Adherence of human enterotoxigenic and bovine mastitis Escherichia coli to rat embryonic fibroblasts was studied. Adhesion of E. coli strains B34289c (human) and 1407 (bovine) was rapid and reached maximum after 30-40min. Strain 1410 (bovine), which binds fibronectin but not its 29K amino-terminal fragment, did not adhere to the fibroblasts. Strain B34289c grown at 25C or below and at 40C or above lost its binding and adhesive properties simultaneously. Maximum binding and adhesion for this strain was achieved when it was grown at 33C. Strains grown at this temperature adsorbed to fibronectin-, 29K fragment-, and Octyl Sepharose, with the exception of bovine strain 1410, which did not adsorb to 29K-Sepharose as expected. None of the strains adsorbed to cross-linked Sepharose 4B. 29K-IgG and Fab fragments thereof specifically blocked both binding (max 55%) and adhesion (>95%). Sonicated and trypsin-treated bacteria were no longer able to bind or adhere. The supernatant of sonicated bacteria inhibited both binding and adhesion. Penicillin G at 0.5μg/ml (1/5 minimal inhibitory concentration: MIC) and tetracycline at 0.2μg/ml (1/5 MIC), when included in the growth medium, suppressed the cell surface components responsible for fibronectin binding and fibroblast adhesion. The presence of fibronectin was demonstrated in the fibroblast extracellular matrix by immunofluorescens with 29K-IgG antibodies.

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The IgG-Fc binding activity and binding sites on the cell surface of streptococci, strains AR1 (group A) and G148 (group G), and Staphylococcus aureus strain Cowan I were examined by hemagglutination (HA) and immunoelectron microscopic methods. No distinct difference was observed in the HA activity among these three strains. However, the strains differed in the distribution of Fc receptor. Cowan I cells (having protein A) were heavily covered with two layers of ferritin particles, whereas AR1 cells were heavily covered with a single, rough layer of ferritin particles. G148 cells (having protein G) were labeled with a relatively thin, rough ferritin layer. The trypsin susceptibility of the Fc receptors of the AR1 strain was much higher than that of the G148 strain. These results suggest that both streptococcal strains are distinctly different in the arrangement or in the conformation of the Fc receptor from the Cowan I strain. It is also suggested that the Fc receptor molecules of the streptococcal strains differ from each other not only in conformation but also in trypsin susceptibility.

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The effects of panose on glucan synthesis and sucrose-dependent cellular adherence by Streptococcus mutans were investigated. Panose effectively inhibited glucan synthesis from sucrose by glucosyltransferases from S. mutans strain 6715, but increasing amounts of panose increased the release of fructose from sucrose by the enzymes. On the other hand, production of a series of oligosaccharides of increasing size by the enzymes was markedly enhanced in the presence of panose. These results indicate that panose activates the enzymes and that the inhibition of glucan synthesis by panose is due to the transfer of the glucosyl group of sucrose to panose. Sucrose-dependent adherence of cells of various S. mutans strains to a glass surface was also inhibited by panose.

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Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPS-responder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.

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Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages.
The SV40-transformed human endothelial cells, designated SV-HUVEC, were positive not only for tumor (T) antigen specific to the SV40-transformed cell, but also for two markers of endothelial cells, Factor VIII-related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40-transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.

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The highly potent and selective anti-herpesvirus agent, (E)-5-(2-bromovinyl)-2'deoxyuridine (BVdU), was examined for its inhibitory effect on the salmonid herpesviruses Oncorhynchus masou virus (OMV) and Herpesvirus salmonis (H. salmonis). Minimum inhibitory concentrations (MIC) of BVdU for OMV and H. salmonis were 1.25 and 3.0μg/ml, respectively; these values were equal to or higher than those obtained for acyclovir or cytarabin. OMV DNA polymerase activity was reduced in a dose-dependent fashion by BVdU 5'-triphosphate (BVdUTP) within the concentration range of 3 to 30μM. However, BVdUTP could also be substituted for the natural substrate, TTP, in the OMV DNA polymerase assay. It is postulated that the inhibitory action of BVdU on the salmonid herpesviruses is more or less similar to that on other herpesviruses and resides with respect to the inhibition of the virus DNA polymerase activity as well as incorporation of BVdU into the viral DNA.

