Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain.

One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The V(H) domain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg(484)-Ile(508), a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The V(H) domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp(712)-Ala(736) in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp(712)-Ala(736) for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr(718719723) for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor. (Blood. 2000;96:540-545)

Longitudinal analysis of factor VIII inhibitors in a previously untreated mild haemophilia A patient with an Arg593-->Cys substitution.

Recent studies suggest that certain missense mutations associated with mild to moderate haemophilia A predispose to inhibitor development. In this study, we present a longitudinal analysis of the epitope specificity of an inhibitor that developed in a mild haemophiliac with an Arg593-->Cys mutation. Immunoprecipitation studies revealed the presence of antibodies directed towards the light chain and A2 domain of factor VIII. Limited reactivity was observed with metabolically labelled C2 domain. Almost complete inhibitor neutralization was achieved upon addition of A2 domain. Binding of the inhibitor was not affected by the presence of the Arg593-->Cys substitution in the recombinant A2 fragment. Evaluation of the epitope specificity of anti-factor VIII antibodies in plasma samples obtained at different time-points of inhibitor development revealed initial development of a low titre inhibitor directed towards the A2 domain and factor VIII light chain. A second period of factor VIII replacement therapy resulted in a dramatic rise in factor VIII inhibitor titre, which maintained their original epitope specificity. Based on the results of this and previous studies (Fijnvandraat et al., 1997; Thompson et al., 1997) it is argued that inhibitor development in patients with the Arg593-->Cys mutation may proceed via a similar mechanism.

Sulfated glycans on oral mucin as receptors for Helicobacter pylori.

Helicobacter pylori is able to colonize gastric epithelia, causing chronic active gastritis, gastric and duodenal ulcers and presumably gastric malignancies. Attempts to identify the natural reservoir for this microorganism other than the stomach have been unsuccessful. It is suspected that H. pylori can be transmitted orally, since the microorganism has been detected at various sites of the oral cavity. The aim of the present study was to determine whether H. pylori can bind to salivary mucins, which in vivo coat the oral epithelia, and characterize further the interaction. Binding of salivary mucins and of synthetic oligosaccharides was studied in ELISA and immunoblotting, using specific mono- and polyclonal antibodies, and synthetic neoglycoconjugates. H. pylori bound most avidly to a highly sulfated subpopulation of high molecular weight salivary mucins, secreted from the palatine salivary glands, and with less avidity to mucin species secreted by the sublingual and submandibular salivary glands, which are less sulfated. Binding was strongly enhanced upon decreasing pH from 6.0 to 5.0. Using synthetic polyacrylamide coupled oligosaccharides it was found that SO3-3-Gal and the SO3-3-Lewis(a) blood group antigen bound to H. pylori. In contrast, binding of sialylated Lewis(a) and Lewis(b) antigens was much weaker. This study indicates that sulfated oligosaccharides on salivary mucins may provide receptor structures for adhesion of H. pylori to oral surfaces.

Diamine oxidase activity in amniotic fluid for diagnosis of ruptured membranes.

Diamine oxidase in vaginal effluent is used as a parameter for ascertaining the state of fetal membranes. A new method using tritiated putrescine as a substrate is described for the determination of diamine oxidase in amniotic fluid and vaginal effluents. A number of tests are used for the diagnosis of premature rupture of fetal membranes. The described procedure for diamine oxidase activity determination can be used in general hospitals and has advantages over other parameters such as pH, glucose or fructose concentration, and the amniotic fluid crystallization test.

The diagnostic value of neuron-specific enolase and carcino-embryonic antigen analyses in patients with carcinoma of the lung.

Neuron-specific enolase and carcino-embryonic antigen were quantified simultaneously in sera of 135 patients attending the Department of Respiratory Diseases for diagnostic bronchoscopy. Fifteen small cell lung carcinomas, 24 non-small cell lung carcinomas and 96 benign pulmonary diseases were investigated. Lung biopsies or bronchial washings were obtained from about 75% of the patients, including all patients with neoplastic diseases. Serum neuron-specific enolase was measured by a recently introduced enzyme-immuno assay (WaKo NS-Enolase EIA-II testkit). The results obtained with this kit were similar to those based on RIA assays. Receiver Operating Characteristic curves (ROC curves) were constructed for comparison of the discriminating ability of neuron-specific enolase and carcino-embryonic antigen in small cell lung carcinomas and non-small cell lung carcinomas. For small cell lung carcinomas the sensitivity and the specificity of neuron-specific enolase (cutoff value: 10 micrograms/l) were 87% and 88%, respectively, and for carcino-embryonic antigen values 60% and 77% were obtained. There was no correlation between neuron-specific enolase and carcino-embryonic antigen in small cell lung carcinoma patients. The diagnostic value of neuron-specific enolase and carcino-embryonic antigen in non-small cell lung carcinomas is illustrated by sensitivities of 13% and 58%, respectively. An extensive literature survey is included to allow comparison with other studies. The use of ROC curves is recommended for the determination of optimal cutoff values for the assays employed.

