Full‐length human von Willebrand factor (vWF) cDNA was assembled from partial, overlapping vWF cDNAs. This cDNA construct includes a coding sequence of 8439 nucleotides which encode a single‐chain precursor of 2813 amino‐acid residues, representing a putative signal peptide, a prosequence and mature vWF of 22, 741 and 2050 amino acids, respectively. This represents the longest coding sequence determined to date. In‐vitro expression of full‐length vWF cDNA revealed the synthesis of a polypeptide with a mol. wt corresponding with that of the unglycosylated precursor. The precursor is a highly repetitive protein which consists of two duplicated (B, C), a triplicated (A), a quadruplicated (D) and a partly duplicated domain (D'), in the following order: H‐D1‐D2‐D'‐D3‐A1‐A2‐A3‐D4‐B1‐B2‐C1‐C2‐OH. Both the prosequence, composed of two D domains (D1, D2), and mature vWF harbor an arg‐gly‐asp (‘R‐G‐D’) sequence which has been implicated in cell‐attachment functions. It is argued that the pro‐sequence is equivalent to von Willebrand Antigen II (vW AgII).

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The humoral immune response against adalimumab is highly restricted and limited to the idiotype of the therapeutic antibody. All antibodies result in functional neutralisation of the drug, thereby providing a mechanism by which AAA formation leads to clinical non-response.

Abstract. In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]; during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF) . In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-lß-or ITNFa-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by -60% (P < 0.005) without interfering with the process of adherence . The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP) . A murine T RANSMIGRATION of neutrophils from the peripheral circulation into inflammatory sites requires neutrophil adherence to the endothelial cell lining of the blood vessels. The CD18 complex proteins are: lymphocyte function-associated antigen-1 (LFA-1),' complement receptor 3 (CR3) or Mac-1, and glycoprotein 150,95 (p150,95) . On neutrophils they are involved in both migration and adherence (Anderson et al ., 1985 ;Harlan, 1985 ;Kuijpers and Roos, 1989). In these processes, endothelial intercellular adhesion molecule-1 (ICAM-1) may function as a cellular ligand for LFA-1 and CR3 (Rotplein et al., 1986 ;Diamond et al ., 1990;Springer, 1990) . Its expression can be increased by the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) Springer, 1990) .Several other adhesion molecules can play an accessory role in the process of neutrophil adherence . The role of a 1. Abbreviations used in this paper: CR3, complement receptor 3 ; EC, endothelial cells ; ELAM-1, endothelial leukocyte adhesion molecule-l ; FMLP, formyl-leucyl-methionyl-phenylalanine ; HSA, human serum albumin ; ICAM-1, intercellular adhesion molecule-1 ; IL-1, interleukin-1 ; LAM-1, leukocyte adhesion molecule-1 ; LFA-1, lymphocyte-function associated antigen-1; MFI, mean fluorescence intensity ; PAF, platelet-activating factor, p150,95, (glyco)protein 150,95 ; rIL-1p, recombinant interleukin-lß ; rIL-8, recombinant interleukin-8 ; rTNFct, recombinant tumor necrosis fkctor-a ; TNF, tumor necrosis factor.

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BackgroundIn a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is however unknown to which extent ADAs are neutralising.ObjectivesTo study which proportion of antibodies to human(ised) anti-TNF (adalimumab, golimumab, certolizumab) as well as chimeric anti-TNF (infliximab) is neutralising.MethodsNeutralising capacity of ADAs was assessed using a TNF competition assay in ADA-positive sera of patients treated with adalimumab (n=21), golimumab (n=4), certolizumab (n=9) or infliximab (n=34) sent in to our diagnostic department.ResultsIn 34 sera with ADAs to adalimumab, golimumab or certolizumab, >97% of the antibodies were neutralising. In 34 sera with ADAs to infliximab >90% of the antibodies were neutralising. Further characterisation of the broader antibody response to infliximab revealed that non-neutralising antibodies to infliximab do not target murine domains, but may bind infliximab-unique domains not involved in TNF binding (located outside the paratope).ConclusionsOur study shows that ADAs to human(ised) as well as chimeric anti-TNF therapeutic antibodies are largely neutralising. This highly restricted ADA response suggests an immunodominant role for the paratope of anti-TNF therapeutics.

Because of its neutrophil-activating properties, interleukin-8 (IL-8) may play an important role in the pathophysiology of sepsis. We measured circulating IL-8 levels in 47 patients with clinical sepsis. Levels on admission were elevated in 42 of the 47 patients (89%o) and were comparable in patients with gram-positive or gram-negative infections. Patients with shock had significantly higher IL-8 levels than normotensive patients (P = 0.0014, Wilcoxon-Mann-Whitney test), whereas no differences in IL-8 levels were found between patients with or without adult respiratory distress syndrome. Patients who died had higher IL-8 levels on admission than the patients who survived. The largest differences in IL-8 levels between survivors and nonsurvivors was found when only patients with positive cultures were considered (P = 0.0342). IL-8 levels appeared to correlate significantly with lactate levels and inversely with leukocyte and platelet numbers and mean arterial pressure. In addition, the IL-8 level in the sepsis patients was found to correlate significantly with levels of IL-6, elastase-al-antitrypsin, and C3a. Serial observations revealed that in most patients IL-8 levels decreased, irrespective of the outcome. Thus, our results demonstrate that IL-8 levels are increased in most patients with sepsis and correlate with some important clinical, biochemical, and inflammatory parameters. These findings suggest a role for IL-8 in the pathophysiology of sepsis.INFECT. IMMUN.

Von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized by vascular endothelial cells as a pre‐pro‐polypeptide with a highly repetitive domain structure, symbolized by the formula: (H)‐D1‐D2‐D′‐D3‐A1‐A2‐A3‐D4‐B1‐B2‐B3‐C1‐C2‐(OH) A heterologous expression system for the synthesis of recombinant vWF protein was developed, consisting of a monkey kidney cell line (COS‐1), transfected with full‐length vWF cDNA. This system was shown to mimic the constitutive secretory pathway of vWF in endothelial cells, since dimerization and multimerization occur similarly. To determine whether the pro‐polypeptide, composed of the domains D1 and D2, is involved in vWF multimerization, a vWF cDNA was constructed that lacked the coding sequence for the pro‐polypeptide. The mutant vWF protein, expressed by COS‐1 cells transfected with this cDNA, did not assemble beyond the dimer stage. From this observation, we conclude that (i) dimerization does not involve the pro‐polypeptide of pro‐vWF and (ii) the presence of the pro‐polypeptide, as part of pro‐vWF, is obligatory for multimerization. It is argued that the interactions, required for interchain binding, are mediated by the D domains.