Effects of cyclosporin A on cell fusion in a monkey kidney cell line persistently infected with measles virus.

The authors studied the effects of immunosuppressive peptide cyclosporin A (CsA) on cell fusion efficiency in cells persistently infected with measles virus (448-PI-Vero cells). Treatment of 448-PI-Vero cells with 5 microM CsA enhanced the infusion. In addition, the expression of measles virus antigen on cell surface was increased by treatment with CsA. The addition of phenothiazine, an anti-calmodulin drug, enhanced the fusion of 448-PI-Vero cells in the presence of CsA, although treatment with phenothiazine alone did not affect polykaryocyte formation. The enhancement of fusion efficiency in 448-PI-Vero cells by CsA was suppressed by oligopeptide Z-D-Phe-Phe-Gly, a synthetic oligopeptide that inhibits fusion induced by measles virus. Since the cell content of major virus-specific polypeptides, such as hemagglutinin, nucleoprotein or matrix protein is the same as in untreated controls, this fusion enhancement may be related to transport and accumulation of measles virus glycoproteins.

Targeted gene transfer for adenocarcinoma using a combination of tumor-specific antibody and tissue-specific promoter.

We have developed a highly specific gene transfer method for adenocarcinoma using a monoclonal antibody against tumor-specific antigen coupled with a plasmid containing the carcinoembryonic antigen (CEA)-specific promoter. The chimeric CEA promoter (CC promoter), which contained an enhancer from the immediate early gene of cytomegalovirus and the CEA promoter, achieved 4- to 5-fold higher transgene expression in CEA-producing cells than the original CEA promoter while maintaining CEA specificity. Furthermore, a complex of a monoclonal antibody against Lewis Y antigen (LYA), the CC promoter-containing plasmid and cationic liposomes (DOTAP) achieved specific gene expression in CEA-producing and LYA-positive adenocarcinoma cell lines that was 200-fold more efficient than in CEA-non-producing and LYA-negative cell lines during a short in vitro incubation. This strategy may be applicable for clinical gene therapy.

[Inhibitory effects of portal venous injection of type II collagen in collagen-induced arthritis].

Collagen-induced arthritis (CIA) is useful animal model for human rheumatoid arthritis. We investigated the inhibitory effects of portal venous (p.v.) injection of type II collagen (CII) in CIA. The arthritis was suppressed by p.v. injection of CII before immunization for CIA induction. The p.v. route was more effective than intravenous or intragastric routes in the induction of tolerance in CIA. The dose of CII necessary for CIA suppression was 10 micrograms/20 g body weight in p.v. injection. Both anti-CII IgG and anti-CII IgG 2 a levels in serum were reduced in mice injected CII before induction of CIA. However, anti-CII IgG 1 levels did not differ between mice injected with CII and mice injected with buffer alone. Thus, the specific reduction in anti-CII IgG 2 a levels in mice treated by p.v. injection before immunization suggests that the suppression of CIA could be responsible for hypofunction of Th 1 cells. Reduction of anti-CII IgG and suppression of arthritis were observed when CII was injected through portal vein after immunization for CIA as well.

The effects of cyclosporin A on the polykaryocyte formation induced by measles virus in a monkey kidney cell line.

The effects of cyclosporin A (CsA) on the polykaryocyte formation induced by measles virus (MV) in a monkey kidney cell line (BSC-1) were studied. CsA inhibited virus-induced polykaryocyte formation as well as the production of infectious MV. The development of polykaryocyte formation in the presence of the CsA varied with virus strains, while pretreatment of the cells with 5 microM CsA for 24 hr before the virus infection enhanced polykaryocyte formation. These data demonstrated that CsA not only inhibits but also enhances virus-induced polykaryocyte formation depending on the conditions of its use.

Production of B cell differentiation factors by mouse parenchymal liver cells.

Previously we reported that most antibody secreting cells secreted IgA in the liver. Here we assessed the possibility that parenchymal liver cells (PLC) produced factors, transforming growth factor (TGF)-beta and IL-5, which participate in the differentiation of B cells to IgA-secreting cells. We showed that TGF-beta activity was present in the culture supernatant of PLC, and IL-5 activity was in the lysate of PLC. Moreover, it was confirmed that IL-5 protein produced by PLC was mainly localized in the cell membrane by histochemical staining. The findings that both TGF-beta and IL-5 were produced by PLC should provide useful information concerning the fact that IgA-secreting cells were dominant in the liver.

