Showing papers in "Microbiology and Immunology in 1980"

Trypsin Action on the Growth of Sendai Virus in Tissue Culture Cells

Abstract: A trypsin-like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC-MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin-like protease which activates the inactive form of Sendai virus. The activation was strongly inhibited by phenylmethylsulfonylfluoride, ethyl-enediaminetetraacetate, antipain, and leupeptin but not by tosyllysylchloromethylketone, suggesting that the activating enzyme in CAF is a protease similar to but not identical with trypsin. The inactive form of the virion was produced in ovo when the seed virus was inoculated along with antipain or leupeptin. In deembryonated chicken eggs in which CAF was substituted for a culture medium, multiple cycle growth occurred, but not when soybean trypsin inhibitor was present. These observations indicate that some activating enzyme, possibly the same one as found in CAF, was secreted from the chorioallantoic membrane.

A Complement Inhibitor Produced by Stachybotrys complementi, nov. sp. K‐76, a New Species of Fungi Imperfecti

Abstract: A complement inhibitor, K-76, was isolated and purified from the culture supernatant of a fungus, Stachybotrys complementi, nov. sp. K-76, isolated from soil of Ishigaki Island, Okinawa. K-76 is a sesquiterpene compound and it can be oxidized to a monocarboxylic derivative (K-76 COOH), the sodium salt of which is very soluble and much less toxic than K-76. K-76 and K-76 COOH both inhibited complement activation by either the classical or alternative pathway. They inhibited generation of the factor chemotactic to human polymorphonuclear leukocytes from human serum by aggregated immunoglobulin. When sensitized erythrocytes were treated with complement in the presence of K-76 COOH, the resulting unlysed cells were found to be in the state of EAC1, 4b, 2a, 3b. Thus K-76 COOH is considered to block mainly the C5 intermediate step. K-76 COOH did not inhibit any proteases or esterases tested, except when tested at high concentration.

Sugar composition of O-antigenic lipopolysaccharides isolated from Vibrio parahaemolyticus

Abstract: Vibrio parahaemolyticus, a causative bacterium of food poisoning unique for its particular primary association with sea products, is now divided serologically into 11 or 12 O-forms based on agglutination and agglutinin-absorption tests. We determined the sugar composition of the somatic O-antigens, i.e., lipopolysaccharides (LPS), of representative strains of each O-form. Of particular interest is the absence of evidence for the presence of 2-keto-3-deoxy-octonic acid (KDO), a regular sugar component of gram-negative bacterial LPS, in any LPS examined, with the exception of 06. Furthermore, 07 and 012 LPS contained a KDO-like compound that is, however, not identical with KDO. Glucose, glucosamine, and L-glycero-D-mannoheptose were found as common sugar constituents. Three unidentified amino sugars, designated here as P1, P2, and P3, were found. Various combinations of each of these unidentified amino sugars, and of galactose, fucose, arabinose, D-glyucero-D-mannoheptose, galactosamine, KDO, and the KDO-like substance were detected in accordance with the O-form of LPS. On the basis of the sugar composition, LPS of the 12 O-forms of V. parahaemolyticus can be classified into nine chemotypes, because 03, 05, and 011 LPS belong to the same chemotype and 07 and 012 to another chemotype.

Adenylate Cyclase Activity of Bordetella Organisms

Abstract: Abundant adenylate cyclase activity was found in the phase I cultures not only of Bordetella pertussis but also of B. parapertussis and B. bronchiseptica. The enzyme activity in the culture fluid increased rapidly and reached a peak during the logarithmic growth phase. B. parapertussis and B. bronchiseptica especially produced a high activity of the enzyme in the culture fluid during the logarithmic phase, but little or no activity was detected in the cells throughout the growth period. In the culture of B. pertussis, the intracellular activity was higher than that in the culture fluid. Phase III cultures of these species lacked both the extracellular and intracellular enzyme activities throughout their growth. In the culture of B. parapertussis, accumulation of cyclic AMP was parallel to that of adenylate cyclase activity through the growth periods, but in B. pertussis there was no parallelism from the stationary through the declining phases. The difference in production patterns of the enzyme activity among the species is discussed.

Macrophage Activation by Muramyl Dipeptide as Measured by Macrophage Spreading and Attachment

Abstract: A synthetic bacterial cell wall constituent, muramyl dipeptide (MDP), was found to induce the enhancement of macrophage spreading and attachment on glass or plastic surfaces. Macrophages exposed to bacterial lipopolysaccharide or lymphokine-containing cell supernatants showed similar enhancement. This finding supports the view that MDP activates macrophages. MDP was also found to enhance the viability of macrophages but to inhibit 3H-thymidine incorporation by macrophages.

