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BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease leading to erosive joint destruction. Although the exact pathogenesis is still elusive, the strong association of certain HLA class II molecules, such as HLA-DRB1*0401 (HLA-DR4), suggest involvement of CD4+ T cells (1,2). Mouse models of RA mimic specific aspects of the disease but are limited by the differences between human and murine immune systems.ObjectivesWe aimed to establish a humanized mouse model (humice) carrying DR4+ RA PBMCs to study its role in the pathogenesis of RA without putting patients at risk.MethodsPeripheral blood mononuclear cells (PBMC) of HLA-DR4 positive RA patients or controls were isolated and injected into NSG-Ab0 DR4 mice (NOD-scid IL2Rgammanull mice lacking MHC class II while expressing the human HLA-DR4) to create humice. Human immune cell composition within humice was profiled using flow cytometry. Development of RA was monitored by examination of the joints and micro computed tomography analysis. Joints were analysed by histology regarding pannus formation, bone erosions, cartilage damage, and human cell infiltration.ResultsTransfer of RA PBMCs induced arthritis in humice recapitulating hallmarks of RA including immune cell infiltration, pannus formation, increased osteoclastogenesis, cartilage damage, and bone erosions. Arthritis was dependent on the implanted human cells as NSG-Ab0 DR4 mice without transfer of human PBMCs did not develop arthritis. T-helper 1 (Th1) cells, dominated the human immune cell composition in humice, while regulatory T cells (Tregs) were diminished compared to donor PBMC composition. Mice humanized with cells from RA patients were more likely to develop inflammatory joint disease, compared to healthy HLA-DR4 positive controls (RA donor 70% vs. healthy control 20%, p=0.00196). CTLA-4 Ig treatment prevented arthritis development in this model (p=0.0055).ConclusionHumice carrying DR4+ RA PBMCs developed an RA-like erosive joint disease driven by the implanted human immune system. The data implies that the disease can be transferred by arthritogenic cells found in the peripheral blood of RA patients. This model will allow new insights into the pathogenesis of RA.References[1]Goulielmos GN, Zervou MI, Myrthianou E, Burska A, Niewold TB, Ponchel F. Genetic data: The new challenge of personalized medicine, insights for rheumatoid arthritis patients. Gene Available from: http://www.ncbi.nlm.nih.gov/pubmed/26869316[2]Holoshitz J. The rheumatoid arthritis HLA–DRB1 shared epitope. Curr Opin Rheumatol Available from: http://www.ncbi.nlm.nih.gov/pubmed/20061955Disclosure of InterestsNone declared.

Placental villous explant culture has been increasingly recognized as suitable model to study secretion of inflammatory and immune modulating factors by human placenta. Most of these factors likely derive from the syncytiotrophoblast, whereas extraplacental sources such as maternal peripheral blood cells are rarely considered. Due to their small size and absence of a nucleus, platelets adhering to perivillous fibrinoid of normal placenta are frequently ignored in routine immunohistochemistry. Here we demonstrate adhering maternal platelets on first trimester placental villi after explant culture and point out that platelet-derived factors must be considered when analysing the inflammatory secretion profile of human placenta.

Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune bleeding disorder, characterized by isolated thrombocytopenia because of destruction of auto-antibody-coated platelets by Fc-receptor-mediated phagocytosis. The destruction of autoantibody-sensitized platelets by FcγR-bearing phagocytic cells and the following antigen presentation are considered to play a key role for the pathophysiology of AITP. Although different isotypes of AITP-mediating autoantibodies, e.g. IgG, IgM and IgA, are frequently found in AITP patients, their role in the pathophysiology of AITP remains unclear. Using a flow cytometric monocyte-based phagocytosis assay, we investigated the impact of disease-associated autoantibody isotype in antibody-mediated phagocytosis of platelets. Platelets, labelled with 5-chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients’ serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incubated with monocytes. Phagocytosis was defined as the product of percentage of CMFDA-positive monocytes and mean fluorescence intensity of CMFDA. Adherence of platelets to monocytes was quantified by anti-CD61-PerCp in a CMFDA+ CD14+ gate. IgG-coated platelets showed a significantly higher phagocytic index than IgM-coated platelets (mean 796 ± 157 versus 539 ± 78, P

Summary Deficiencies in early complement components are associated with the development of systemic lupus erythematosus (SLE) and therefore early complement components have been proposed to influence B lymphocyte activation and tolerance induction. A defect in apoptosis is a potential mechanism for breaking of peripheral B cell tolerance, and we hypothesized that the lack of the early complement component C4 could initiate autoimmunity through a defect in peripheral B lymphocyte apoptosis. Previous studies have shown that injection of a high dose of soluble antigen, during an established primary immune response, induces massive apoptotic death in germinal centre B cells. Here, we tested if the antigen-induced apoptosis within germinal centres is influenced by early complement components by comparing complement C4-deficient mice with C57BL/6 wild-type mice. We demonstrate that after the application of a high dose of soluble antigen in wild-type mice, antibody levels declined temporarily but were restored almost completely after a week. However, after antigen-induced apoptosis, B cell memory was severely limited. Interestingly, no difference was observed between wild-type and complement C4-deficient animals in the number of apoptotic cells, restoration of antibody levels and memory response.