Changes in M-CSF-like activity during chicken embryonic development.

We have established a method for separation of chicken bone-marrow cells using Percoll density gradient centrifugation, and have developed a new method for determining chicken M-CSF-like activity employing a liquid culture. Using this method, we determined M-CSF-like activities in egg yolk, chorioallantoic fluid (CAF) and amniotic fluid (AmF), and studied the effects of M-CSF on development of chicken embryos. M-CSF-like activity in egg yolk was at a high level before the incubation of the egg; it began to decrease on the third day of incubation and rapidly decreased on the fourth day, and no significant activity was detected after the tenth day of incubation. M-CSF-like activity in CAF was very low, and it exhibited almost no change during development. No M-CSF-like activity was detected in AmF throughout the experimental period.

Patterns of lymphokine production by Semliki Forest virus-specific T-cell hybridomas stimulated with different antigen-presenting cells.

The development of infection seems to be influenced by the characteristics of antigen-presenting cells (APC) in the infection site. Thus, we compared the Semliki Forest virus (SFV)-antigen-presenting capacity of spleen cells, B-cell lymphomas, bone marrow-derived mast cells and nonparenchymal liver cells by measuring the production of lymphokines in SFV-specific T-cell hybridomas. Spleen cells were able to provide the signals needed to stimulate the production of IL-2, IL-4, IL-6 and IFN-gamma, while B lymphomas the signals leading to only IL-2 production. When bone marrow-derived mast cells were used as APC, SFV-specific T-cell hybridomas produced IL-2, IL-4 and IL-6 in the presence of soluble anti-CD3 antibody. However, no lymphokine production was detected when the SFV antigen was used instead of the antibody. Nonparenchymal liver cells containing liver endothelial cells and Kupffer cells have an APC function stimulating the production of IL-2 and IL-6. These findings confirmed that the T-cell hybridomas can be selectively stimulated by different APC to produce different lymphokines, and it would influence the development of the immune-mediated inflammatory response.

[An improved colorimetric assay for chicken M-CSF like activity using chicken bone marrow cells cultured on micro-culture plates].

The role of M-CSF in the process of mammalian development has been drawing attention. Unlike mammalian embryonic development, in which M-CSF is supplied by the maternal body, avian embryonic development begins and proceeds under the influence of a certain amount of M-CSF present in each fertilized egg, and its concentration can be experimentally controlled while observing the embryo's developmental state. Apart from this fact, aves provide us with many other advantages in the study of embryonic development. It is, however, not easy to separate multipotent hematopoietic stem-cell fractions from avian bone marrow. In addition, because a macrophage colony forming assay in soft agar, though it has been widely used, requires a large number of cells and a long culture period, the method of assay is not adequate for the sensitive detection of M-CSF activity in small scale samples. We have established a method for the depletion of nucleated erythrocytes from chicken bone marrow cell suspensions using percoll density gradient centrifugation, and have also developed a new method for determining chicken M-CSF-like activity employing a liquid culture. In this method, a 100 microliters aliquot of fractionated hematopoietic stem cells, 1 x 10(5), was placed in a well of 96-well flat-bottom culture plate, 100 microliters of sample was then added to each well, and the uptake of neutral red was measured after 4 days of culture. These procedures represent a simple and sensitive means of detecting M-CSF-like activity in chicken serum of x60 to x32 dilutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth control of primary culture hepatocytes by nonparenchymal liver cells. Role of interferon produced by liver sinusoidal cells.

Using the primary culture of liver cells, we showed that interferon produced by nonparenchymal liver cells inhibits the proliferation of cultured parenchymal liver cells. DNA synthesis of parenchymal liver cells was suppressed not only by their coculture with nonparenchymal liver cells but also by the addition of the culture supernatant of nonparenchymal liver cells. The suppressive activity of the supernatant correlated closely with the interferon (alpha + beta) level in the supernatant and was reduced by anti interferon (alpha + beta) serum. Furthermore, purified interferon (alpha + beta) also suppressed parenchymal liver cell proliferation in a dose-dependent manner and the suppression was released by anti interferon (alpha + beta) serum. The interferon level of the supernatant necessary for suppressing parenchymal liver cell proliferation, however, was extraordinarily low compared with purified interferon. The possibility exists that IFN in the culture supernatant of nonparenchymal liver cells works synergistically with other factors in the supernatant to suppress the cell proliferation.

