Assessment of validity and reliability of Drug Hypersensitivity Quality of Life Questionnaire: The Dutch experience.

SUMMARY: Drug Hypersensitivity Quality of Life Questionnaire (DrHy-Q) is the first questionnaire that captures health related quality of life impact in patients with drug hypersensitivity. The aim of this study was to translate and validate the original Italian 15-item DrHy-Q for use among Dutch-speaking residents. We also compared the DrHy-Q scores obtained across countries. In a prospective cohort study, the Dutch DrHy-Q was completed by 124 patients (65.3% female, age 56.8 ± 14.0) with a confirmed drug hypersensitivity. Median DrHy-Q score was 12 [0-88]. Validity and reliability of the DrHy-Q was confirmed through, 1, confirmatory factor analysis; 2, concurrent validity with a generic health related quality of life questionnaire (RAND-36); 3, internal consistency; and 4, test-retest reliability. A country specific difference in scores was observed.

Simian immunodeficiency virus lentivector corrects human X-linked chronic granulomatous disease in the NOD/SCID mouse xenograft.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the phagocyte nicotinamide dinucleotide phosphate oxidase catalytic subunit gp91(phox). Gene therapy targeting hematopoietic stem cells (HSCs) can correct CGD, but permanent correction remains a challenge. Lentiviral vectors have become attractive tools for gene transfer, and they may have the potential to transduce very primitive HSCs. We used a self-inactivating RD114/TR-pseudotyped simian immunodeficiency virus (SIVmac)-based vector encoding human gp91(phox) for ex vivo transduction of peripheral blood-mobilized stem cells (PBSCs) from patients with X-CGD. In PBSCs from two patients, ex vivo transduction efficiencies of 40.5 and 46% were achieved, and correction of oxidase activity was observed in myeloid cells differentiating in culture. When transduced PBSCs from these patients were transplanted into nonobese diabetic/severe combined immunodeficient mice and compared to normal control, 10.5 and 7.3% of the human myeloid cells in bone marrow developing at 6 weeks from the human xenografts expressed the gp91(phox) transgene. Sustained functional correction of oxidase activity was documented in myeloid cells differentiated from engrafted transduced PBSCs. Transgene marking was polyclonal as assessed by vector integration site analysis. These data suggest that RD114/TR SIVmac-based vectors might be suitable for gene therapy of CGD and other hereditary hematologic diseases.

Effects of elastic property of the wall on flow characteristics through arterial stenoses.

Hemodynamic characteristics of blood flow through arterial stenoses are numerically investigated in this work. The blood is assumed as a Newtonian fluid and the pulsatile nature of flow is modeled by using measured values of the flowrate and pressure for the canine femoral artery. An isotropic elastic and incompressible material is assumed for the wall at each axial section, but a non-uniform distribution of the shear modulus in axial direction is used to model the high stiffness of the wall at the stenosis location. Full Navier equations for a thick wall are used as the governing equations for the wall displacements. A continuous grid extending over the flow field and the wall is considered and governing equations are transformed for use in the computational domain. Discretized forms of the transformed wall and flow equations, which are coupled through the boundary conditions at their interface, are obtained by control volume method and simultaneously solved using the well-known SIMPLER algorithm. To study the effects of wall deformability, solutions are obtained for both rigid and elastic walls. The results indicate that deformability of the wall causes an increase in the time average of pressure drop, but a decrease in the maximum wall shear stress. Displacement and stress distributions in the wall are presented.

Recognition of alpha 2-macroglobulin by the low density lipoprotein receptor-related protein requires the cooperation of two ligand binding cluster regions.

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds several ligands including the activated form of the pan-proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M*) and amyloid precursor protein, two ligands genetically linked to Alzheimer's disease. To delineate the contribution of LRP to this disease, it will be necessary to identify the sites on this receptor which are responsible for recognizing these and other ligands to assist in the development of specific inhibitors. Structurally, LRP contains four clusters of cysteine-rich repeats, yet studies thus far suggest that only two of these clusters (clusters II and IV) bind ligands. Identifying binding sites within LRP for certain ligands, such as alpha(2)M*, has proven to be difficult. To accomplish this, we mapped the binding site on LRP for two inhibitors of alpha(2)M* uptake, monoclonal antibody 8G1 and an amino-terminal fragment of receptor-associated protein (RAP D1D2). Surprisingly, the inhibitors recognized different clusters of ligand binding repeats: 8G1 bound to repeats within cluster I, whereas the RAP fragment bound to repeats within cluster II. A recombinant LRP mini-receptor containing the repeats from cluster I along with three ligand binding repeats from cluster II was effective in mediating the internalization of (125)I-labeled alpha(2)M*. Together, these studies indicate that ligand binding repeats from both cluster I and II cooperate to generate a high affinity binding site for alpha(2)M*, and they suggest a strategy for developing specific inhibitors to block alpha(2)M* binding to LRP by identifying molecules capable of binding repeats in cluster I.

