Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.     

Antigenic characterization of the salmonid pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis, the etiological agent of salmonid rickettsial septicemia, was purified from infected immortal chinook salmon (Oncorhynchus tshawytscha) embryo cells by a combination of differential and Percoll density gradient centrifugation. Immune sera from rabbits immunized with purified whole cells of P. salmonis reacted with four protein antigens and two carbohydrate antigens with relative molecular sizes of 65, 60, 54, 51, 16, and approximately 11 kDa, respectively. The carbohydrate antigens appear to be mainly core region lipo-oligosaccharide with lesser amounts of lipopolysaccharide. Serum from convalescent rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) reacted with several minor immunoreactive protein antigens between 10 and 70 kDa in size and a carbohydrate antigen with a relative molecular size of approximately 11 kDa. The salmonid immune system did not appear to elicit a strong humoral response against this intracellular pathogen. Indirect immunofluorescence microscopy, immunogold transmission electron microscopy, and biotin labeling of intact P. salmonis cells suggest that the immunoreactive antigens identified with rabbit antisera are surface exposed and differ significantly from those identified with salmonid antisera.     

Selected Properties of Formaldehyde-Free Polymer-Straw Boards Made from Different Types of Thermoplastics and Different Kinds of Straw

This research investigated the effects of different thermoplastics types and different kinds of straw on selected properties of polymer-straw boards. Polyethylene, polyethylene, and polystyrene of virgin and of recycled origin were used for bonding the boards. Three kinds of straw were used: rape (Brassica napus L. var. napus), triticale (Triticosecale Witt b m.), and rye (Secale L.). Five-layer polymer-straw boards were produced. The obtained boards differed in both the materials they were made of and the moisture content (7, 25, and 2% for the core, the middle, and the face layers, respectively), and 30% of straw particles were substituted with thermoplastics added to the face layers. It was found that properties of polymer-straw boards strongly depend on both the kind of straw and the type of polymer used. The best mechanical properties were obtained for rye straw and polystyrene or recycled polymers, whereas the best hydrophobic properties were observed for rape straw combined with recycled polyethylene or polypropylene. Although recycled polymers improved the hydrophobic properties of the boards, they impaired their mechanical properties in comparison with the reference ones. However, in terms of bending strength, they still met the requirements for heavy duty load-bearing boards for use in humid conditions (20 MPa for P7 boards according to EN 312).     

Total hip arthroplasty in patients with fibrous dysplasia: a modern update

BackgroundFibrous dysplasia (FD) results from an abnormality in lamellar bone formation and most frequently involves the proximal femur. This can lead to the development of osteoarthritis requiring total hip arthroplasty (THA). Such cases are challenging, and there is a lack of information guiding best management. As such, we devised a study assessing the outcomes and complications in patients with FD undergoing THA with modern implant technology, and we outlined our preferred surgical technique.MethodsA search of our institutional arthroplasty database was performed to identify patients who underwent THA for FD between January 2001 and July 2018 at Mount Sinai Hospital in Toronto, Canada. Data regarding implants used and the use of allograft material or metal augments or both were obtained. Complications and revision requirements were noted. Radiographic and clinical leg length discrepancies were assessed.ResultsA total of 10 hips in 9 patients who underwent THA for FD were identified. Mean follow-up time was 6.0 years (range 0.5 to 10.3 yr). The majority of patients underwent THA using uncemented femoral and acetabular components with large femoral heads on highly cross-linked polyethylene liners. Most cases (80% of hips) required allograft to the proximal femur. A single complication requiring revision was noted. In 90% of hip surgeries, the patient required transfusion of packed red blood cells. Mean radiographic and clinical leg length discrepancies were 0.9 cm (range −2.4 to 2.4 cm) and 0.9 cm (range −4 to 0 cm), respectively.ConclusionContrary to previous reports, low complication and revision rates were observed with cementless components and routine use of allograft material. The challenging nature of such cases warrants use of an experienced arthroplasty treatment team.     

Treatment of Failed Allograft Prosthesis Composites Used for Hip Arthroplasty in the Setting of Severe Proximal Femoral Bone Defects

This study assessed failures of allograft prosthesis composites (APC) and revisions with a new APC. Twenty-one patients with failed APC’s after revision hip arthroplasty with severe proximal femoral bone loss underwent revision with a new APC. Causes of failure were aseptic loosening (18 patients), infection (3 patients). Of these 21 APC revisions, two patients failed (after 60, 156 months). The 5 and 10 year survival rates were 83.5% (95% CI, 79–100%, number at risk 12 and 6 accordingly). In addition, two patients had non-union at the host-allograft bone junction and were augmented with bone autograft and plate. These results suggest that failed APCs may be revised to a new APC with a predictable outcome.     

Discovery of Marburg virus neutralizing antibodies from virus-naïve human antibody repertoires using large-scale structural predictions

Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. We computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a full-length native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.

With the advent of high-throughput immune repertoire sequencing, the number of available human antibody (Ab) sequences is exploding rapidly from thousands to billions. These datasets provide a resource for understanding the natural process of Ab maturation through somatic mutations, quick identification of novel functional Abs, and engineering of improved Abs. Ultimately, such Ab repertoires may provide or add templates for developing epitope-focused (1) and germline-targeting (2, 3) vaccines. While Ab function sometimes can be predicted from sequence homology, often Abs of very different sequence have the same function by virtue of adopting a similar structure. However, despite substantial progress in computational methods of modeling Abs (4, 5) and Ab–antigen interactions (6–10), and the use of high-performance computing, it is still beyond the available computational resources to predict and study the structure of billions of Abs one by one. Therefore, in order to take advantage of the rapidly increasing Ab sequence databases, it is essential to find more effective ways of relating Ab sequence to function.The recently proposed position-specific structure scoring matrix (P3SM) approach is a new computational method specifically designed for rapid screening of large Ab sequence libraries (11, 12). This approach aims to predict whether a given Ab sequence can adopt the desired 3D conformation and thus correctly place critical-for-activity functional groups. This prediction is based on modeling of a small subset of structures using Rosetta (13) followed by evaluation of the compatibility of each of 20 amino acids in each of the analyzed positions with the desired structure and function. The resulting P3SM then can be used for rapid screening of the remaining Ab sequences. The best-scoring candidate sequences are fed back into Rosetta for a detailed energetic analysis and prioritization for experimental validation.The human monoclonal Ab MR78 was isolated previously from a B cell in the peripheral blood of an otherwise healthy individual with a prior history of naturally acquired Marburg virus (MARV) infection (14). A crystal structure of MARV glycoprotein (GP) in complex with MR78 (Protein Data Bank [PDB] ID 5UQY) revealed that the Ab heavy-chain complementarity determining region 3 (HCDR3) contacts the receptor binding domain (RBD) on MARV GP that interacts with the natural receptor on human cells (Niemann–Pick disease, type C1 [NPC1] protein) (15). Here, we applied the P3SM approach to search for structurally homologous Abs to MR78 based on this Ab–antigen cocrystal structure.     

Aortic dissecting aneurysms: clinical masks,differential diagnosis

101 necropsies of aortic dissection (AD) made in 1991-2000 have been analysed. Among the deceased men prevailed (76.3%), 23(22.8%) of them were under 50 years of age. An accurate intravital diagnosis was made only in 40.6 patients. The most common clinical masks were the following: coronary, valvular, vascular, gastrointestinal, pulmonary, thromboembolic, mediastinal, cerebral, renal and anemic. Most often AD masks as acute myocardial infarction. The knowledge of encountered clinical masks of AD promoted improvement of early AD diagnosis to correct AD surgically and reduce lethality.