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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.

HLA-DRB3*02:61Q , a novel HLA-DRB3 allele identified in a volunteer bone marrow donor.

Die Versorgungslage mit Blutkomponenten und Plasmaderivaten ist in Deutschland, Osterreich und der Schweiz als sehr gut zu bezeichnen. Viele gemeinsame Anstrengungen der Blutspendedienste, der pharmazeutischen Industrie und der nationalen Uberwachungsbehorden haben dazu beigetragen, dass aus menschlichem Blut hergestellte „Arzneimittel“ heute sehr sicher bezuglich der transfusionsassoziierten Ubertragung von Krankheitserregern sind. Allogene Bluttransfusionen bergen jedoch auch andere als infektiose Risiken. Aktuelle Hamovigilanzdaten aus dem deutschsprachigen Raum und europaischer Nachbarstaaten zeigen, dass allergische Transfusionsreaktionen, hamolytische Transfusionsreaktionen und kardiale Nebenwirkungen (z. B. transfusionsassoziierte Volumenuberladung) die haufigsten schwerwiegenden Nebenwirkungen sind. Diese freiwilligen Uberwachungssysteme vermogen jedoch nicht solche „Nebenwirkungen“ zu erfassen, die durch eine – wie auch immer geartete – Immunmodulation verursacht sind, und zu einer hoheren postoperativen Infektionsrate, hoherer Morbiditat und gegebenenfalls hoherer Mortalitat kausal beitragen. Dieser wichtigen Fragestellung widmen sich weltweit Initiativen zum „Patient Blood Management“. Hierbei stehen patientenzentrierte Behandlungskonzepte „am Krankenbett“ im Vordergrund. Die Vermeidung von allogenen Bluttransfusionen durch Therapie einer praoperativen Anamie, durch Minimierung des diagnostischen bzw. intraoperativen Blutverlusts und durch Ausschopfung einer groseren, postoperativen Anamietoleranz sind wichtige Ziele der Anasthesiologie und aller chirurgischen Disziplinen im perioperativen Umfeld. Die rational begrundete Indikation zur Transfusion allogener Blutkomponenten sollte nach Masgabe aktueller evidenzbasierter Daten in aller Regel tatsachlich restriktiv, jedoch unter sorgfaltiger Vermeidung hierdurch verursachter lebensbedrohlicher Zustande und auch unter Berucksichtigung des individuellen Patientenwillens erfolgen. Neben dem wissenschaftlichen Erkenntnisgewinn ist es sowohl der Transfusionsmedizin als auch Experten des „Patient Blood Management“ besonders wichtig, die krankenhausinterne Implementierung klinischer Qualitatssicherungssysteme zu fordern und hierfur geeignete Masnahmen zu konzeptionieren (Schulungen, Berichtswesen, Transfusions- und Gerinnungsalgorithmen, praoperative Anasthesiologie-Sprechstunde, Dokumentation der Transfusionsindikation, Soll-Ist-Abgleich mit den Querschnittsleitlinien, interne Auditierung). Eine durch Transparenz und Teamgeist getragene „Patient-Blood-Management“-Initiative ist der beste Garant fur den erwunschten Erfolg.

A*68:02:11 differs from A*68:02:01 by a silent mutation in codon 122.

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.

Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.

Optimal mobilization and harvest of hematopoietic progenitors are essential for peripheral blood stem cell transplantation after myeloablative high-dose chemotherapy. Conflicting data have been published concerning the most useful, cost-effective collection strategy which is also convenient for patients.A total of 66 leukaphereses in 20 patients were retrospectively evaluated. We assessed the predictive value of the number of white blood cells, mononuclear cells (MNCs) and CD34+ cells in peripheral blood for the yield of CD34+ cells in leukapheresis products. The concentrations of MNCs and CD34+ cells were quantified simultaneously by a flow cytometric procedure using fluorescent microparticles. Their collection efficiencies were calculated based on a newly developed formula.The collected hematopoietic progenitor concentration could be predicted only by the number of peripheral blood CD34+ cells prior to apheresis (r = 0.902; p0.01). Furthermore, the mobilization of at least 30 CD34+ cells/microl peripheral blood was a good predictor that a single leukapheresis would yield a minimum of 2.0x10(6) CD34+ cells/kg body weight. The collection efficiencies calculated by the new formula were 55.2+/-10.7% and 57.7+/-11.2% for MNCs and CD34+ cells, respectively.The precise quantification of MNCs and CD34+ cells by a direct flow cytometric assay, as well as the new formula to determine the collection efficiencies, has an impact on optimizing high-quality stem cell products.

