[Coronavirus infection and risk of postoperative venous thromboembolic events]. / Koronavirusnaya infektsiya i risk posleoperatsionnykh venoznykh tromboembolicheskikh sobytii u khirurgicheskikh bol'nykh.

OBJECTIVE: To determine whether patients with perioperative or previous coronavirus infection (CVI) have a greater risk of venous thromboembolic events (VTE). MATERIAL AND METHODS: A multiple-center regional prospective retrospective cohort study included elective and emergency patients who underwent surgery in November 2020. The primary endpoint was VTE (PE/DVT) within 30 days after surgery. CVI was stratified as perioperative (7 days before surgery - 30 days after surgery), recent (1-6 weeks before surgery) and remote (≥7 weeks before surgery) infection. There was no information about prevention or preoperative anticoagulation at baseline data collection. RESULTS: Incidence of postoperative VTE was 1.5% (10/650) in patients without CVI, 33.3% (3/9) in patients with perioperative CVI, 18.1% (2/11) in patients with recent CVI and 8.3% (1/12) in patients with remote CVI. After adjusting the confounders, patients with perioperative and recent CVI remained at a higher risk of VTE. In general, VTEs were independently associated with 30-day mortality. In patients with CVI, mortality rate among ones without VTE was 21.7% (5/23), with VTE - 44.4% (4/9). CONCLUSION: Patients with perioperative CVI have a higher risk of postoperative VTE compared to those without CVI and patients with previous CVI and no residual symptoms. Mortality in this group is also higher than in other cohorts.

Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.

The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb-restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10-fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response.

T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity.

The critical discriminatory event in the activation of T lymphocytes bearing alpha beta T cell receptors (TCRs) is their interaction with a molecular complex consisting of a peptide bound to a major histocompatibility complex (MHC)-encoded class I or class II molecule on the surface of an antigen-presenting cell. The kinetics of binding were measured of a purified TCR to molecular complexes of a purified soluble analog of the murine MHC class I molecule H-2Ld (sH-2Ld) and a synthetic octamer peptide p2CL in a direct, real-time assay based on surface plasmon resonance. The kinetic dissociation rate of the MHC-peptide complex from the TCR was rapid (2.6 x 10(-2) second-1, corresponding to a half-time for dissociation of approximately 27 seconds), and the kinetic association rate was 2.1 x 10(5) M-1 second-1. The equilibrium constant for dissociation was approximately 10(-7) M. These values indicate that TCRs must interact with a multivalent array of MHC-peptide complexes to trigger T cell signaling.

Studying interactions involving the T-cell antigen receptor by surface plasmon resonance.

T-lymphocyte activation is initiated by the interaction of the alpha beta TCR with a complex consisting of a class I or class II MHC-encoded molecule and an antigenic peptide, displayed on the surface of an antigen-presenting cell. Real-time binding measurements using surface plasmon resonance have revealed kinetic and equilibrium parameters for the interactions between purified MHC molecules and peptides, between TCR and MHC-peptide complexes, and between TRC and superantigens. The MHC-peptide interaction is characterized by its high affinity and long half-life, the TCR-MHC/peptide interaction by its low affinity and short half-life, and the TCR-superantigen interaction by its low-to-moderate affinity, which is dependent on the particular superantigen involved. The consistent finding is that both MHC-peptide complexes and superantigens interact with TCR with a low affinity attributable to rapid dissociation. That an MHC-peptide complex that encounters a single TCR only briefly can still deliver the necessary activation signals offers a mechanistic conundrum for which several solutions have been proposed.

Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector.

