Objectively assessing bioartificial organs.

The metrics used, thus far, to assess bioartificial organ function are shown to be subjective and requiring validation. Therefore, four categories of correlations are proposed based on, respectively, device, in vitro and in vivo evaluations, and clinical function. Examples are presented whereby the correlations among individual indicators are used as a means to expedite the development of immunoisolated cells. Specifically, a case study illustrating the validation of in vitro indicators of in vivo graft function for the bioartificial pancreas (microencapsulated islets) is summarized. This has revealed thresholds with respect to given metrics relating to in vivo device function, the necessity to couple bioartificial organ design with transplant site selection, as well as the lack of objectivity involved in the evaluation and establishment of hypotheses. Specific quantitative indicators illustrate the need for quality-controlled measures, for example, relating to the tolerance of microcapsule diameter and membrane thickness distributions. Qualitative indices representing fibrosis and device properties (e.g., sphericity) are also used to describe the need for in vitro experiments in the development of bioartificial organs.

New multicomponent capsules for immunoisolation.

A new generation of microcapsules based on the use of oligomers which participate in polyelectrolyte complexation reactions has been developed. These freeze-thaw stable capsules have been applied as a bioartificial pancreas and have resulted in normoglycemia for periods of six months in concordant xenotransplantations. The new chemistry permits the control of permeability and mechanical properties over a wide range and can be adapted both to microcapsule and hollow fiber geometries rendering it a robust tool for encapsulation in general. Methods, and metrics, for the characterization of the mechanical properties and permeability of microcapsules are presented.

Neutralizing and protective antibodies directed against vaccinia virus envelope antigens.

The infection mechanism of vaccinia virus is largely unknown. Neither the attachment protein of extracellular enveloped virus (EEV), the biologically relevant infectious form of the virus, nor its cellular receptor has been identified. Surprisingly, all former attempts using antibodies to block EEV infection of cells in vitro had failed. Here, we report the production of an anti-envelope hyperimmune serum with EEV neutralizing activity and show that a polyclonal antiserum against the extraviral domain of protein B5R also inhibited EEV infection. In vivo, mice vaccinated with B5R protein were protected against a lethal vaccinia virus challenge. This protectivity is likely to be mediated by neutralizing antibodies. Protein A33R, but not A34R and A36R, also proved to be protective in active and passive vaccination experiments. However, in contrast to B5R, A33R protectivity did not correlate with antibody titers. Because anti-A33R antibodies did not neutralize EEV in vitro, the protectivity mediated by A33R protein probably involves a mechanism different from simple antibody binding. Taken together, our results suggest that antibodies to a specific protective epitope or epitopes on protein B5R are able to prevent EEV infection. The protein encoded by the B5R gene is therefore likely to play a crucial role in the initial steps of vaccinia virus infection-binding to a host cell and entry into its cytoplasm.

Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Lack of association of secretory component with IgA in J chain-deficient mice.

J chain has been proposed to play a role in the mucosal transport of polymeric Igs (pIg) by the polymeric Ig receptor (pIgR). We have previously reported the generation of J chain-deficient mice. These mice exhibited elevated serum IgA and depleted biliary and fecal IgA levels compared with wild-type mice. We report here that, unlike the IgA levels in bile and feces, IgA levels in local mucosal and glandular secretions were not depressed in J chain-deficient mice. Breast milk, intestinal mucosal surface, and nasal wash IgA levels in the mutant mice were similar to wild-type mice while bronchoalveolar lavage IgA levels were higher in the J chain-deficient animals. Western blot analysis with an Ab to secretory component (SC), the portion of the pIgR that remains bound to pig in secretions, and immunoprecipitation with Abs directed against IgA showed that secreted IgA was associated with SC in wild-type but not J chain-deficient mice. The IgA in wild-type secretions was polymeric while the secretions of J chain-deficient mice contained IgA monomers and other nonpolymeric IgA forms. Thus, J chain is not essential for IgA transport by intestinal, mammary, or respiratory epithelia but is necessary for the stable association of pIgA with SC. Further, we suggest that J chain-deficient IgA is transported into secretions by a different mechanism than wild-type IgA.

Biochemical analysis of the major vaccinia virus envelope antigen.

The major envelope antigen of vaccinia virus is an acylated protein of M(r) 37,000 (p37K) which is required for the formation of extracellular enveloped virions (EEV). Despite its important role in the wrapping process, p37K has not been studied in much detail. In order to better characterize this protein we have undertaken a detailed biochemical analysis. Sodium carbonate treatment showed that p37K is tightly bound to the viral envelope. Its resistance to proteinase K digestion indicates that it is not exposed on the surface of EEV but lines the inner side of the envelope. Since p37K does not contain a signal peptide characteristic of most membrane proteins, we examined the possibility that the protein acquires its membrane affinity through the addition of fatty acids. Indeed, Triton X-114 phase partitioning experiments demonstrated that p37K is hydrophobic when acylated, but hydrophilic in the absence of fatty acids. Three other viral proteins have been shown to be required for virus envelopment and release from the host cell and we therefore tested whether p37K interacts with viral proteins. In EEV and in absence of reducing agents, an 80-kDa complex reacting with an anti-37K antiserum was found. Analysis of this complex showed that it most likely consists of a p37K homodimer. Interestingly, only a small amount of p37K occurs as a complex, most of it is present in the viral envelope as monomers.

Production of human secretory component with dimeric IgA binding capacity using viral expression systems.

The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.

A repetitive protein from Trypanosoma brucei which caps the microtubules at the posterior end of the cytoskeleton.

The major structural component of the cyto-architecture of Trypanosoma brucei is a microtubular array which envelopes the entire cell body and which is in close contact with the overlying cell membrane. A cytoskeletal protein has been identified which is associated with the microtubules of this array at their posterior ends exclusively. This protein, Gb4, is coded for by a very large gene which consists of numerous, tandemly linked repeat units of 0.6 kb length. Despite the large size of the gene, and also of the corresponding mRNA, the mature Gb4 protein has a size of only 28 kDa. Gb4 is well conserved between different species of African trypanosomes.

The tubulin gene cluster of Trypanosoma brucei starts with an intact beta-gene and ends with a truncated beta-gene interrupted by a retrotransposon-like sequence.

In Trypanosoma brucei the genes coding for alpha- and beta-tubulin are arranged in a single cluster comprising about ten alternating alpha and beta genes. The cluster starts at its 5' end with an intact beta gene. In this area, a 1.1-kb segment upstream of the mini-exon addition site has been sequenced. Several potential transcription signals were seen. The 3' end of the cluster terminates in the middle of a beta gene, making it an inactive truncated pseudogene. The adjoining 1.7-kb sequence shows a high degree of similarity with a retrotransposon-like (RTnL) element previously characterized elsewhere in the trypanosome genome. This is the first report of an RTnL element interrupting a protein-coding gene of T. brucei.