The prospects for using the genetically engineered surface antigen of the hepatitis B virus (HBsAg) expressed by recombinant poxvirus strains as the basis for vaccines against hepatitis B

The use of a recombinant poxvirus (RPV) strain, expressing HBsAg in the process of reproduction in different bioreactor systems under stationary and bioreactor conditions of cultivation, made it possible to obtain highly purified HBsAg. The identity and purity of HBsAg was confirmed by the analysis of its amino acid composition, SDS electrophoresis in polyacrylamide gel, electron microscopy and high-performance liquid chromatography. Good prospects of the use of RPV-expressed gene engineering HBsAg as the basis vaccines against hepatitis B was demonstrated in 10 experimental batches of vaccine. All batches of the preparation had pronounced immunogenicity and were safe and nontoxic in animal experiments. The ID50 of experimental batches did not exceed 211 ng/ml, which, according to the data of comparative experiments, was lower than, or equal to, corresponding values of analogous foreign commercial preparations, based on plasma or yeast HBsAg.     

In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus

Rotaviruses are the leading cause and coronaviruses are the major contributors of acute gastroenteritis in the young of various mammalian and avian species. Despite numerous trials and decades of research, vaccines have limited efficacy particularly for calves. As an alternative method of controlling infection, we have investigated broad spectrum antiviral agents that are not discriminatory among various viruses. This report involves testing a variety of adsorbent agents including charcoal, clay, and clay minerals to adsorb rotavirus and coronavirus in vitro. Results revealed that all the adsorbent agents had good to excellent capability of adsorbing rotavirus and excellent capability of adsorbing coronavirus. Percent adsorptions ranged from 78.74% to 99.89% for rotavirus and 99.99% for coronavirus; while sand (negative control) was < 0.01%. A high affinity binding was present as determined by a low percent desorption (0.06-3.09%). However, the adsorbent bound virus complex retained, and may have actually enhanced, infectivity.     

Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes

We recently described a novel population of blood-borne cells, termed fibrocytes, that display a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and contribute to scar formation. To further characterize the role of these cells in vivo, we examined the expression of type I collagen and cytokine mRNAs by cells isolated from wound chambers implanted into mice. Five days after chamber implantation, CD34+ fibrocytes but not CD14+ monocytes or CD90+ T cells expressed mRNA for type I collagen. Fibrocytes purified from wound chambers also were found to express mRNA for IL-1beta, IL-10, TNF-alpha, JE/MCP, MIP-1alpha, MIP-1beta, MIP-2, PDGF-A, TGF-beta1, and M-CSF. The addition of IL-1beta (1-100 ng/ml), a critical mediator in wound healing, to fibrocytes isolated from human peripheral blood induced the secretion of chemokines (MIP-1alpha, MIP-1beta, MCP-1, IL-8, and GRO alpha), hemopoietic growth factors (IL-6, IL-10, and macrophage-CSF), and the fibrogenic cytokine TNF-alpha. By contrast, IL-1beta decreased the constitutive secretion of type I collagen as measured by ELISA. Additional evidence for a role for fibrocytes in collagen production in vivo was obtained in studies of livers obtained from Schistosoma japonicum-infected mice. Mouse fibrocytes localized to areas of granuloma formation and connective matrix deposition. We conclude that fibrocytes are an important source of cytokines and type I collagen during both the inflammatory and the repair phase of the wound healing response. Furthermore, IL-1beta may act on fibrocytes to effect a phenotypic transition between a repair/remodeling and a proinflammatory mode.     

Characterization of mutated transforming growth factor-beta s which possess unique biological properties

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation. On the basis of the crystal structure of TGF-beta 2, we have designed and synthesized two mutant TGF-beta s, TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73). Although both of these molecules inhibited the growth of Mv1Lu mink lung epithelial cells and LS1034 colorectal cancer cells, which are affected equally by TGF-beta 1 and TGF-beta 2, TGF-beta 1 (delta 69-73) was much less potent than TGF-beta 1 or TGF-beta 1 (71 Trp) at inhibiting the growth of LS513 colorectal cancer cells which are growth-inhibited by TGF-beta 1 but not TGF-beta 2. Both TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73) increased levels of mRNAs for fibronectin and plasminogen activator inhibitor with Mv1Lu cells, whereas only TGF-beta 1 (71 Trp) and not TGF-beta 1 (delta 69-73) up-regulated the mRNA level of carcinoembryonic antigen in LS513 cells. The expression level of carcinoembryonic antigen mRNA in LS1034 cells was not altered by either wild-type or mutant TGF-beta s. Receptor labeling experiments demonstrated that TGF-beta 1 (71 Trp) bound with high affinity to the cell-surface receptors of Mv1Lu, LS1034, and LS513 cells while TGF-beta 1 (delta 69-73) bound effectively to the receptors of Mv1Lu and LS1034 cells but much less to the receptors on LS513 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural co-localization of calmodulin and B-50/growth-associated protein-43 at the plasma membrane of proximal unmyelinated axon shafts studied in the model of the regenerating rat sciatic nerve

Calmodulin and de-phosphorylated B-50/growth-associated protein-43 (GAP-43) have been shown to bind in vitro in a molecular complex, but evidence for an in situ association in the nervous system does not exist. Previously, we have reported that, in the model of the regenerating rat sciatic nerve, the B-50/GAP-43 immunoreactivity is increased and concentrated at the axolemma of unmyelinated axons located proximal to the site of injury and axon outgrowth. To explore a putative function of B-50/GAP-43, namely, the capacity of binding calmodulin to the plasma membrane, we examined the ultrastructural distribution of calmodulin in the proximal unmyelinated axon shafts of this model, using double immunolabelling and detection by fluorescent or gold probes conjugated to second antibodies. Immunofluorescence showed that seven days post-sciatic nerve crush the calmodulin immunoreactivity, similar to B-50/GAP-43 immunoreactivity, was intense in unmyelinated axon shafts located proximal to the site of injury of the regenerating nerve. Ultrastructurally, calmodulin was located at the axolemma of these regenerating unmyelinated axon shafts and inside the axoplasm, where it was associated with vesicles and microtubules. The plasma membrane labelling (approximately 69%) was significantly higher than the axoplasmic labelling. Over 60% of the plasma membrane-associated calmodulin co-localized with B-50/GAP-43 in a non-random distribution. Since normally calmodulin is largely present in the cytoplasm, these data suggest that calmodulin has been concentrated at the plasma membrane of unmyelinated axons, most probably by B-50/GAP-43. If the concentrating effect is due to B-50/GAP-43, then there is a possibility that these proteins may be present as a molecular complex in situ. The physiological significance could be that this association regulates the local availability of both B-50/GAP-43 and calmodulin for other interactions.