We describe a stable chelating linkage for the reversible immobilization of oligohistidine tagged proteins in the flow cell of the 'BIAcore' surface plasmon resonance (SPR) biosensor. The carboxymethylated dextran surface of the flow cell was covalently derivatized with N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA ligand) via its single primary amino group, and the derivatized surface charged with Ni2+. 6His-VP55, an N-terminally tagged derivative of the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was immobilized to this surface in a manner that was dependent upon the immobilized NTA ligand, the prior injection of Ni2+ at a concentration of > 10(-5) M and the 6His tag, and which was reversible upon injection of EDTA. The stability of immobilization varied inversely with the amount of 6His-VP55 immobilized and was greatest in buffer of pH 8.0 or greater, containing NaCl at a concentration of 0.1 M. Utilizing these conditions, 6His-VP55 remained stably immobilized during 60 min of buffer flow at moderate flow rates. VP39, the stimulatory subunit of vaccinia poly(A) polymerase, interacted with the immobilized 6His-VP55. Approximately 99% of immobilized 6His-VP55 molecules were available for VP39 binding, in contrast to the approximately 40% availability for 6His-VP55 molecules immobilized covalently, via primary amino groups. Three additional proteins, tagged at either the N- or C-terminus with oligohistidine, were shown to be stably immobilized via the chelating linkage. This simple method permits immobilization of proteins in the BIAcore biosensor via a commonly employed affinity tag, in a stable and reversible manner, and requires only a single biosensor flow cell for the iterative generation of immobilized protein surfaces.

Measuring interactions of MHC class I molecules using surface plasmon resonance.

To examine the molecular interactions between major histocompatibility complex (MHC)-encoded molecules and peptides, monoclonal antibodies (mAbs), or T cell receptors, we have developed model systems employing genetically engineered soluble MHC class I molecules (MHC-I), synthetic peptides, purified mAbs, and engineered solubilizable T cell receptors. Direct binding assays based on immobilization of one of the interacting components to the dextran modified gold biosensor surface of a surface plasmon resonance (SPR) detector have been developed for each of these systems. The peptide binding site of the MHC-I molecule can be sterically mapped by evaluation of a set of peptides immobilized through the thiol group of cysteine substitutions at each peptide position. Kinetic binding studies indicate that the MHC-I/peptide interaction is characterized by a low to moderate apparent kass (approximately 5000-60000 M-1 s-1) and very small kdis (approximately 10(-4)-10(-6) s-1) consistent with the biological requirement for a long cell surface residence time to permit engagement with T cell receptors. Several mAb directed against different MHC-I epitopes were examined, and kinetic parameters of their interaction with MHC molecules were determined. These showed characteristic moderate association rate constants and moderate dissociation rate constants (kass approximately 10(4)-10(6) M-1 s-1 and kdis approximately 10(-2)-10(-4) s-1), characteristic of many antibody/protein antigen interactions. The interaction of an anti-idiotypic anti-TCR mAb with its purified cognate TCR was of moderate affinity and revealed kinetic binding similar to that of the anti-MHC mAbs. The previously determined interaction of a purified T cell receptor with its MHC-I/peptide ligand is characterized by kinetic constants more similar to those of the antibody/antigen interaction than of the MHC-I/peptide interaction, but is remarkable for rapid dissociation rates (apparent kdis approximately 10(-2) s-1). Such binding studies of reactions involving the MHC-I molecules offer insight into the mechanisms responsible for the initial specific events required for the stimulation of T cells.

Study of avian adenovirus DNA infectivity in chick embryos.

Inoculation of CELO adenovirus deproteinized DNA into the allantoic cavity of 9-day-old chick embryos (CE) induced the synthesis of infectious viral particles. The produced virions appeared to be identical with CELO adenovirions in terms of morphology, electrophoretic and immunochemical properties of hexon major capsid protein and also of DNA dot-hybridization. High infectivity of CELO DNA (minimal infective dose equaled 40 ng) may be also related, at least in part, to the absence of deoxyribonuclease activity in the allantoic fluid (AF).

Brain casein kinase 2: affinity purification procedure using immobilized polyethylenimine.

A simplified procedure for casein kinase 2 purification from bovine brain is described. The purification procedure consists of two affinity chromatography steps, using heparin and polyethylenimine immobilized on a synthetic matrix (Toyopearl 650M). The adsorption and elution conditions for each column were optimized, resulting in a simple elution protocol for each column. A stable, highly purified casein kinase 2 preparation was obtained in 4 h using this procedure. Polyethylenimine was shown to stimulate the casein kinase 2 activity using exogeneous substrates (casein, calmodulin, MAP2, and tau) but not the enzyme's autophosphorylation activity. The polyethylenimine stimulation could be overcome by applying a mass excess of the casein kinase 2 inhibitor, heparin.

Comparison of adenoviral hexon polypeptides (monomers) and of native hexons (trimers) by SDS-polyacrylamide gel electrophoresis.