Transfusion-associated graft-versus-host disease (TA-GvHD) is an infrequent, but fatal, complication associated with transfusion of any cellular blood component. At present, gamma-irradiation of cellular blood components is the only acceptable method for preventing TA-GvHD. All blood components can be subjected to gamma-irradiation, which irreversibly inactivates leukocytes, especially T lymphocytes, while preserving the functional integrity of the pharmaceutically effective cellular blood components. Pathogen inactivation technologies have been developed to eliminate the minimal transfusion-associated risks caused by viral or bacterial contaminants. The INTERCEPT™ Blood System for platelets is based on the use of amotosalen HCl and UVA-irradiation. It sufficiently inhibits the replication of parasitic, bacterial, and viral genomes and inactivates T cell replication and cytokine generation on at least an equivalent level to gamma-irradiation. The INTERCEPT Blood System for platelets shows great robustness in inactivating viable T lymphocytes with more than a 5 log10 reduction in platelet concentrates, and it may have the potential to replace gamma-irradiation of platelet concentrates.

SummaryImmune thrombocytopenia is due to platelet destruction by circulating glycoprotein-specific antibodies and is found in various disorders. Methods for the detection of platelet-associated IgG (PAIgG) are generally sensitive but unspecific, whereas glycoprotein-specific assays are highly specific but less sensitive. Usefully, a sensitive screening method for PAIgG detection would also provide information for differential diagnosis. We developed a quantitative direct Platelet Immunofluorescence Test (PIFT) by flow cytometry and studied 79 thrombocytopenic patients with immune thrombocytopenia and other disorders. The sensitivity of the assay was 94%, its specificity 66% for the detection of a clinically obvious immune thrombocytopenia. PAIgG levels of patients with immune thrombocytopenia differed significantly from those of other patients with low platelet counts (p

In the last few years, recommendations, guidelines, and laws have all served as important motivators, not just for improving the quality of medical products such as blood components but also for carefully and comprehensively guaranteeing the safety of blood donors. The German Guidelines for the Collection of Blood and Blood Components from 2000 [1] allow 26 thrombocytaphereses per year without the need for a threepart white blood cell (WBC) differentiation, whereas the apheresis frequency is limited to 24 procedures in the yet to be updated FDA guidelines from 1988 [2] which point out the magnitude of potential risks, especially lymphocyte depletion. Nowadays, this old-fashioned recommendation can only be understood by bearing in mind that the first generation of cell separators generated platelet concentrates with WBC contaminations in the order of 109 cells per concentrate [3]. Over the last three decades, the question of immune deficiency arising from frequent blood donations has been raised repeatedly. The paper of Carrero et al. [4] published in this issue also deals with the theme of analyzing the cellular immune status amongst thrombocytapheresis donors. These results and many other published contributions provide clear and definitive evidence that a slight loss of leukocytes, especially lymphocytes, does not impair the donor’s immune system in its

Reactivation of latent Epstein-Barr virus (EBV) infection is considered to exert substantial immunomodulating activities. Little is known about EBV's modulating activities on cytokine production upon primary, in contrast to reactivated, infection. Therefore, we investigated the cytokine production of latently infected EBV-positive (EBV-pos) adults upon in vitro EBV stimulation compared to nonimmune age-matched EBV-negative (EBV-neg) donors. Production of interleukin (IL)-1beta and IL-6 was strongly decreased in peripheral blood mononuclear cell (PBMC) cultures of EBV-pos adults; in contrast, IL-10 and IL-1 receptor antagonist exhibited significantly higher levels. As expected, interferon (IFN)-gamma production was almost exclusively observed in EBV-pos donors; it was accompanied by a significantly higher IL-12 synthesis. Experiments employing T cell-depleted PBMC showed similar cytokine levels between EBV-pos and EBV-neg individuals suggesting that reactivation of EBV-specific memory T cells was responsible for the divergent cytokine profiles. Production of viral IL-10 was excluded as a reason for higher IL-10 levels in EBV-pos individuals. In conclusion, our results do not appear to relate to any primary immunological differences between EBV-neg and EBV-pos adults, but show that the EBV-specific memory response to latent EBV infection is characterized by some anti-inflammatory effects. This might be of relevance upon reactivation of latent EBV infection in vivo and provides further evidence that EBV infection acts in an immunomodulating fashion.