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

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 0.01). There were no significant differences regarding platelet adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP-mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prognostic approaches in AITP.

As part of the bone marrow niche, cellular and acellular components like mesenchymal stem cells (MSCs) and extracellular matrix (ECM) proteins influence human haematopoiesis. To identify factors able to improve the in vitro generation of red blood cells (RBCs), we investigated the effect of these factors on proliferation and differentiation of human haematopoietic stem cells (HSCs) into erythroid cells.Granulocyte colony-stimulating factor-mobilized CD34(+) HSCs were cultured for 16 days using an in vitro erythropoiesis assay as described previously (by our group). The HSCs were co-cultured with MSCs in either direct or indirect contact and with different ECM proteins (fibronectin, laminin, collagen and a mixture of ECM proteins, called ECM gel).Co-culturing of HSCs with ECM gel improved cell viability, and the presence of laminin slightly increased the maturation into enucleated RBCs. HSC expansion could not be improved by addition of any of the ECM proteins investigated. In contrast, fibronectin inhibited erythroid formation. Co-culturing of HSCs with MSCs generally stimulated cell viability and HSC proliferation, however, in favour of the myeloid lineage. In summary, of all investigated factors, only laminin and ECM gel had a supportive effect on RBC development under the described in vitro culture conditions.

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

Background: Neovascularization plays a critical role during matrix based dermal regeneration, since blood vessels have to supply nutrients, oxygen and immunologically competent cells for the tissue in regeneration to ensure therapeutic success. Bone marrow derived stem cells have not yet been employed for dermal matrix bioactivation. In this work we combined the use of a bioartificial template for dermal regeneration with bone marrow derived cells to improve neodermal vascularization in vivo. Materials and Methods: Apheresis samples were obtained from G-CSF mobilized human donors. Total bone marrow derived cells (TBMDC) were isolated by ficoll gradients and early hematopoietic stem cells (CD133+) were isolated by the use of magnetic microbeads. Cells were characterized by FACS. After that, each 250,000 cells were seeded in a matrix (Integra™) for dermal regeneration of bilateral 15 mm diameter full skin defects of nu/nu mice (TBMDC in Group A, CD 133+ in Group B). Viability was analysed by MTT assay. Control matrices received cell medium only (Group C). Matrices were analysed after 2 weeks for total amount of angiogenesis. Each subgroup comprised 6 animals. Results: CD133+ cells were isolated and FACS results showed more than 95 % of puricity in the sample. In vitro, distribution of the cells was analyzed by confocal microscopy and interaction with the matrix was shown by SEM. MTT assay showed that both cell populations can survive, for at least two weeks in normal culture conditions. After 2 weeks of transplantation in a full skin defect model, microangiographical matrix analysis showed that both TPBMNC and CD133+ cells significantly improved vascularization levels compared to control matrices (P < 0.05). No statistical differences were found between both Groups A and B. Conclusions: In this work we show for the first time that direct engraftment of total human derived bone marrow mononuclear cells or early hematopoietic stem cells significantly improve neodermal vascularization in vivo. These results suggest that stem cell therapy could be used to develop new therapeutical approaches to improve vascularization during matrix based dermal regeneration.

To investigate the stability of a BHK-21 A cell line transfected with human EPO (huEPO) and the human α2,6-sialyltransferase ST6N (huST6N), the cell line was cultivated over a time period of 18 days in a 2.5 L perfused bioreactor in protein- and serum-free medium and under physiological stress after addition of ammonium. The stability of gene expression and the activity of the newly introduced huST6N and the endogenous α2,3-sialyltransferase (α2,3-ST) were analyzed during the different cultivation conditions by RT-PCR and in vitro assays. Furthermore, the purified secreted huEPO was analyzed by western blot and the liberated N-glycans were characterized by HPAEC-PAD analysis. The results confirmed the stability of the cell line with respect to the expression of the newly introduced human proteins even under physiological stress. The elevated ammonium concentration led to reversible changes on the EPO N-glycans.