[Morphology and function of intra-hepatic lymphocytes derived mast cells].

We have examined the functional characteristics of mast cells grown in tissue culture from intra-hepatic lymphocytes of mice (IHL-MC) and compared them with mast cells grown from bone marrow (BMC-MC). Intrahepatic lymphocyte derived mast cells had the functional characteristics of cultured mast cells. These cells were stained by alcian blue, had a lower histamine content than rat peritoneal mast cells (considered to be a model of connective tissue type mast cells) and responded to Ca2+ ionophore and IgE receptor mediated stimulation. However, there were some differences between IHL-MC and BMC-MC. IHL-MC had a higher histamine content and lower growth activity by T-cell derived factor than BMC-MC.

T-cell hybridomas recognizing the envelope proteins of Semliki forest virus: their sensitivity to endo/lysosomal protease and the antigenicity.

Eight T-cell hybridomas were established from the draining lymph node of C3H mice immunized with Semliki forest virus (SFV). Six of them showed specificity toward viral-structure protein E2, while the remaining two clones included one with specificity to an other structural protein E1 and the other with specificity to C. The production of IL-2 by the E2 protein-specific T-cell hybridomas in the presence of SFV was suppressed by treating the antigen-presenting cells (APC) with ammonium chloride raising pH of the acidic compartments. It was found also that treatment of APC with a thiol protease inhibitor, leupeptin or E64, resulted in a reduced response of some of the E2-specific T-cell hybridomas. The E2 protein of SFV proved to be resistant at pH 7.0, and sensitive at pH 5.0 to in vitro cathepsin B treatment. In contrast, the E1 and C proteins proved to be resistant to both pH values. These results indicate that the thiol protease, probably cathepsin B, works as one of the enzymes group involved in antigen processing.

The liver and the hematolymphoid system: I. The regulation of nylon-passed spleen cell proliferation by active factors released from syngeneic nonparenchymal liver cells.

Nylon-passed spleen cells were found to proliferate when cultured with syngeneic nonparenchymal adherent liver cells and their culture supernatants. The supernatants contained IL-1, IL-6, GM-CSF, and IFN (alpha + beta) activities but not IL-2 and IL-3 activities. The IFN level was higher in early culture sup (2-24 hr) than in later culture sup (48-72 hr). Proliferation was greatly increased by anti-IFN (alpha + beta) serum in the spleen cells cultured in the earlier sup. This antiserum increased the spleen cell proliferation only slightly in the later culture sup. This suggests that nonparenchymal liver cells produce two factors, one having a suppressor, and the other an enhancer action, with IFN being one of the suppressor factors. With culture time, DNA synthesis of spleen cells increased and IL-2 and IL-3 activities were generated in the culture sup. Cells proliferated during culture were found to be morphologically lymphocytes, granulocytes, and macrophages. The mechanisms by which nonparenchymal liver cells regulate the hematolymphoid system are discussed based on our observations.

A synthetic peptide induces long-term protection from lethal infection with herpes simplex virus 2.

Immunization against viral pathogens is generally directed toward the induction of virus neutralizing antibody (VNA) and the maintenance of the potential for a second-set (IgG) response. Indeed, an elevated level of specific antibody is considered a reliable clinical indicator that a state of immunity exists in the host. However, in the case of herpes simplex virus (HSV), the presence of circulating VNA does not necessarily correlate with protection. Thus, it has been found that secondary infections occur in individuals even with high neutralizing titers to HSV, suggesting that antibody to the virus may be useless or even deleterious. In consideration of these facts, we were interested in inducing a T cell response to HSV. We had already shown that synthetic peptides corresponding to the NH3-terminal region of the glycoprotein D (gD) molecule of HSV could induce a strong T cell response when injected into mice, but did not, by themselves, confer protection. In this report, we examined the ability of peptides, covalently coupled to palmitic acid and incorporated into liposomes, to induce virus-specific T cell responses that confer protection against a lethal challenge of HSV-2. We have demonstrated that long-term protective immunity is achieved with a single immunization in the absence of neutralizing antibody when antigen is presented in this form. Furthermore, T cells but not serum from such immune mice can adoptively transfer this protection.