• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.398-403
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• 1999

In our previous studies, we reported that hybridoma B-lymphocytes can be used to determine the virulence of Listeria species in an in vitro cytotoxicity assay. Here, we examined the cytopathic effect, i.e., membrane damage and the nature of cell death induced by Listeria monocytogenes on murine hybridoma B-lymphocytes (Ped-2E9). Membrane damage was assessed by microscopic analyses and by measuring the release of intracellular alkaline phosphatase(AP) and lactate dehydrogenase (LDH). Cell death was determined by DNA fragmentation analyses using agarose gel electrophoresis. Infection by listeriolysin O (LLO)-producing L. monocytogenes strains induced substantial amounts of AP and LDH release from Ped-2E9 hybridoma B-cells, suggesting severe membrane damage in these cells, while an LLO-negative L. monocytogenes mutant strain had no effect. An LLO-producing recombinant L. innocua ($prifA^+hly^+$) strain also induced high AP and LDH release and cytopathic changes in Ped-2E9 cells. Light or scanning electron microscopic examination revealed L. monocytogenes mediated membrane destabilization, pore formation, intense cytoplasmic granulation, bleb formation, and lysis of Ped-2E9 cells. LLO-producing L. monocytogenes and L. innocua ($prifA^{+}hly{^}+$) also induced ladder-like DNA fragmentation in Ped-2E9 cells. Collectively, these results suggest that L. monocytogenes, specifically LLO-producing strains, can induce a severe cytopathic effect leading to apoptosis in hybridoma B-lymphocytes (Ped-2E9).

• Journal of Periodontal and Implant Science
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• v.24 no.3
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• pp.472-482
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• 1994

Cytokines expressed specifically in leukocytes subsets and in activated cells, which are involved in chemotaxis and activation of leukocytes, are recently defined as chemokines. Macrophage inflammatory $protein-1{\alpha}(MIP-1{\alpha})$ and $MIP-1{\beta}$ are members of C-C chemokine subfamily which produces wide immunomodulatory, proinflammatory, and hematopoietic modulatory actions. We have studied their gene expression by using Northern blot analysis in various blood cells such as cytolytic T lymphocyte(CTL), helper T lymphocyte(HTL), macrophage, and B lymphocyte. Resting CTL line CTLL-R8 expressed $MIP-1{\alpha}$ mRNA which was downregulated by ConA stimulation. Both of resting and ConA stimulated HTL line Hut78 and Jurkat did not express $MIP-1{\alpha}$ mRNA. There was detectable $MIP-1{\alpha}$ transcript in HTL hybridoma 2B4.11 which was a little upstimulated by ConA stimulation. B cell line 230, and macrophage cell line RAW264.7 and WR19M.1 showed distinct $MIP-1{\alpha}$ message which were induced after LPS stimulation. Expression pattern of $MIP-1{\beta}$ in all cell lines or cell were almost identical to that of $MIP-1{\alpha}$. Also strategies employed to identify and characterize the biological functions was preceded by receptor cloning to trace the shorcut to the final goal of cytokine research. For the cloning of $MIP-1{\alpha}$ receptor(R), we used synthetic oligonucleotides of transmembrane(T) conserved sequences of already cloned human(h) IL-8-R, and performed reverse transcription-polymerase chain reaction(RT-PCR) amplification using murine(m) macrophage cell line mRNA. Among 5RT-PCR products, we isolated a homologous cDNA with hIL-8-R which were shown to be putative mIL-8-R cDNA.

• BMB Reports
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• v.33 no.1
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• pp.92-95
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• 2000

The HIV-1 p24(202-221) sequence ETINNEEEWDRVHPV HAGP contains a B-cell epitope with the earliest immune response and the highest antibody titer against anti-mouse sera obtained by immunization with p24 antigens. A novel mouse monoclonal antibody (mAb) was generated against the immunodominant B-cell epitope of the HIV-1 p24 capsid protein, p24(202-221). BALB/c mice were immunized with the four branched multiple antigenic peptide (MAP) containing the HIV-1p24(202-221) sequence, and antibody-secreting hybridoma were produced by fusion of mouse splenocytes with P3X63Ag8.653, mouse myeloma cells. One clone which produced the antigen-specific mAb named KI-24 (Isotype IgG1, light chain: ${\kappa}$) was identified. mAb KI-24 was highly specific for both the p24(202-221) and p24 proteins when analyzed by ELISA and Western blotting. Since p24(202-221) also contains a cytotoxic T-lymphocyte epitope, this specfic peptide epitope and the monoclonal antibody with specific reactivity against the p24 protein and p24(202-221) can be used in peptide vaccine development and p24 antigen detection from HIV patients.