Comparison of physiological and simple pulsatile flows through stenosed arteries.

Most experimental and numerical studies of pulsatile flow through stenosed arteries have been performed for a first harmonic oscillatory flow. In this paper, numerical solutions are presented for a physiological pulsatile flow as well as for an equivalent simple pulsatile flow, having the same stroke volume as the physiological flow, and the differences in their flow behavior are discussed. The analysis is restricted to laminar flow, Newtonian fluid and axisymmetric rigid stenosis. Comparison of results shows that the behaviors of the two flows are similar at some instances of time, however, important observed differences indicate that for thorough understanding of pulsatile flow behavior in stenosed arteries, the actual physiological flow should be simulated.

Pathogenicity of an enterotoxigenic Escherichia coli hemolysin (hlyA) mutant in gnotobiotic piglets.

Pigs infected with hemolytic F4(+) strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4(+) enterotoxigenic E. coli to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.

Prelytic and lytic conformations of erythrocyte-associated Escherichia coli hemolysin.

Flow cytometry was developed as a method to assess the conformation of erythrocyte-bound Escherichia coli hemolysin polypeptide (HlyA). Topology of membrane-associated hemolysin (HlyA(E)) was investigated by testing surface accessibility of HlyA regions in lytic and nonlytic bound states, using a panel of 12 anti-HlyA monoclonal antibodies (MAbs). Hemolysin associates nonlytically with erythrocytes at 0 to 2 degrees C. To test the hypothesis that the nonlytic HlyA(E) conformation at 0 to 2 degrees C differs from the lytic conformation at 23 degrees C, MAb epitope reactivity profiles at the two temperatures were compared by flow cytometry. Four MAbs have distinctly increased reactivity at 0 to 2 degrees C compared to 23 degrees C. HlyA requires HlyC-dependent acylation at lysine residues 563 and 689 for lytic function. Toxin with cysteine substitution mutations at each lysine (HlyA(K563C) and HlyA(K689C)) as well as the nonacylated form of hemolysin made in a HlyC-deficient strain were examined by flow cytometry at 0 to 2 and 23 degrees C. The three mutants bind erythrocytes at wild-type toxin levels, but there are conformational changes reflected by altered MAb epitope accessibility for six of the MAbs. To test further the surface accessibility of regions in the vicinity of MAb-reactive epitopes, HlyA(E) was proteolytically treated prior to testing for MAb reactivity. Differences in protease susceptibility at 0 to 2 degrees and 23 degrees C for the reactivities of three of the MAbs further support the model of two distinct conformations of cell-associated toxin.

Battling against host phagocytes: the wherefore of the RTX family of toxins?

The RTX family of bacterial exotoxins is a group of related cytolytic proteins produced by a wide variety of gram-negative human and animal pathogens. While diverse in their associated diseases and in their target cell specificities, there remain several themes common to RTX toxins, including genetic organization, structural and functional features, and effects on target cells. In this review, we summarize and discuss the genetics, regulation, epidemiology, structure/function relationships, and in vivo and in vitro activities of the best characterized RTX toxins, and speculate on their roles in pathogenesis and their use in immunotherapy.

Effects of temperature, time, and toxin concentration on lesion formation by the Escherichia coli hemolysin.

We performed osmotic protection experiments to test the hypothesis that the Escherichia coli hemolysin forms a discrete-size pore in erythrocyte membranes. The effects of toxin concentration, assay time, temperature, and protectant concentrations were examined. The results we present here raise doubts about the existing model of pore formation by hemolysin. We demonstrate that osmotic protection by various sugars of different sizes is a function of hemolysin concentration and assay time. The data indicate that under various conditions, lesion sizes with a diameter ranging from < 0.6 to > 1.2 nm can be inferred. Quantification of hemolysin permitted the estimation of the number of HlyA structural protein molecules required per erythrocyte for lysis in the presence of each protectant. It appears that hemolysin induces heterogeneous erythrocyte lesions which increase in size over time. Influx experiments utilizing radioactive sugar markers indicated that time-dependent osmotic protection patterns are independent of the diffusion rates of individual protectants. We demonstrate that the rate of the putative growth in the size of hemolysin-mediated lesions is temperature dependent. The erythrocyte membrane lesions formed at 37 degrees C can be stabilized in size when shifted to 4 degrees C. On the basis of these data, new models for the nature of the hemolysin-mediated erythrocyte membrane lesions are presented.