Demonstration of Akabane virus antigen using immunohistochemistry in naturally infected newborn calves.

Eight newborn calves showing ataxia were necropsied and examined histologically. Six of seven cerebrospinal fluid samples collected from these animals had neutralizing antibody for Akabane virus (AKV). All examined calves had nonsuppurative encephalomyelitis, localized mainly in the midbrain and spinal cord. Corresponding to the encephalitic lesion, AKV antigen was demonstrated in neuroglial cells in the brain stem and neuronal cells in the ventral horn of the spinal cord. This is the first study to demonstrate AKV antigen by immunohistochemistry in naturally infected newborn calves.

Characteristic cytokine products of Th1 and Th2 cells in hemodialysis patients.

Dysfunction of the host defense against infection in hemodialysis (HD) patients has major clinical and socioeconomic implications. T helper type 1 (Th1) and type 2 (Th2) cytokines are implicated in regulating the immune responses and, therefore, may be involved in impaired status. The present study was designed to examine Th1 and Th2 cytokine profiles in 22 stable HD patients (aged 63 +/- 11 years) and 22 healthy controls (aged 60 +/- 6 years). The T cell activity was significantly retarded in HD patients as compared with normal persons. The proportions of T cytotoxic/suppressor cells and natural killer cells were significantly higher in HD patients than in controls. In contrast, the proportions of T helper/inducer and B cells were significantly lower in HD patients than in controls. The production of interleukin (IL) 2, which is involved in cell-mediated immune responses, and the production of IL-4 and IL-10, which affect humoral immunity, were significantly lower in patients than in controls. The production of IL-12 by macrophages and of interferon gamma by Th1 cells was significantly higher in HD patients than in controls. The concentration of plasma sIL-2R was significantly higher in patients than in controls. These results suggest that both cellular immunity induced by Th1 and humoral immunity induced by Th2 decrease in HD patients, but that improved IL-12 secretion by macrophages activated natural killer cells to produce interferon gamma, which in turn induced macrophage activity.

Effects of astragali radix extract on carcinogenesis, cytokine production, and cytotoxicity in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine.

We studied the effects of astragali radix extract, a Chinese herb and one of eight components in Shikaron, on carcinogenesis, natural killer (NK) cell activity, and the cytokine production of lymphocytes in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine (BBN). We found a significantly lower incidence of urinary bladder carcinoma in mice treated with BBN plus 10 mg/kg/day or more of Astragalus extract (7, 2, and 3 mice among 15 mice in 10, 20, and 40 mg/kg/day group, respectively, vs. 14 of 15 mice treated with BBN alone). Astragalus extract prevented the cytotoxic activity of lymphocytes against YAC-1 cells from the depression by BBN. It also protected the production of interleukin-2 and gamma-interferon of lymphocytes from the depression by BBN. These results, including our previous findings, suggest that the Astragalus extract exerts an anticarcinogenic effect in carcinogen-treated mice through activation of cytotoxic activity and the production of cytokines.

Anticarcinogenic effects of shikaron, a preparation of eight Chinese herbs in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine.

We studied the effects of Shikaron, which is composed of 8 Chinese herb extracts, on carcinogenesis and the cytotoxic activity and cytokine production of lymphocytes in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine (BBN). We found a significantly lower incidence of urinary bladder carcinoma in mice treated with BBN plus Shikaron 200 mg/kg/day (5 of 20 mice, 25%), than in mice treated with BBN alone (16 of 20 mice, 80%). Shikaron protected the cytotoxic activity of lymphocytes against YAC-1 cells from suppression by BBN. Cytotoxic activity against P-815 cells significantly increased in mice treated with BBN plus Shikaron, as compared with normal mice and BBN-treated mice. Shikaron also protected production of interleukin-2 and interferon-gamma by lymphocytes from suppression by BBN. These findings strongly suggest that Shikaron exerted an anticarcinogenic effect in carcinogen-treated mice through activation of NK and LAK cells and cytokine production by T lymphocytes.