Screening urine specimen populations for normality using different dipsticks: evaluation of parameters influencing sensitivity and specificity.

The reagent test strip Combur-9 Test-RL (Boehringer Mannheim) and the 8-SG Multistix (Ames) were simultaneously evaluated as a rapid method for screening urines for normality. Differences between the two methods are for a considerable part determined by adjustment of the lowest detection limits of the leukocyte and erythrocyte dipstick fields. Patient populations (243 specimens presented to the routine laboratory and 230 specimens submitted for microbiological culture), sediment analysis (routine or standardized) and composition of the screening protocol strongly influence values obtained for the sensitivity, specificity and predictive values, whereas use of a different dipstick is of minor importance on the final results. Higher sensitivity and specificity are observed when relating positive dipstick screening to positive culture than when relating positive standardized sediment to positive culture. Evaluation of dipstick method, using microscopic sediment analysis as a reference parameter appears to be very dependent on the quality of the latter, which is therefore relatively unsuitable for this purpose. Apart from standardization, additional clinically significant findings are obtained using dipstick screening.

Differentiation of functional adrenoceptors in human and guinea pig airways.

The nature of the beta-adrenoceptor population(s) mediating direct smooth muscle relaxation, inhibition of antigen-induced histamine release and inhibition of antigen-induced (leukotriene-mediated) smooth muscle contraction of human and guinea pig central and peripheral airways was investigated. Preferential blockade by beta 1- and beta 2-selective antagonists of the relaxation induced by beta 1- and beta 2-selective agonists, respectively, revealed the guinea pig tracheal smooth muscle relaxation to be mediated by both beta 1- and beta 2-adrenoceptors. Using a highly beta 2-selective antagonist, the NE-induced relaxation was split up biphasically into a beta 1- and a beta 2-component. In contrast, no such differential blockade was observed with the relaxation of the guinea pig lung parenchyma strip, neither with the human tracheal, main bronchus and respiratory bronchiolus smooth muscle, which are all mediated by homogeneous beta 2-adrenoceptor populations. Only in the guinea pig trachea did neuronal and extraneuronal uptake inhibitors produce pronounced left shifts of the NE- and ISO-induced relaxation curves, respectively, suggesting a causal relationship between noradrenergic innervation and the presence of the beta 1-adrenoceptor subpopulation in the airways. Using the same techniques, it was established that inhibition of antigen-induced histamine release from guinea pig lung and tracheal mast cells is mediated by homogeneous beta 2-adrenoceptor populations as well. In contrast to catecholamines, non-catecholamine beta-agonists such as fenoterol, clenbuterol and zinterol had a substantially higher apparent affinity for the inhibition of the anaphylactic (leukotriene-mediated) guinea pig tracheal contraction than for the inhibition of histamine release; the same was true for lung tissue, though the difference was less pronounced. With some non-catecholamine beta-agonists considerable selectivity both in central and peripheral airway preparations was observed for the inhibition of anaphylactic contraction as compared with smooth muscle relaxation.

Comparison of functional beta-adrenoceptor heterogeneity in central and peripheral airway smooth muscle of guinea pig and man.

The nature of the beta-adrenoceptor population(s) mediating the relaxation of guinea pig and human airway smooth muscle was investigated. On the basis of a preferential blockade by beta 1- and beta 2-selective antagonists of the relaxation induced by beta 1- and beta 2-selective agonists, guinea pig tracheal strip relaxation was found to be mediated both by beta 1- and beta 2-adrenoceptors, the relative participation of which depending on the relative affinities of the agonist towards these two receptors. With highly selective antagonists the noradrenaline (NA)-induced relaxation could be split up biphasically into a beta 1- and a beta 2-component. In contrast, no such differential blockade was observed with the guinea pig lung parenchyma strip relaxation which is mediated by a homogenous beta 2-adrenoceptor population. On comparison of the tracheal, the spirally cut main bronchus- and intrapulmonary airway smooth muscle strips it could be shown that both the sensitivity of NA for neuronal uptake and the apparent affinity of the relaxation by NA decreased in the direction of the lung periphery. Using the same techniques it was ascertained that the relaxation of human tracheal smooth muscle (autopsy, obtained within 6 hours after death), main bronchus and intrapulmonary smooth muscle (operation) are mediated by homogenous beta 2-adrenoceptor populations. In addition, neuronal and extraneuronal uptake sites were not operative in these preparations, whether obtained from operation or from autopsy.