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Bacteria isolated from two salmonid hatcheries were screened for antiviral activity against infectious hematopoietic necrosis virus (IHNV) to ascertain the presence of bacteria with anti-IHNV activity in the aquatic environment. Out of 710 bacterial isolates from the water and sediment samples, 190 strains showed anti-IHNV activities of more than 50% plaque reduction. These antiviral activities were detected predominantly in Pseudomonas, Aeromonas/Vibrio, and coryneforms. In one hatchery, the bacteria with antiviral activities were more prevalent in sediment samples than in water samples. Seventy-seven percent of the isolates with higher antiviral activities (>90% plaque reduction) belonged to Pseudomonas.

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Campylobacter may be one of the most common causes of bacterial gastroenteritis (GE) in children. It has recently been suggested that it is one of the bacterial pathogens most likely to infect immune-compromised children, and it may facilitate colonization of enteric pathogens. The immune system response was studied in 12 children with Campplobacter fetus subspecies jejuni (CBJ) infections. Serum concentrations of IgA, IgM, and IgG were analyzed using a Beckman autoanalyzer. Sera specific Ab to CBJ were tested with CBJ specific enzyme-linked immunosorbant assay (ELISA). Mitogen stimulation of lymphocytes was performed to three lectins: Con A, PWM, and PHA. The lymphocyte blast transformation to Campylobacter was studied using the Campylobacter antigen. T-cell subsets were studied using the monoclonal antibodies Leu 2, 3, and 4 (Becton Dickinson). Chemotaxis was measured in modified Boyden chambers; chemotactic stimulants were the Formyl Met Leu Phe, Campylobacter antigen virion, and E. coli 0111 B. Immunoglobulins were normal in nine cases and abnormal in two children previously diagnosed as agammaglobulinemic and one diagnosed as hypoagammaglobulinemic. Specific serum Ab level was significantly higher in the CBJ group, except in the agammaglobulinemic group. Stimulation indices to mitogens and monoclonal subset were in the normal range. The blastogenic transformation to CBJ Ag was decreased compared to normal lectins, and positive and high compared to controls. The chemotactic activity to campylobacter Ag was decreased in comparison to other stimulants. Most CBJ infections are self-limiting due to a normal immune response and collaboration of all cellular limbs. When, however, the immune response is disturbed, we may find a prolonged and complicated course of CBJ.

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We measured histamine concentration and its metabolizing enzymes in the skin of MRL/Mp-lpr/lpr (MRL/l) and BXSB mice to clarify the contribution of histamine metabolism to the mechanisms of the development of lupus dermatoses. The concentration of histamine seemed to differ with the mouse strain. The activity of histamine-N-methyltransferase (HMT), one of two major metabolizing enzymes, was significantly lower in the tail and back skin of MRL/l mice at the age of 5 months than in the control MRL/Mp-+/+(MRL/n) mice, although there were no characteristic differences among several mouse strains of 1 mo of age. In the back skin of MRL/l mice, an age-dependent decrease of HMT activity was observed along with a corresponding decrease in histamine concentration, whereas an age-dependent increase of both HMT activity and histamine concentration was demonstrated in BXSB mice and other control mouse strains. Autoimmune-prone male BXSB mice and non-autoimmune female BXSB mice at 5 mo of age showed similar HMT activity. Corticosteroid treatment restored HMT activity in the skin of MRL/l mice but not in MRL/n mice. In addition, the change in HMT activity in MRL/l mice treated with corticosteroid appeared earlier than changes in clinicopathological examinations including skin eruptions, dermatopathology and proteinuria. Diamine oxidase (DAO) activity, another major metabolizing enzyme, was not detected in the skin of any autoimmune or control mouse strains. These findings suggest that the low activity of HMT in the skin of MRL/l mice plays a significant pathological role in the development of spontaneous lupus-like eruption. In other mouse strains, it is assumed that HMT activity is regulated by genetic factors.

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Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte-derived macrophages and a subclone of the myelomonocytic cell line, U937-F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14⁺, OKM5⁺, Mac-1⁺ (equivalent to OKM1 and Mo1), OKT9⁺, HLA-DR^- and CD20⁺. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met-Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti-tumor necrosis factor, anti-lymphotoxin and anti-interleukin 1 antisera.

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Escherichia coli showed the chemotactic response for methamphetamine, a biogenic stimulative amine. The response was qualitatively detected by using a paper disk filter. The quantitative assay showed that E. coli responded to methamphetamine at 10^-5M or more.

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