Immunological characteristics of the effector cells induced by a combination therapy with cyclophosphamide and allogeneic lymphocytes.

A significant inhibition of tumor growth was observed when sarcoma 180 (S180)-bearing ICR strain mice were treated by a combination therapy, with a low dose of cyclophosphamide (CY) and an inoculation of allogeneic lymphocytes collected from C57BL/6 mice. The growth-inhibitory effect was significantly increased by an inoculation of a relatively lower dose of allogeneic lymphocytes (1 x 10(5) cells) and CY. The effector cells induced in the mice treated with CY and allogeneic lymphocytes expressed the Lyt 1.2, Lyt 2.2, IL-2R antigens on their membrane surface and did not express the H2KbDb (donor H-2) antigen, and they showed a specific cytostatic activity against S180 cells. These results strongly suggested that a combination therapy with a low dose of CY with an inoculation of allogeneic lymphocytes augmented an induction of specific cytotoxic T lymphocytes in the tumor-bearing recipient mice.

Effects of Lentinus edodes, Grifola frondosa and Pleurotus ostreatus administration on cancer outbreak, and activities of macrophages and lymphocytes in mice treated with a carcinogen, N-butyl-N-butanolnitrosoamine.

ICR mice were treated with a carcinogen, N-butyl-N'-butanolnitrosoamine BBN), every day for 8 consecutive weeks and the effects of oral administration of edible mushrooms on the induction of urinary bladder carcinoma and on the activities of macrophages and lymphocytes were studied. Bladder carcinoma were found in all 10 mice (100%) treated with BBN alone, while we observed carcinoma only in 9 of 17 mice (52.9%), in 7 of 15 mice (46.7%) and 13 of 20 mice (65.0%) treated with Lentinus edodes, Grifola frondosa and Pleurotus ostreatus, respectively. Chemotactic activity of macrophages was suppressed in mice treated with BBN alone but maintained almost the normal level in mice treated with BBN plus Lentinus, Grifola or Pleurotus. Lymphocytes collected from mice treated with BBN plus each mushroom showed almost normal blastogenic response against concanavalin A, although those from mice treated with BBN alone completely retarded their response. Cytotoxic activity of lymphocytes against Yac-1 cells was also maintained at a normal level in mice treated with BBN plus each mushroom. Whereas in mice treated with BBN alone significant depression of NK cell activity occurred. Significantly higher cytotoxic activity against P-815 cells was observed in lymphocytes from mice treated with BBN plus each mushroom than that in lymphocytes from normal mice or mice treated with BBN alone.

Release of superoxide anion from polymorphonuclear leukocytes stimulated by rubella viral antigen-antibody complex in vitro.

Using a highly sensitive and specific chemiluminescence (CL) method, we detected the release of superoxide anion (O2-.) from human polymorphonuclear leukocytes (PMNLs) stimulated by rubella viral antigen-antibody complex (VAAC) adsorbed on latex particles. The amount of superoxide anion produced by PMNLs was proportional to the amount of VAAC. Neither rubella virus alone nor antibody alone, adsorbed on latex particles, stimulated production of superoxide anion by PMNLs. It is likely that rubella virus requires the presence of antibody in order to be recognized by PMNLs, and that the superoxide anion is somehow involved in the disease process of rubella.

Effects of Chinese herbs on macrophage functions in N-butyl-N-butanolnitrosoamine treated mice.

We investigated the effect of Chinese herbs Lithospermi radix, Astragali radix and Cnidii rhizoma on the functions of macrophages obtained from mice treated with the carcinogen N-butyl-N-butanolnitrosoamine (BBN). The chemotactic activity of murine macrophages was significantly decreased by 17 weeks of treatment with BBN compared with controls. Production of IL-1 and TNF was also markedly reduced. Treatment with Lithospermi radix, Astragali radix, and Cnidii rhizoma significantly inhibited BBN-induced suppression chemotactic activity and production of IL-1 and TNF-alpha by macrophages. Moreover, we found that Astragali radix treated macrophage chemotaxis, it or Cnidii rhizoma induced productions of TNF-alpha were in excess of control.