Background : White cell reduction of blood products minimizes the risks of alloimmunization against HLA-antigens, the transmission of viral diseases and the incidence of platelet transfusion reactions. One modern strategy is leukocyte depletion with an integrated filter system immediately after preparation and prior to storage. Study design and methods : We evaluated the efficiency of a novel in-line filter system Sepacell ® PLX-5 BPS for leukocyte reduction of platelet concentrates (PC) from pooled buffy-coats. A total of 44 PCs were investigated with regard to different filtration flow rates (25–110 ml/min) and leukocyte depletion and thrombocyte recovery rates were analysed. Furthermore, we studied the influence of filtration on PCs over a storage period of 6 days ( n =12) by investigation of pH, lactate and glucose. Platelet function was determined by means of hypotonic shock response, external shape change and expression of CD62p. Results : The mean leukocyte depletion rate was >log 5. After filtration the mean leukocyte count was 0.12 ± 0.21 × 10 6 . In 60% of the PCs the leukocyte count lay below the detection level of the Nageotte chamber, which is 5 . The flow rate correlates significantly with the leukocyte count in the PCs ( r =0.325; p =0.033) and therefore with the leukocyte depletion rate ( r =−0.422; p =0.01). Flow rates under 40 ml lead to a significantly lower leukocyte contamination. Only in one PC, at a flow rate of 84 ml/min, was the leukocyte threshold of 1 × 10 6 exceeded. We did not find a significant correlation between filtration flow rate and thrombocyte recovery ( r =0.315; p =0.069). The mean platelet count in the PC was 2.88 ± 0.47 × 10 11 . Compared with the thrombocyte count in the pooled buffy coat, the recovery was 68.6%. We observed a decrease of pH, glucose, external shape change and hypotonic shock response over the storage period while lactate and the expression of CD62p increased. Conclusion : The filter system Sepacell ® PLX-5 BPS proved to be suitable for in-line filtration of platelet concentrates prior to storage. Filtration flow rates of up to 40 ml/min allowed efficient leukocyte depletion without significant loss in the quality of the platelet concentrates and the platelet function in vitro.

t~tiology of inflammatory bowel disease is still unknown. There are several hints speaking for an immunological background. We wanted to investigated this Thl / Th2 hypothesis for the respective types of colitis in IBD patients.We therefore investigated T-cell cytokines in peripheral blood mononuclear cells (PBMC) and lamina propria mononuclaer ceils (LPMC) from 6 Crohn' disease (CD), 6 ulcerative colitis(UC) patients and 10 healthy controls. PBMC were isolated using standard procedures, then stimulated for five hours with PMA and ionomycin. The ceils were fixed in 4% paraformaldehyd, then lysed in saponin-buffer. Anti-cytokine antibodies and antibodies to surface markers were added at the same time. Cells were finally measured in the FACS. Cytokines of interest were interferon (IFN)r, interleukin (IL) 2 and tumor necrosis factor (TNF) a as Thl cytokines and IL4, IL5 and IL 10 as Th2 cytokines. For staining of surface markers we used CD3 (T-cells), CD8 (cytotoxic T-cells, CD8neg helper T-cells), CD45RA and CD45R0 for naive and memory cells, respectively. As to surface markers, ratio of CD8pos/CD8neg T-cells was not different between PBMC and LPMC. PBMC mainly revealed the naive phenotype (CD45RApos) whereas these cells were greatly decreased in LPMC which mainly displayed the memory phenotype (CD45R0pos). CD45RA pos cells were significantly increased in UC mucosa when compared to controls and CD patients. All measured cytokines were produced to greater extends in memory cells than in naive cells. IL2 was the main cytokine produced in PBMC and reduced in LPMC. It was produced primarily in CD8 neg cells. IL2 production was significantly reduced in CD45RApos and CD8 pos cells in UC mucosa. IFNr was the main cytokine produced in mucosal cells and percentages of positive cells were always higher than in PBMC, it was produced to similar amounts in CDSpos and CD8neg cells. IN LPMC, CD patients produced highest amounts of IFNr, controls intermediate amounts and UC showed weakest production. Th2 cytokines were produced by very few cells, we did not find any differences between LPMC and PBMC nor between disease groups. IL4 was produced to some higher extents than IL5 and IL 10. In our investigations we could find interesting differences between mucosal and blood derived lymphocytes. As to comparison of disease groups, CD patients showed highest amounts of IFNr production and reduction in IL4 which supports the Thl hypothesis.