Comparison between conventional systems and a newer system of combined cultures for isolation of influenza viruses

Summary The sensitivity of conventional systems was compared with the sensitivity of the newer system of combined cultures for the isolation of influenza viruses. Both systems were comparable. It was found, that certain isolates were obtained only by conventional systems, and conversly, others by combined cultures only. These results showed that the joint usage of both the conventional and newer systems resulted in a significantly higher isolation rate.     

Innervation of human nasal glands

Summary The innervation of human nasal glands was investigated with the electron microscope in 14 males and 11 females using osmium and permangante fixed material. Acetylcholinesterase activity was localized electron microscopically in formalin fixed tissues.It was found that the tubuloalveolar glands possessed myoepithelial cells which extended over the basal surfaces of the secretory cells and were attached to them by desmosomes. The glands were supplied by fenestrated capillaries. Periacinar and intraepithelial nerves of the glands exhibited high positive acetylcholinesterase and negative butyrocholinesterase activity. The reaction product was localized in the interval between the axolemma and the cell membranes of the secretory, myoepithelial or Schwann cells, respectively. The axon varicosities of the periacinar nerves and the intraepithelial endings contained accumulations of agranular vesicles in preparations fixed with osmic acid, formalin or permanganate solutions, signifying cholinergic nerves. The blood vessels of the nasal glands were supplied by cholinergic and adrenergic nerves.     

Construction and Immunological Evaluation of Multivalent Hepatitis B Virus (HBV) Core Virus-Like Particles Carrying HBV and HCV Epitopes

A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcΔ protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-γ) production profile.Genetically engineered virus-like particle (VLP)-based vaccines are one of the most promising tools in modern vaccinology. VLPs from almost all classes of viruses are being evaluated now or have just been adopted to use as carriers for presentation of foreign immunological epitopes (for a review, see references 29 and 31). VLP technologies possess obvious advantages for the generation of safe and efficacious vaccines. First, the repetitive antigenic structure of VLPs makes them highly immunogenic. Second, VLPs lack viral genomes or genes and are noninfectious, although they mimic infectious viruses in their structural and immunological features. Third, VLPs are generated by highly efficient heterologous expression of the cloned viral structural genes with subsequent quantitative in vivo or in vitro self-assembly of their products. Fourth, VLPs can be obtained by simple and efficient purification procedures. VLPs can be used for a broad range of applications, but the generation of vaccines against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is of special interest.The HBV core (HBc) protein was first reported as a promising VLP carrier in 1986 and was published in 1987 (6, 10, 24). In many ways, HBc occupies a unique position among the VLP carriers because of its high-level synthesis and efficient self-assembly in virtually all known homologous and heterologous expression systems, including bacteria (for a review, see references 29 to 31). The major HBc B-cell epitopes (c and e1) are localized within the major immunodominant region (MIR), whereas the next important epitope, e2, is localized around amino acid position 130, close to the C-terminal histone-like region (for a review, see reference 30).The high-resolution spatial structure of HBc icosahedrons (11, 43) shows that the MIR is located on the tip of the spike, around the most protruding region between amino acids (aa) 78 and 82. For this reason, the MIR is generally accepted as the target site of choice for insertion of foreign epitopes (30). The other widely accepted site for insertions is C-terminal position 144, a short stretch after the e2 epitope. For C-terminal insertions, so-called HBcΔ vectors lacking a 39-aa-long positively charged C-terminal histone-like fragment are preferred for their high insertion capacities (up to 741 amino acid residues) (30).Here, we present the construction and preliminary immunological characterization of a first multivalent HBV and HCV vaccine candidate. As an HBV epitope, we chose the pre-S1 sequence aa 20 to 47, which alone is able to elicit HBV-neutralizing and protective antibodies (23), for insertion into the HBc MIR. Concurrently, we inserted at the C terminus of the HBcΔ vector the N-terminal 60-aa fragment of the HCV core, which is highly conserved among various HCV genotypes with amino acid homology exceeding 95% (12, 14) and therefore is an attractive target for the generation of an HCV vaccine (19, 41). Such a combination of foreign epitopes did not prevent correct self-assembly of chimeric HBc-based particles and provided them with specific HBV and HCV antigenicity and immunogenicity in mice.     