Effect of shi-ka-ron on cytokine production of lymphocytes in mice treated with cyclophosphamide.

The effect of Shi-ka-ron (CX) on cytokine production of lymphocytes in mice treated with cyclophosphamide was investigated. Shi-ka-ron, a traditional Chinese prescription, consists of 8 crude Chinese herbal extracts. Its main efficacy is to strengthen the body resistance. We observed that each CX component stimulated interleukine-2 and interferon-gamma production of murine splenic lymphocytes both in vitro and in vivo. IL-2 and IFN-gamma production of splenic lymphocytes were also examined in mice treated with CX combined with cyclophosphamide (CY) in vivo. We found that CX not only could increase IL-2 and IFN-gamma production in vitro, but also in vivo at suboptimal concentration.

Release of superoxide radicals by mouse macrophages stimulated by oxidative modification of glycated low density lipoproteins.

Diabetic patients have high levels of glycated LDL. Although glycated LDLs are implicated in the development of atherosclerosis in such patients, convincing data are lacking. We observed release of superoxide radicals (O2-) from mouse resident peritoneal macrophages stimulated by an oxidized/glycated LDL by using a highly sensitive and specific chemiluminescence method. Oxidized/glycated LDL was achieved by an addition of low concentration of Fe3+ to glycated LDL. Macrophages took up an appreciable amount of the glycated LDL oxidized by iron, leading to the development of foam cells, while they did not take up untreated glycated LDL or the native LDL. These observations clearly indicate that the oxidized/glycated LDL reacts well with macrophages. Since an oxidation of glycated LDL may occur in vivo, the oxidized/glycated LDL might play an important role in atherogenesis.

[Immunomodulative effects of Chinese herbs in mice treated with anti-tumor agent cyclophosphamide].

Extracts of Chinese herbs were administered with antitumor agent, cyclophosphamide (CY), and their effects on macrophages and lymphocytes were studied. Number of peritoneal macrophages significantly decreased and their chemotactic activity was suppressed by treatment with CY. Blastogenic responsiveness to Concanavalin A and NK cell activity of spleen lymphocytes were suppressed significantly in CY-treated mice. Extracts of Lithospermi radix, Astragali radix and Glycyrrhizae radix showed protective effects on immunosuppressive mice. The number of macrophages, chemotactic activity of macrophages and blastogenic response of lymphocytes were recovered to the same or more than that of normal levels. An extract of Ginseng radix showed protective effects on the number and functions of macrophages by treatment with CY but did not show any effects on the lymphocytic blastogenesis. On the contrary it showed a strong inhibitory effect on the NK cell activity. These results suggest that Chinese herbs could modulate cellular immune response, especially in the activation of macrophages and splenic lymphocytes.

Epidemiologic analysis of spatial clustering of bovine ephemeral fever outbreaks. II. Principal component analysis.

The principal component analysis (PCA) was applied to analyze a correlation matrix of three variables on epidemic data of bovine ephemeral fever (BEF) outbreaks. These original data were summarized from the official outbreak report of Fukuoka Prefecture. The first and the second principal components of the PCA were interpreted as the infectious potency due to BEF virus and the prevention against BEF virus infection, respectively. The BEF outbreak areas were able to be classified epidemically into 4 groups by using the two principal components. The valuable epidemiological insights can be reasonably obtained from an application of the PCA. The results provided an important information for a further BEF vaccination campaign in the western part of Japan.

Effect of shi-ka-ron and Chinese herbs on cytokine production of macrophage in immunocompromised mice.

Shi-Ka-Ron is a prescription composed of 8 crude extracts of Chinese herbs. It reduces suppression of cytokine production by peritoneal macrophages in mice Immunocompromised by the anti-tumor agent, cyclophosphamide (CY), in vivo. Although it dose not increase IL-1 production in vitro, it enhances TNF production. We found that Ginseng radix, Lithospermi radix, Astragli radix and Glycyrrhizae radix somewhat reduced suppression of cytokine production in CY treated macrophages. Especially, Glycyrrhizae radix shows an active immune response both in vivo and in vitro. Our results suggested that the mechanism underlying immunomodulation of Shi-Ka-Ron is closely related to cytokine production: each herb stimulating macrophages.

