The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

Effect of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein (rBPI23) on cerebrospinal fluid inflammation induced by endotoxin

Endotoxin triggers the subarachnoid inflammation of gram-negative meningitis. This study examined the ability of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein (rBPI23) to block endotoxin-induced meningitis in rabbits. Intracisternal (ic) injection of 10-20 ng of meningococcal endotoxin induced high cerebrospinal fluid (CSF) concentrations of tumor necrosis factor (TNF) and CSF pleocytosis and increased CSF lactate concentrations. ic administration of rBPI23 significantly reduced meningococcal endotoxin-induced TNF release into CSF (P < .005), lactate concentrations (P < .001), and CSF white blood cell counts (P < .01). No such effect was observed in animals receiving intravenous rBPI23. Concentrations of rBPI23 in CSF were high after ic administration but low or undetectable after systemic administration. Thus, high concentrations of rBPI23 can effectively neutralize meningococcal endotoxin in CSF, but low CSF concentrations after systemic administration currently limit its potential usefulness as adjunctive drug treatment in gram-negative meningitis.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Detection of muscle wasting in patients with chronic heart failure using C-terminal agrin fragment: results from the Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF).

AIMS Skeletal muscle wasting affects 20% of patients with chronic heart failure and has serious implications for their activities of daily living. Assessment of muscle wasting is technically challenging. C-terminal agrin-fragment (CAF), a breakdown product of the synaptically located protein agrin, has shown early promise as biomarker of muscle wasting. We sought to investigate the diagnostic properties of CAF in muscle wasting among patients with heart failure. METHODS AND RESULTS We assessed serum CAF levels in 196 patients who participated in the Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF). Muscle wasting was identified using dual-energy X-ray absorptiometry (DEXA) in 38 patients (19.4%). Patients with muscle wasting demonstrated higher CAF values than those without (125.1 ± 59.5 pmol/L vs. 103.8 ± 42.9 pmol/L, P = 0.01). Using receiver operating characteristics (ROC), we calculated the optimal CAF value to identify patients with muscle wasting as >87.5 pmol/L, which had a sensitivity of 78.9% and a specificity of 43.7%. The area under the ROC curve was 0.63 (95% confidence interval 0.56-0.70). Using simple regression, we found that serum CAF was associated with handgrip (R = - 0.17, P = 0.03) and quadriceps strength (R = - 0.31, P < 0.0001), peak oxygen consumption (R = - 0.5, P < 0.0001), 6-min walk distance (R = - 0.32, P < 0.0001), and gait speed (R = - 0.2, P = 0.001), as well as with parameters of kidney and liver function, iron metabolism and storage. CONCLUSION CAF shows good sensitivity for the detection of skeletal muscle wasting in patients with heart failure. Its assessment may be useful to identify patients who should undergo additional testing, such as detailed body composition analysis. As no other biomarker is currently available, further investigation is warranted.

Periacetabular osteotomy through the pararectus approach: technical feasibility and control of fragment mobility by a validated surgical navigation system in a cadaver experiment

PURPOSE The pararectus approach has been validated for managing acetabular fractures. We hypothesised it might be an alternative approach for performing periacetabular osteotomy (PAO). METHODS Using four cadaver specimens, we randomly performed PAO through either the pararectus or a modified Smith-Petersen (SP) approach. We assessed technical feasibility and safety. Furthermore, we controlled fragment mobility using a surgical navigation system and compared mobility between approaches. The navigation system's accuracy was tested by cross-examination with validated preoperative planning software. RESULTS The pararectus approach is technically feasible, allowing for adequate exposure, safe osteotomies and excellent control of structures at risk. Fragment mobility is equal to that achieved through the SP approach. Validation of these measurements yielded a mean difference of less <1 mm without statistical significance. CONCLUSION Experimental data suggests the pararectus approach might be an alternative approach for performing PAO. Clinical validation is necessary to confirm these promising preliminary results.