Polymers with sulfinyl and oxyethylene constitutional units as catalysts of nucleophilic substitution reactions

The catalytic activity of polystyrene- and polyphosphazene-supported monosulfoxides and oligo(oxyethylene)s was investigated in the Williamson alkylation of sodium phenoxide with 1-bromooctane in 1,4-dioxane at 75°C. Polymer-supported monosulfoxides are less efficient than low-molecular-weight species. Polymer-supported oligo(oxyethylene)s with pseudo-crown-ether structures show a polymer effect, which may even be stressed by the presence of onium units in the side chains.     

The influence of the length of alkoxy side groups on the phase state of polydialkoxyphosphazenes

Polydialkoxyphosphazenes with different substituents varying from methoxy up to octoxy which contain only minor amounts (below 0,1 mol-%) of various defective units in their macromolecules were synthesized. It was shown that such polyphosphazenes with propoxy, butoxy and pentoxy side groups can exist in the mesomorphic state and that a defect content as low as about 2 mol-% prevents mesophase formation. In spite of high flexibility of the main chain, the polydialkoxyphosphazenes were found to gain the ability to crystallize only if the number of C atoms in the alkoxy substituents is higher than six.     

Determination of the minimal length of preS1 epitope recognized by a monoclonal antibody which inhibits attachment of Hepatitis B virus to hepatocytes

The minimal amino acid sequence sufficient to be recognized efficiently by virus-attachment inhibiting murine monoclonal anti-preS1 antibody MA 18/7 has been determined. We have constructed a recombinant gene library using the cloned coat protein gene of Escherichia coli RNA bacteriophage fr as a carrier. Different fragments of preS1 region from cloned hepatitis B virus (HBV) genomes, subtype ayw and adw, were inserted at position 2 of the 129 amino acid-long fr coat protein gene in the appropriate E. coli expression vectors. Fine mapping of preS1 epitope recognized by MA18/7 was accomplished by bidirectional shortening of the preS1 within original recombinant preS-fr coat protein genes with Bal31 exonuclease. Immunoblot analysis of the obtained recombinant protein library revealed that the tetrapeptide Asp-Pro-Ala-Phe (DPAF), located at the position preS(31–34) and conserved in all known HBV genomes, is sufficient to bind MA18/7 antibody. Recognition of the preS1 region by MA18/7 occurred irrespective of the amino acid context surrounding this DPAF tetrapeptide. Further shortening of this minimal epitope from the left or from the right side completely prevented antibody binding in immunoblots.     

Fine-mapping of the B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen

In this study, we report the exact localization and substitutional characterization of a B-cell epitope domain at the N-terminus of the preS2 region of the hepatitis B surface antigen. A set of deletion variants containing preS2 sequences of different length was generated on the basis of frCP as a carrier. It was found after Western blot analysis that three monoclonal antibodies (MAbs) (2-11B1, 3-11C2, HB.OT10) recognized the linear preS2 sequence within the amino acid (aa) stretch 3-WNSTTFHQTLQDP-13. The importance of each aa residue of the epitope was proved by comparison of antibody binding to alanine-substituted peptides in both free-peptide and Pepscan variants.     

The fine structure and innervation of the cushion veins of the human nasal respiratory mucosa

Cushion veins of the human nasal lining were studied in eight patients of both sexes ranging in age from 11 to 59 years. It was found that the subendothelial cushions were part of the tunica media and consisted of smooth muscle cells, collagen and elastic fibers and occasional fibrocytes. The muscle fibers of the cushion nearest to the endothelium were circular. They extended processes towards the endothelium through gaps in the endothelial basement membrane and formed appositional junctions with the endothelial cells. The rest of the cushion consisted of longitudinal muscle fibers. The sarcoplasm of the muscle cells was characterized by large areas filled with vesicles of various sizes. In addition, these cells possessed cytoplasmic processes which were devoid of a basement membrane and which did not show the regular structure of sarcoplasm. The subendothelial cushion possessed a rich, intrinsic nerve supply of adrenergic and cholinergic axons. It is suggested that the cushion veins regulate the drainage of the cavernous tissue and are under nervous and humoral control. The increase in girth of the subendothelial cushion is effected by contraction of the longitudinal muscle cells and probably by uptake of extracellular fluid by means of the specialized cytoplasmic processes. The single layer of circular muscle cells situated between the endothelial lining and the longitudinal musculature, may provide protection to the endothelium against distension when the cushion expands.