[In vitro induction of chlorhexidine- and benzalkonium-resistance in clinically isolated Pseudomonas aeruginosa].

A study was made on the MIC distributions of chlorhexidine and benzalkonium chloride against clinically isolated 178 strains of Pseudomonas aeruginosa to find out the existence of strains resistant to those disinfectants and also on the in vitro induction of resistance to both drugs. The MIC of chlorhexidine gluconate was found to be distributed from 78 to 625 micrograms/ml with a single peak at 312 micrograms/ml. All 178 strains of clinical isolates were sensitive to chlorhexidine and none could be induced to become chlorhexidine resistant in vitro, suggesting that P. aeruginosa can not easily acquire chlorhexidine resistance. On the other hand, the MIC of benzalkonium chloride was distributed in two peaks; one peak was benzalkonium sensitive at 625 micrograms/ml (150 strains/178 strains: 84.3%) and the another peak was benzalkonium resistant at 5,000 micrograms/ml (28 strains/178 strains; 15.7%). Six (4.0%) of the 150 benzalkonium sensitive strains acquired benzalkonium resistance by in vitro induction of resistance; the MIC of 5 strains increased from 625 micrograms/ml to 2,500 micrograms/ml and that of the residual 1 strain increased from 312 micrograms/ml to 1,250 micrograms/ml. However, no change of MIC was observed in 28 benzalkonium-resistant strains of clinically isolated P. aeruginosa by in vitro resistance induction. Strains with MIC more than 5,000 micrograms/ml could not be obtained at all. The results suggest that the benzalkonium resistance can be introduced in P. aeruginosa whereas the resistance-acquiring rate is low. These results suggest that chlorhexidine gluconate is the first choice for prevention of Pseudomonas infection in the hospital and benzalkonium is also useful in 0.5% solution is used.

Epidemiologic analyses of spatial clustering of bovine ephemeral fever outbreaks. I. Application of the negative binomial distribution.

An epidemic that propagates through a population results in a contagious spatial pattern. The incidence per farm of bovine ephemeral fever (BEF) outbreaks in the 22 areas of Fukuoka Prefecture in 1988 was well explained by the negative binomial distribution, which is generally considered, as the clustering of the outbreaks. Furthermore, the clustering was analyzed with three categories of vaccination rate (No. 1-40%, and 40-80%). As a result, the no vaccination and high vaccination rate (41-80%) groups tended to shift to similarly the Poisson distribution. It may be speculated that an insufficient vaccination control of the BEF was influenced with the clustering.

Immunological properties of TtT/M-87 cell line established from murine pituitary tumor-associated macrophages.

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-alpha and IL-1 alpha, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-gamma. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.

[Relationship between hand-washing activity of disinfectants and in vitro bactericidal activity in the presence of peptone].

A study was made on the relation between in vitro bactericidal activity against Staphylococcus epidermidis which is the main bacterial flora of skin and the disinfecting activity of hand-washing with povidone-iodine, sodium hydrochloride, chloramin-T, chlorhexidine gluconate and benzalkonium-chloride. Although the bactericidal activity of povidone-iodine and sodium hydrochloride was significantly high as compared with those of the other three disinfectants, both povidone-iodine and sodium hydrochloride showed no or a little hand-washing effects. In vitro the bactericidal activity of both povidone-iodine and sodium chloride were significantly retarded by the addition of peptone in the reaction mixture. On the other hand, chloramin-T, chlorhexidine gluconate and benzalkonium chloride showed a high disinfecting activity in hand washing in spite of low in vitro bactericidal activity. Moreover, the bactericidal activity of these three disinfectants were not suppressed by the addition of peptone in the reaction mixture. These results strongly suggested that hand washing effect of disinfectant is affected by the protein on the hand and that hand-washing activity of disinfectant should be determined by in vitro bactericidal activity in the presence of peptone.

