Trimerization of murine TNF ligand family member LIGHT increases the cytotoxic activity against the FM3A mammary carcinoma cell line.

LIGHT is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. Recombinant soluble human LIGHT produced by mammalian cells or Escherichia coli is functional at nanomolar concentrations. However, there is little information about the biological activity of mouse LIGHT (mLIGHT) because of the difficulty in producing bioactive soluble mLIGHT. In this study, recombinant trimeric soluble mLIGHT, or Foldon-mLIGHT, was produced by fusing mLIGHT with the trimerization domain foldon from bacteriophage T4 fibritin. Foldon-mLIGHT was secreted from 293F cells as a 68-kDa trimeric protein. The recombinant protein potently inhibited the growth of the FM3A mouse mammary carcinoma cell line with an IC(50) of 77 pM; however, the monomer or dimer forms of mLIGHT produced by E. coli or mammalian cell systems showed weak or no inhibitory activity. These data clearly indicated that trimerization of soluble mLIGHT is essential for its biological activity.

The ultrastructural morphology of native hepatitis B virus.

Cell lines (2.2.15 cells) capable of supporting the replication of hepatitis B virus (HBV) DNA and intact viral particles have been established by HBV DNA transfection into HepG2 cells. The purpose of this study was to determine the ultrastructural morphology of native HBV particles without purification in the culture supernatants and in sera from patients. Electron microscopy (EM) and immunogold EM of the samples were carried out using polyclonal and monoclonal anti-hepatitis B surface antigen antibodies. HBV particles in the purified samples from the culture supernatants by density-gradient centrifugation were examined to compare the morphology with that of unpurified samples. EM and immunogold EM studies demonstrated the presence of Dane particles (41.8 nm in diameter), cobra-shaped (head diameter, 42.4 nm), and horn-shaped (head diameter, 43.5 nm) particles in the culture supernatants and in the sera from two patients. The tail of the cobra-like particles had a diameter of 21.0 nm and a length of 214 nm. The hornlike particles had a long branch 20.1 nm in diameter with a length of 189 nm, and a short branch 21.4 nm in diameter with a length of 112 nm. The ratio of Dane particles and cobra- and horn-shaped particles in the supernatants was 5 : 4 : 1. After ultracentrifugation, the cobra- and horn-shaped particles completely disappeared; there were only Dane particles together with spheres of 22 nm and filaments. In conclusion, this study showed for the first time that the native replicative form of HBV is cobra- and horn-shaped.

Renewal of EBV-hybridoma method: efficient generation of recombinant fully human neutralizing IgG antibodies specific for tetanus toxin by use of tetroma cells.

We have generated tetroma cell lines by the fusion of newly developed parental 6JC5.2 cells with a human B cell line, which was transformed with Epstein-Barr virus (EBV) and enriched with antibody-forming cells that produce neutralizing antibodies to tetanus toxin (TT) by a limiting dilution method using IL-6. The resultant two tetroma cell lines stably produced different monoclonal antibodies (mAbs), TT1 (IgG1.lambda) and TT2 (IgG4.kappa) reactive with TT after three-times consecutive cell cloning. Although weak to almost nonexistent neutralizing activities against TT were detected in TT1 and TT2 mAbs, respectively, mixing of them resulted in a dramatic increase in the neutralizing activity and complete protection from the toxin was observed in vivo. Moreover, functional immunoglobulin (Ig) genes were cloned from at least 10 cells in the first cloning step of tetromas after the cell fusion. None of the endogenous Ig genes, derived from the parental cell that hinders functional Ig gene cloning, was amplified. In addition, the EBV genome derived from the B cells was eliminated from the antibody producing tetroma lines. This classical but revised EBV-hybridoma method using fusion partner 6JC5.2 may become one alternative method for production of fully human antibodies useful for prevention and treatment of infectious diseases and cancer.

Mutual and directional allogeneic cytotoxic reaction of hemocytes in the solitary ascidian Halocynthia roretzi revealed by one-step quantitative fluorimetric assay.

A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls alloreactivity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with "effector cells" and those from the other animal of the pair with "target cells". In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.

An immunodominant neutralization epitope on the 'thumb' subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies.

An antibody phage display library was produced from the splenocytes of mice immunized with an infectious vaccinia virus recombinant (WRRT) expressing the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The library was panned against HIV-1 RT. Two clones, 5F and 5G, which produced Fab fragments specific for RT, were isolated. Surprisingly, both 5F and 5G Fab fragments were capable of strongly inhibiting the RNA-dependent DNA polymerase activity of HIV-1 RT. A hybridoma cell line that produces the monoclonal antibody 7C4, which strongly inhibits RT activity, was established previously using splenocytes from mice immunized with WRRT by the same immunization protocol. The epitope recognized by 7C4 exists in the region of the template primer-binding sites (or the 'helix clump') of RT. By epitope mapping and competitive ELISA analysis, it was shown that the 5F and 5G Fab fragments were directed against the same, or a very closely related, epitope that is recognized by 7C4. The neutralizing activities of the 5F, 5G and 7C4 Fab fragments correlated with their affinities for HIV-1 RT. DNA sequencing indicated that the immunoglobulin genes of the heavy chains of 5G and 7C4, as well as those of the light chains of 5F and 5G, had the same origin. These results suggest that the neutralizing epitope, which is recognized by these antibodies, becomes immunodominant after repeated immunization of mice with WRRT. This unique epitope, HIV-1 RT-specific and immunodominant neutralizing epitope (HRSINE), is a logical target for new types of HIV-1 RT inhibitors and gene therapy.