Anatomic and functional results of macular holes using vitrectomy and platelet-concentrate (PC) or autologous whole blood (WB) were investigated without peeling any epiretinal membrane in order to minimize retinal trauma.44 patients with macular holes stage II, III and IV underwent a vitrectomy with removal of posterior cortical vitreous (in stage II and III) and C2F6 gas endotamponade. No peeling of any epiretinal membrane or the ILM was performed. In 14 patients autologous WB was injected and in 30 patients autologous PC. The mean follow-up time was 9.3 months.An anatomic closure was observed in 36.4% of patients in the WB-group and in 93.9% of patients in the PC-group. SLO-microperimetry showed a reduction of absolute scotomas in 80% of the WB- and in 75% of the PC-group. In the WB-group 1 retinal detachment and 1 branch vein occlusion occurred; in the PC-group 3 retinal detachments, 1 central artery occlusion, 1 macular pucker, 1 cystoid macular edema, 1 late reopening and 1 secondary glaucoma were observed. In both groups no endophthalmitis occurred.If autologous platelet concentrate is available, high anatomic success rates can be achieved without aggressive membrane removal, which may cause retinal damage. Autologous whole blood cannot be recommended as an alternative adjunct for the treatment of macular holes without membrane removal.

Inflammatory cytokines in platelet concentrates (PC) may cause side-effects such as febrile non-haemolytic transfusion reactions. The maximum white blood cell (WBC) content tolerable to avoid the accumulation of cytokines, and whether these cytokines originate from degranulating leucocytes or de novo synthesis during storage, had not been investigated prior to this study.We investigated the secretion of interleukin (IL)-1beta, IL-2, IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) and quantified the appropriate expression of corresponding mRNA in PC with regard to different levels of WBC contamination and storage times. In addition we tested the viability of WBCs during PC storage (by staining with 7-aminoactinomycin D) and their ability to perform de novo cytokine synthesis (by using superantigen stimulation).We detected a statistically significant increase of IL-1beta, IL-6, IL-8 and TNF-alpha in PC withor = 108 WBCs. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed increasing mRNA expression of the respective cytokines depending on the number of WBC present. On day 5 of storage, WBC viability was80% and the leucocytes were still able to produce cytokines de novo.These data show clear evidence for de novo synthesis of cytokines in PC. The cytokine pattern supports the hypothesis that activated monocytes are responsible for this cytokine synthesis. PC with a WBC contamination ofor = 108 contain inflammatory mediators in clinically relevant concentrations.

Immune 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 (p0.001). The quantitative PIFT proved to be a sensitive method for PAIgG detection and should therefore be used as a screening method. In addition, it could be helpful for differential diagnosis in marked thrombocytopenia where a MAIPA is not feasible.

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.

To determine the frequency of in vivo activated T(H)1 lymphocytes, T-cell subsets of 9 multiple sclerosis patients with active disease and 17 healthy controls were analyzed by immunostaining for CCR5, CD26, and their expression of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha. The numbers of CCR5+ interferon-gamma- and tumor necrosis factor-alpha-producing T cells were significantly increased in the peripheral blood of multiple sclerosis patients. CCR5 expression may be a useful marker to identify effector cells in multiple sclerosis and could be used as a tool for monitoring disease activity.

The analysis of many natural glycoproteins and their recombinant counterparts from mammalian hosts has revealed that the basic oligosaccharide structures and the site occupancy of glycosylated polypeptides are primarily dictated by the protein conformation. The equipment of many frequently used host cells (e.g. BHK-21 and CHO-cells) with glycosyltransferases, nucleotide-sugar synthases and transporters appears to be sufficient to guarantee complex-type glycosylation of recombinant proteins with a high degree of terminal alpha2-3 sialylation even under high expression conditions. Some human tissue-specific terminal carbohydrate motifs are not synthesized by these cells since they lack the proper sugar-transferring enzymes (e.g. alpha1-3/4 fucosyltransferases, alpha2-6 sialyltransferases). Glycosylation engineering of these hosts by stable transfection with genes encoding terminal human glycosyltransferases allows to obtain products with tailored (human tissue-specific) glycosylation in high yields. Using site-directed mutagenesis, unglycosylated polypeptides can be successfully converted in N- and/or O-glycoproteins by transferring glycosylation domains (consisting of 7-17 amino acids) from donor glycoproteins to different loop regions of acceptor proteins. The genetic engineering of glycoproteins and of host cell lines are considered to provide a versatile tool to obtain therapeutic glyco-products with novel/improved in-vivo properties, e.g. by introduction of specific tissue-targeting signals by a rational design of terminal glycosylation motifs.

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.