Characterization of a tick isolate of Borrelia burgdorferi that possesses a major low-molecular-weight surface protein.

An unusual strain of Borrelia burgdorferi (DN 127 cl 9-2) that was isolated from an Ixodes pacificus tick did not react with monoclonal antibodies (MAbs) to OspA and OspB surface proteins, which are found in most U.S. strains. The strain exhibited an abundant protein with an apparent molecular weight of 25,000 (25K protein). A MAb, 86 DN-1, that was prepared to the 25K protein was used in studies on the effect of proteases on the intact spirochetes, immune electron microscopy, and Western blot (immunoblot) analyses; the results indicated that the low-molecular-weight protein was an apparent surface protein that was loosely attached to the spirochete. Five tick isolates from California possessed low-molecular-weight proteins in the 20,000- to 25,000-molecular-weight range that reacted with the 86 DN-1 MAb. The 25K protein of DN 127 cl 9-2 was unaffected by prolonged in vitro passage of cultures in BSK II medium, while the low-molecular-weight proteins of the other strains of B. burgdorferi from California either decreased in quantity or became undetectable on long-term in vitro passage.

Anti-tumor effects of the oral administration of the streptococcal preparation OK-432 (PICIBANIL)--the inhibition of carcinogenesis and growth in rats with ENNG-induced gastrointestinal tumors.

We examined whether the Streptococcal preparation OK-432, an immunopotentiating agent, increases immunocompetence of the gut-associated lymphoid system (GALS), inhibits gastrointestinal carcinogenesis, and has an anti-tumor effect. 14C-labelled OK-432 was orally and intraperitoneally administered to rats, and the distribution of the agent in various organs then serially evaluated. The concentration of OK-432 in Peyer's patches and mesenteric lymph nodes was higher after oral administration than after intraperitoneal administration, and showed a biphasic pattern peaking at 30 minutes and 5 hours following administration, in the Peyer's patches. With regard to immunocompetence, PHA- and PWM-stimulated blastogenesis of lymphocytes derived from the mesenteric lymph nodes and peripheral blood enhanced, and the helper/suppressor T-cell ratio was elevated after the oral administration of OK-432. Moreover, chemotactic activity of peritoneal macrophages was also increased. ENNG-induced gastrointestinal carcinogenesis was observed in 60 per cent of the rats orally administered OK-432 as compared with 88 per cent of the controls. The 13-month survival rate of the rats with gastrointestinal cancer was 50 per cent in those administered OK-432 as compared with 25 per cent in those administered OK-432 as compared with 25 per cent in the controls. When administered orally, the agent prevented reduction in immuno-competence in the course of carcinogenesis, suppressed carcinogenesis, and prolonged the survival of animals with cancer without any of the side effects associated with injection. The oral administration of OK-432 is thus considered to be an effective non-specific immunotherapy against gastro-intestinal malignancies.

Effect of vitamin B complex on the immunodeficiency produced by surgery of gastric cancer patients.

We studied the effect of vitamin B complex (vitamin B1, B6 and B12 complex) on the immune responsiveness in gastric cancer patients who underwent surgery. The depression of blastogenic responses to both PHA and PWM was observed 2 weeks after surgery in half of the patients treated with Vitamedin but the degree was significantly less than that in the control patients without vitamin B treatment whose lymphocyte responses were depressed. Moreover, the blastogenic responses were induced by vitamin B administration 2 or 4 weeks after surgery in 5 of the 8 stage III-IV patients whose lymphocytes had not responded prior to surgery. Four weeks after surgery, the patients without vitamin B treatment showed only a tendency of recovery of their lymphocyte responses, whereas the recovery of blastogenic responses in the patients treated with vitamin B was significant. Essentially similar results were obtained with skin reactions to PHA and PPD. These results suggest that the administration of vitamin B1, B6 and B12 complex is useful for the protection against and the recovery of immune dysfunction produced by surgery in cancer patients.