to completion. Various degrees of overt intravascular hemolysis were observed in all the patients, and lysed erythrocyte membranes were recovered from the blood samples. Immunoassays performed with use of antibodies to C5b-9 neoantigens demonstrated the presence of C5b-9 on red-cell ghosts but not on intact erythrocytes. The appearance of ghosts carrying C5b-9 always coincided with hemolysis. Furthermore, granulocytes isolated from 20 patients during bypass were all found to carry C5b-9 complexes, whereas cells isolated before or 24 hours after surgery carried no C5b-9. The neoantigen-positive material present in detergent extracts of granulocytes sedimented in a broad peak (25 to 40 sedimentation coefficient [S]) in sucrose-density gradients, exactly as did pore-forming C5b-9 complexes. Deposition of C5b-9 on blood cells during cardiopulmonary bypass may be partly responsible for the hemolysis and may augment granulocyte activation by the stimulation of arachidonate metabolism in those cells.

High-level serum B-cell activating factor and promoter polymorphisms in patients with idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which platelets are opsonised by autoantibodies and destroyed by macrophages. Therefore, ITP represents a prototype of a B‐cell‐mediated autoimmune disorder. B‐cell activating factor (BAFF) is a member of the tumour necrosis factor family and an important regulator of B‐cell development. BAFF levels were determined in serum samples from 53 patients with ITP. Serum BAFF levels in patients with an active ITP were increased when compared with the healthy control group (median 1620 pg/ml vs. 977 pg/ml; P < 0·001). Moreover, immunosuppressive treatment was associated with strongly suppressed BAFF levels (median 629 pg/ml; P < 0·01). In addition, a polymorphic site was detected in the BAFF promoter region (−871) that appeared to occur more frequently in ITP patients than in healthy persons. This promoter variant was associated with very high BAFF levels in ITP patients. Our data suggest that BAFF is an important pathogenetic factor in the development of ITP.

Rapid detection of heparin-induced platelet antibodies with particle gel immunoassay (ID-HPF4)

Early and acute diagnosis is needed to prevent thromboembolic complications in heparin-induced thrombocytopenia. We developed a particle agglutination assay that allows the detection of such antibodies within 20 min.

Detailed analysis of the variability of peptidylarginine deiminase type 4 in German patients with rheumatoid arthritis: a case–control study

Peptidylarginine deiminase type 4 (PADI4) genotypes were shown to influence susceptibility to rheumatoid arthritis (RA) in the Japanese population. Such an association could not previously be confirmed in different European populations. In the present study, we analysed exons 2–4 of PADI4 in 102 German RA patients and 102 healthy individuals to study the influence of PADI4 variability on RA susceptibility by means of haplotype-specific DNA sequencing. Analyses of the influence of PADI4 and HLA-DRB1 genotypes on disease activity and on levels of anti-cyclic citrullinated peptide antibodies were performed.Comparing the frequencies of PADI4 haplotype 4 (padi4_89*G, padi4_90*T, padi4_92*G, padi4_94*T, padi4_104*C, padi4_95*G, padi4_96*T) (patients, 14.7%; controls, 7.8%; odds ratio = 2.0, 95% confidence interval = 1.1–3.8) and carriers of this haplotype (patients, 27.5%; controls, 13.7%; odds ratio = 2.4, 95% confidence interval = 1.2–4.8), a significant positive association of PADI4 haplotype 4 with RA could be demonstrated. Other PADI4 haplotypes did not differ significantly between patients and controls. Regarding the individual PADI4 variants, padi4_89 (A→G), padi4_90 (C→T), and padi4_94 (C→T) were significantly associated with RA (patients, 49.5%; controls, 38.7%; odds ratio = 1.6, 95% confidence interval = 1.1–2.3). Considering novel PADI4 variants located in or near to exons 2, 3, and 4, no quantitative or qualitative differences between RA patients (8.8%) and healthy controls (10.8%) could be demonstrated. While the PADI4 genotype did not influence disease activity and the anti-cyclic citrullinated peptide antibody level, the presence of the HLA-DRB1 shared epitope was significantly associated with higher anti-cyclic citrullinated peptide antibody levels (P = 0.033).The results of this small case–control study support the hypothesis that variability of the PADI4 gene may influence susceptibility to RA in the German population. Quantitative or qualitative differences in previously undefined PADI4 variants between patients and controls could not be demonstrated.

Antibodies to high-frequency antigens may decrease the quality of transfusion support: an observational study

BACKGROUND:  There is only little information on the transfusion support of patients with antibodies to high‐frequency RBC antigens.

Drug-induced immune hemolytic anemia

Background: Drug-induced immune hemolytic anemia is frequently associated with serious complications. Objective: To identify which drugs are implicated in drug-induced immune hemolytic anemia, how they stimulate the production of antibodies and how they could be identified. Methods: Individual studies were selected from Pubmed and Scholar databases without any time restrictions placed on the studies. All English language reports including those from the cross-references (∼ 560) were carefully studied. The results of the most relevant clinical and serological studies in this field are summarized and discussed in this review. Results: Drugs may lead to the production of two types of antibodies. One type reacts with red blood cells (RBCs) only in the presence of the drug and/or its metabolites, and predominantly causes complement-mediated intravascular hemolysis. The second type reacts with RBCs, also in the absence of the drug, belongs to the IgG class, does not activate complement and causes Fc-mediated extravascular hemolysis. Some drugs may stimulate the production of both types of antibodies. Meanwhile, > 130 drugs have been reported to be the cause of immune hemolytic anemia (IHA) but the data are frequently incomplete or implausible. Conclusion: The number of drugs involved in inducing IHA is increasing. However, the vast majority of these drugs seem to cause IHA only in isolated cases. Based on the number of reports in the literature, new drugs that most frequently cause IHA are the new generation of cephalosporins, diclofenac, oxaliplatin and fludarabin.

Daily doses of 20 mg of elemental iron compensate for iron loss in regular blood donors: a randomized, double-blind, placebo-controlled study.

BACKGROUND:  A considerable number of regular blood donors develops an iron deficiency, and the exact amount of iron required to compensate for the iron loss from whole‐blood donation in males and females is still unknown.