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BackgroundPre-transplant donor specific antibodies (DSA), directed at non-self human leukocyte antigen (HLA) protein variants present in the donor organ, have been associated with worse outcomes in kidney transplantation. The impact of the mean fluorescence intensity (MFI) and the target HLA antigen of the detected DSA has, however, not been conclusively studied in a large cohort with a complete virtual cross-match (vXM).MethodsWe investigated the effect of pre-transplant DSA on the risk of antibody-mediated rejection (ABMR), graft loss, and the rate of eGFR decline in 411 DSA positive transplants and 1804 DSA negative controls.ResultsPre-transplant DSA were associated with a significantly increased risk of ABMR, graft loss, and accelerated eGFR decline. DSA directed at Class I and Class II HLA antigens were strongly associated with increased risk of ABMR, but only DSA directed at Class II associated with graft loss. DSA MFI markedly affected outcome, and Class II DSA were associated with ABMR already at 500-1000 MFI, whereas Class I DSA did not affect outcome at similar low MFI values. Furthermore, isolated DSA against HLA-DP carried comparable risks for ABMR, accelerated eGFR decline, and graft loss as DSA against HLA-DR.ConclusionOur results have important implications for the construction and optimization of vXM algorithms used within organ allocation systems. Our data suggest that both the HLA antigen target of the detected DSA as well as the cumulative MFI should be considered and that different MFI cut-offs could be considered for Class I and Class II directed DSA.

Thrombotic thrombocytopenic purpura (TTP) either occurs in a congenital form caused by ADAMTS13 gene mutations or it is acquired and most often due to ADAMTS13 inhibitory autoantibodies. In congenital TTP siblings are often affected, while acquired TTP occurs sporadically and familial clustering has not been described so far. We report identical twin sisters suffering from acquired TTP due to immunoglobulin G (IgG) autoantibodies inactivating ADAMTS13, suggesting an important role of hitherto unidentified genetic determinants of ADAMTS13 inhibitor formation. These cases also demonstrate that familial clustering is not sufficient for unambiguously diagnosing hereditary ADAMTS13 deficiency and congenital TTP. (Blood. 2004;103:4195-4197)

Human interferon-inducible protein 10 (IP-10), a member of the family of the small secreted proteins called intercrine cytokines or chemokines, is secreted by interferon gamma-stimulated T cells, monocytes, endothelial cells, and keratinocytes. We have begun to explore the biological properties of IP-10 by cloning and overexpression in baculovirus and in bacterial protein expression systems. A 9.9-kD protein was secreted by infected insect cells, which on sodium dodecyl sulfate-polyacrilamide gel electrophoresis comigrated with keratinocyte IP-10 and with f(22-98), a bacterial recombinant fragment lacking the signal sequence but containing all other residues of IP-10. All three reacted with antibodies recognizing residues 10-98 (alpha IP-10) and 77-98 of IP-10 (alpha 22), demonstrating that it is secreted by keratinocytes and insect cells after removal of the signal sequence but without proteolysis of the COOH-terminal end. Purified rIP-10 suppresses in vitro colony formation by early human bone marrow progenitor cells which need r-steel factor (rSLF) and rGM-CSF or rSLF and r-erythropoeitin (rEPO). The inhibition is dose dependent, is complete at concentrations > or = 50 ng/ml, is prevented by preincubation of rIP-10 with alpha IP-10, but not by alpha 22, and is seen with highly purified CD34+ cells, suggesting direct effect of rIP-10 on the progenitors. Combination of rIP-10 and other chemokines at inactive concentrations inhibited colony formation in a synergistic manner. rIP-10 did not affect colony formation in the absence of any growth factors or in the presence of rEPO or rGM-CSF but in absence of rSLF. The effects of IP-10 may be relevant to normal marrow function and might be harnessed to protect human hematopoietic progenitors from the cytotoxic effects of chemotherapy.

We examined the production of PAF and LTB4 by PMN in response to NAP-1 IL-8 alone, or after preincubation with GM-CSF, which has been shown to enhance PMN responsiveness and to prime PMN for production of those bioactive lipids. NAP-1 IL-8 does not induce the synthesis of PAF and LTB4 from endogenous phospholipid precursors, even after preincubation with GM-CSF. In addition and again in contrast to fMLP and C5a, NAP-1 IL-8 fails to induce an enhanced oxidative burst in GM-CSF primed PMN. Exogenously added PAF or LTB4 can mimic the priming effect of GM-CSF for an enhanced oxidative burst in response to all examined chemotactic peptides including NAP-1 IL-8 . Our data reveal a possible autocrine role of PAF and LTB4 in the enhanced responsiveness of GM-CSF primed PMN towards fMLP or C5a, but not NAP-1 IL-8 .

Phospholipases and protein kinases are critical for the intracellular transmission and amplification of signals induced by extracellular ligands. Chemotactic activation of phagocytes through G protein coupled receptors leads to inflammatory responses of the immune cells. Downstream of G proteins, phospholipases generate precursors for eicosanoid synthesis and are involved in the functional responses. Recently, the molecular characterization of specific enzymes of the signalling cascades has gained much attention in research.

GM-CSF regulates the growth of hemopoietic progenitor cells, enhances the responsiveness of mature PMN and primes these cells for synthesis of leukotrienes and PAF in response to secondary stimuli. The biochemical requirements for PAF production in GM-CSF primed PMN was examined using different metabolic inhibitors. GM-CSF stimulates uridine incorporation into RNA and inhibitors for RNA and protein synthesis decrease PAF synthesis in our model. This suggests a role for gene expression and de novo synthesis of proteins in the action of GM-CSF. Different PLA2 inhibitors, including a 9 amino-acid peptide derived from a conserved region of hte calpactin superfamily, decrease PAF production, indicating that in GM-CSF primed PMN the chemotactic peptide fMLP triggers lipid mediator synthesis by activating PLA2.

Publisher Summary The chapter presents a study on release and metabolism of leukotriene A 4 (LTA 4 ) in neutrophil-mast cell interactions. The chapter reviews the findings that LTA 4 formed by Ca 2+ ionophore-stimulated neutrophils (PMN) does not appreciably decay within the cell, but is released into the extracellular medium as the intact epoxide in relatively large amounts. Low concentrations of albumin-bound LTA 4 are efficiently and rapidly taken up by mast cells and metabolized into LTC 4 . More recent studies showed that neutrophil-derived LTA 4 can be metabolized into biologically active leukotrienes even by cells (like erythrocytes, platelets, and endothelial cells), which are lacking the 5-1ipoxygenase (5-LOX) and thus are unable to form LTA 4 . Therefore, interactions between various cell types that release or utilize LTA 4 may provide an important metabolic pathway for the production of leukotrienes. In neutrophils LTA 4 is formed in excess of what is enzymatically hydrolyzed to LTB 4 . This is clearly indicated by the fact that whenever LTB 4 is formed in vitro , the all-trans stereoisomers of LTB 4 are also present.