Improving Step by Step

Throughout the past years we stepwise modified our immunosuppressive treatment regimen for patients with antibody-mediated rejection (ABMR). Here, we describe three consecutive groups treated with different regimens. From 2005 until 2008, we treated all patients with biopsy-proven ABMR with rituximab (500 mg), low-dose (30 g) intravenous immunoglobulins (IVIG), and plasmapheresis (PPH, 6x) (group RLP, ). Between 2009 and June 2010, patients received bortezomib (1.3 mg/m2, 4x) together with low-dose IVIG and PPH (group BLP, ). In July 2010, we increased the IVIG dose and treated all subsequent patients with bortezomib, high-dose IVIG (1.5 g/kg), and PPH (group BHP, ). Graft survival at three years after treatment was 73% in group BHP as compared to 45% in group BLP and 25% in group RLP. At six months after treatment median serum creatinine was 2.1 mg/dL, 2.9 mg/dL, and 4.2 mg/dL in groups BHP, BLP, and RLP, respectively (). Following treatment, a significant decrease of donor-specific HLA antibody (DSA) mean fluorescence intensity from to () was observed in group BHP, but not in the other groups. Our results indicate that graft survival, graft function, and DSA levels could be improved along with stepwise modifications to our treatment regimen, that is, the introduction of bortezomib and high-dose IVIG treatment

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related V(L) chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS

an In Vitro Study

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP- BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration

Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1β, TNF-α, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology

Fine Mapping of Posttranslational Modifications of the Linker Histone H1 from Drosophila melanogaster

The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous

Failure of bedside ABO testing is still the most common cause of incorrect blood transfusion in the Barcode era

Background and objectives. ABO-incompatible red blood cell (RBC) transfusions are a major risk in transfusion medicine. Identification of factors leading to this hazard is important to improve transfusion safety. Material and methods. All consecutive erroneous ABO-incompatible transfusions occurring from January 1997 to December 2004 at the Charité University Hospital in Berlin, Germany were analysed. Results. A total of 343,432 RBC units were transfused, and eight patients erroneously received 13 ABO-incompatible RBC concentrates. The most frequent error was incorrect bedside testing (n = 7). Intensive care treatment was required in two cases, but there were no fatal mistransfusions. Four patients had no or only mild reactions. Conclusion. Mistransfusions are still a considerable risk in transfusion medicine despite quality control systems and electronic data processing. An increase in transfusion safety may require the introduction of further systems, e.g. radio-frequency identification (RFID) tags

Coexistence of autoantibodies and alloantibodies to red blood cells due to blood transfusion

BACKGROUND: Autoantibodies (AUTO) to red blood cells (RBCs) are frequently associated with alloantibodies (ALLO). The mechanism for the coexistence of these antibodies is obscure. STUDY DESIGN AND METHODS: Between August 1998 and June 2006, all in- and outpatients of the Charité University Hospital, Berlin, with detectable AUTO were included in this study. Serologic examination was performed with standard techniques for the detection of RBC antibodies. RESULTS: A total of 717 patients were found to have AUTO, with ALLO observed in 200 of these patients (28%). The history of antibody production could be evaluated in 98 of the 200 patients. Both AUTO and ALLO were due to RBC transfusion in 73 cases (75%) and peripheral blood progenitor cell transplantation in 6 cases (6%). Nine (9%) patients were primarily alloimmunized and subsequently developed AUTO. The remaining 10 (10%) patients were primarily autoimmunized and later developed ALLO. Only 6 of these patients had chronic idiopathic autoimmune hemolytic anemia. CONCLUSION: The majority of AUTO associated with ALLO appears to be due to RBC transfusion that must be recognized as a major cause for autoimmunization