Purified hexons of 27 serotypes of human, simian, bovine and avian adenoviruses were analysed by SDS-PAGE. The apparent molecular weights of hexon polypeptides calculated by comparison with 5 non-hexon and 3 sequenced hexon polypeptide markers ranged from 98 kDa (for bovine adenovirus Ad bos7) to 118 kDa (for simian adenovirus Ad sim13; SV36). A stability of native hexon capsomers (trimers) in SDS at room temperature permitted us to resolve native (trimeric) hexon by SDS-PAGE and to distinguish them from denatured (monomeric) hexon polypeptides by electrophoretic mobilities. Hexon trimer bands with slow mobility in SDS-PAGE (unlike hexon monomer polypeptide bands) retained native hexon antigenicity as revealed by immunoblot analyses. Possible applications of simultaneous analyses of hexon trimers and monomers by SDS-PAGE are discussed.

MHC class I/peptide interactions: binding specificity and kinetics.

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class I molecules as well as in the application of real time biosensor technology have permitted the direct analysis of the binding of MHC class I molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as 'pepsicles', as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class I peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Detailed analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides reveals that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kd values for antigenic peptide/class I interactions at 25 degrees C are in the range of ca 10(-4)-10(-6)/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.

Direct detection of major histocompatibility complex class I binding to antigenic peptides using surface plasmon resonance. Peptide immobilization and characterization of binding specificity.

We have developed model systems in which the binding of purified, genetically engineered, soluble analogues of major histocompatibility complex (MHC) class I molecules to immobilized antigenic peptides can be monitored in real time using surface plasmon resonance (SPR). Synthetic analogues of several peptides known to bind different mouse and human MHC class I molecules were prepared with cysteine residues substituted at appropriate positions. The analogue peptides were immobilized via the bifunctional reagent N-gamma-maleimidobutyryloxy-succinimide to amino groups generated on the dextran-modified gold surface of a biosensor flow cell. Using this approach, each position in the sequence of an H-2Ld-specific viral peptide, pMCMV (YPHFMPTNL), was used for coupling, and the resulting surfaces were tested for binding of the soluble analogue of H-2Ld, H-2Lds. In accord with our previously described H-2Ld/pMCMV three-dimensional structural model, only those residues of the peptide that remain exposed following binding (positions 4-8) can be replaced by cysteine and used for coupling. Stable binding of soluble MHC class I molecules, H-2Lds, H-2Dds, H-2Kbs, and HLA-A2s to their respective immobilized cognate peptides was detected by SPR. Specificity of the peptide/MHC interaction was characterized both by direct binding using immobilized peptides and by competition with peptides in solution, and in general was consistent with known immunological reactivity. Some peptides bound not only their cognate MHC molecule, but others at lower apparent affinity. Measurement of real time binding of MHC class I molecules to peptides immobilized through specific side chains suggests the application of a similar approach to the study of the interaction of peptides with a wide variety of peptide-binding macromolecules.

Characterization of a series of monoclonal antibodies specific for bovine adenovirus serotype BAV3 hexon antigen and their use in an ELISA for detection of adenoviral hexons.

After immunization of mice with purified hexon (the main capsid antigen) of bovine adenovirus serotype BAV3 we have obtained a set of 16 individual hybridoma clones producing MAb's against BAV3 hexon. All MAb's were shown to belong to immunoglobulin G class. Specificity of the most avid MAb marked B3Hx-1 was tested on a panel of representative hexon antigens from 16 adenovirus serotypes of human and animal origin using several immunoassays. In Western blot analysis the MAb B3Hx-1 reacted only with native (trimeric) form of hexon protein and not with denaturated hexon polypeptide chains. The epitope defined by B3Hx-1 appeared stable against SDS at ambient temperature and against chloramine-promoted iodination. The specificity of the epitope was characterized as almost genus-crossreactive: it was absent from hexons of avian and of bovine subgroup 2 adenovirus serotypes and present in most hexons of bovine, canine, simian and human adenoviruses tested. Within the latter group its expression was weak or absent only for human subgenus C serotypes. Several variants of sandwich-type ELISA were developed using MAb B3Hx-1 and different polyclonal antibodies against hexons of mammalian adenoviruses. The level of hexon detection for different adenovirus serotypes varied in range 10(-9) to 10(-8) g per ml.