ABSTRACT
<p><b>OBJECTIVE</b>To generate an oncolytic herpes simplex virus (oHSV) permissive mouse melanoma cell line B16RHSV, preserving the tumorigenic ability in syngeneic mice.</p><p><b>METHODS</b>The herpes simplex virus entry mediator (HVEM) gene was amplified by PCR from human melanoma cell line A375, and cloned into pGEM-T Easy vector for sequencing. The HVEM gene was then cloned into pcDNA3 vector to generate pcDNA3-HVEM for transfection of mouse melanoma cell line B16-F10 cells. After that, the putative transfected cells were selected in full growth medium containing G418. The HVEM-expressing cells were isolated by immunomagnetic bead separation. The mouse melanoma cell line expressing oHSV receptor-HVEM, designated as B16RHSV, was generated. The permissibility of B16RHSV cells to oHSV infection was examined with green fluorescence protein (GFP)-expressing oHSV (oHSVGFP). To investigate the tumorigenic ability of both cells in vivo, 2×10(5) cells in 100 µl were subcutaneously inoculated into the right flanks of C57/BL mice.</p><p><b>RESULTS</b>In vitro, the B16RHSV mouse melanoma cells were shown by fluorescence microscopy capable of being infected by oHSVGFP. In vivo, the B16RHSV cells, like their wild type counterpart, grew to form melanoma in syngeneic mice.</p><p><b>CONCLUSION</b>A herpes simplex virus-permissive mouse melanoma cell line was established. Its tumorigenicity remained unchanged.</p>
Subject(s)
Animals , Female , Humans , Mice , Cell Line, Tumor , Gene Amplification , Genetic Vectors , Herpesvirus 1, Human , Genetics , Physiology , Melanoma , Pathology , Virology , Mice, Inbred C57BL , Neoplasm Transplantation , Plasmids , Receptors, Tumor Necrosis Factor, Member 14 , Genetics , Metabolism , Transfection , Tumor BurdenABSTRACT
<p><b>OBJECTIVE</b>To develop a recombinant vaccinia virus vaccine expressing HPV58 E7 and to determine its immuno-protective activity in mice bearing HPV58 E7+ tumor.</p><p><b>METHODS</b>E7 DNA was amplified and cloned from a plasmid containing HPV58 E7 genome by PCR. To abolish its transforming activity, the nucleotides coding for amino acid residues at positions 24 and 92 were modified by site-directed mutagenesis so that cysteine was substituted by glycine. Balb/c 3T3 cells were transfected with mE7. The expression of E7 protein by the mE7-transfected Balb/c cells was confirmed by immunofluorescence staining. The transfected cells were observed in vitro for anchorage-independent growth and tumorigenesis in nude mice. Recombinant E7 vaccinia virus vaccine was constructed by homologous recombination of HPV58 E7 vaccinia expression plasmid and vaccinia virus (Tiantan stain). The immuno-protective activity of the vaccines was determined by tumor growth inhibition and cytotoxic T lymphocytes (CTL) induction in vaccine-immunized syngeneic mice.</p><p><b>RESULTS</b>Substitution of cysteine by glycine at both positions 24 and 92 of HPV58 E7 abolished its transforming activity. Growth of HPV E7+ tumor in mice immunized with the recombinant vaccinia virus expressing HPV58 E7 was inhibited, and the surviving time of the immunized mice was prolonged. CTL activity was induced as revealed by in vitro cytotoxicity assay using E7+ tumor cells as target cells.</p><p><b>CONCLUSIONS</b>HPV58 E7, with its transforming potential abolished, may be used as vaccine for immunotherapy of patients with HPV 58 related cancers.</p>
Subject(s)
Animals , Mice , Mice, Inbred BALB C , Mutagenesis, Site-Directed , Oncogene Proteins, Viral , Genetics , Papillomaviridae , Genetics , Allergy and Immunology , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Transfection , Vaccines, Synthetic , Allergy and Immunology , Vaccinia virus , Genetics , Allergy and ImmunologyABSTRACT
TNP-conjugated spleen adherent cells (SAC) from normal BALB/c mice could stimulate lymph-node T lymphocytes (LNT) of 1, 3, 5-trinitrobenzene (TNCB) -primed syngeneic mice to proliferate in vitro. The proliferative response was suppressed when phorbol myristic acetate.(PMA) was present in the culture. SAC pretreated with PMA also suppressed markedly the response. Furthermore, PMA was shown to inhibit interleukin-1 (IL-1) production and/or secretion by macrophages in response to LPS stimulation in vitro. Therefore, the suppressive effect of PMA on antigen presentation seems to be due to its inhibitory effect on IL-1 production and/or secretion.
ABSTRACT
Peritoneal macrophages from thioglycollate-treated C57BL/6 mice were separatedby centrifugation on Percoll discontinuous gradients.Macrophages were so separ-ated into four subpopulations and their tumor cytotoxicity and the effect of sodi-um selenite on different subsets were studied.There was no marked difference inphagocytic activity and Fc recepter activity among the four subpopulations.High-density macrophages activated by MAF were kighly cytostatic and cytolyticto tumor cells,while low-density macrophages were not Peritoneal injection of sodiumSelenite(lmg/kg)augmented macrophage-mediated cytotoxic activity by increasingtheir MAF responsiveness which occurred mainly in the low-density macrophages.sodium selenite did not affect macrophage maturation as the percentage of theperoxidasepositive macrophages remained unchanged,
ABSTRACT
The inhibitory effect of Fusarin C,a new mutagen isolated from Fusarium moniliforme,on M? activation was investigated.The inhibitory effect of Fusarin C disappeared partially after 24 hr culture in the absence of the mycotoxin and completely after 72 hr.In addition,it could be overcome by high concentrations of M? activating factor or anti-serum to the target cells.
ABSTRACT
The effect of Fusarin C, a new mutagen extracted from Fusarium moniliforme,on murine peritoneal M? activation by measuring M?-mediated MTC ADCC and CS,Fusarin C could inhibit M? activation by both MAF and FDP. The inhibitory effect was dose-and time-dependent and the curves of dose-and time-response were similar in all three assays. There is, however, apparent difference in Fusarin C dose and time needed to induce significant in hibition among these assays. Significant inhibition occured at 0.4?g/ml and 0.5?g/ml for MTC and CS respectively, but 1.6?g/ml for ADCC. Similarly, the minimal period of time necessary to bring about significant inhibition was 2 hr for MTC, but 3 hr for ADCC. Finally, significance of the inhibitory effect of Fusarin C on M? anti-tumor activity in relation to carcinogenesis was discussed.
ABSTRACT
Target cells K562 were labeled using two different isotopes,~(51)Cr and ~(125)I-UdR,for detecting NK activity of peripheral blood mononuclear cells in normal subjects.The NK activity was higher in ~(51)Cr release assay in comparison with ~(125)I-UdR release assay. After six-hour incubation,the percentage release of ~(51)Cr was around 60% whereas that of ~(125)I-UdR was only 30% in 20 hours. ~(125)I-UdR release could be enhanced by trypsin treatment.