Reassembly of the Regularly Arranged Subunits in the Cell Wall of Lactobacillus brevis and Their Reattachment to Cell Walls

Abstract: The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in the native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamide. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L. plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.

Production and Properties of Immune Interferon from Spleen Cell Cultures of Toxoplasma‐Infected Mice

Abstract: In response to antigenic stimulation, spleen cells from Toxoplasma-infected mice produce a factor showing inhibitory activity against vesicular stomatitis virus infection in L cell cultures. When BALB/C and ICR mice were inoculated intraperitoneally with the low-virulent S-273 strain of T. gondii, such activity was first detected in 4 and 7 days and reached maximum levels at 10 and 14 days respectively, and retained these levels for at least three weeks. However, BALB/C mice, which are considerably more sensitive to Toxoplasma infection than ICR mice, produced significantly smaller amounts of interferon (IF) after challenge with the high virulent strain. The IF produced in this system possessed certain known properties of immune (type II) IF and was not neutralized by rabbit antiserum against mouse type I IF. The immune IF preparation also inhibited multiplication of Toxoplasma within nonphagocytic L cells in an IF-like fashion, whereas Newcastle disease virus-induced (type I) IF had no effect on this parasite. The antiviral and anti-Toxoplasma activity in immune IF preparations could not be distinguished solely on the bases of their molecular weight and isoelectric point. The experiments with anti-θ serum plus complement and with nylon wool column effluent cells strongly suggest that immune IF was produced by T lymphocytes and required the assistance of macrophages.

The Pathogenicity of Newcastle Disease Virus Isolated from Migrating and Domestic Ducks and the Susceptibility of the Viral Glycoproteins to Proteolytic Cleavage

Abstract: Besides the chicken, other poultry are known to be susceptible to Newcastle disease virus (NDV) (9), and the virus appears to be distributed widely in various wild birds as indicated by isolation of new NDV strains from such birds (10, 11). We considered it to be of ecological interest to learn the properties of such virus strains since there is a possibility that some species of wild birds act as a reservoir from which the infection in fowls arises. Recently evidence that pathogenic and apathogenic strains of NDV differ from each other with respect to the susceptibility of the viral glycoproteins to proteolytic cleavage was obtained (4, 6). The glycoproteins of pathogenic strains are readily cleaved to become biologically active molecules in all types of host cells tested, whereas those of apathogenic strains are cleaved only in the endoderm of the chorioallantoic membrane of the chick embryo but not in most other cells. On the basis of these studies we have characterized five new NDV strains, D-13, D-26, D-28, D-43, and D-49, which were recently isolated from various migrating and domestic ducks in Japan by Yamane et al (11). The stock viruses were grown in the allantoic cavity of 11-day-old chick embryos. The capacity of the viruses to kill chick embryos was determined by measuring the mean death time (MDT) of the embryo after inoculation of 102.5 EID50 of the viruses into the allantoic cavity of 11-day-old chick embryos (9). Labeling of virus with [3H]-glucosamine (New England Nuclear Corp., 10-30 Ci/mmol) in MDBK cells and isolation of the labeled virions were carried out as described previously (4, 5). To analyze proteins and glycoproteins, virions were dissociated with 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol and subjected to electrophoresis on polyacrylamide slab gels in Tris-glycine buffer with SDS (2). [3H]-glucosamine on the gels was detected by scintillation autography (1). Hemagglutinin, neuraminidase and hemolytic activities were determined as described previously (4). Infectivity titers were determined by infective doses for MDBK cells as indicated by the production of detectable amounts of hemagglutinin (8). First we examined the virulence of the newly isolated strains for chick embryos by determining the MDT. All of the five strains were found to have an MDT of

Adjuvant action of capsular polysaccharide of Klebsiella pneumoniae on antibody response. VIII. Its effect on the size and the number of cells of regional lymph node and other lymphoid organs.

Abstract: Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control) Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus Although LPS prepared by Westphal's method from Escherichia coli 055 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E coli O111 or that by Boivin's method from E coli O55 was similar to that of CPS-K It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action

An Outbreak of Acute Enteritis Due to Campylobacter fetus Subspecies jejuni at a Nursery School in Tokyo

Abstract: An outbreak of acute enteritis due to Campylobacter fetus subspecies jejuni involving a total of 35 out of 74 children occurred at a nursery school in Tokyo in January 1979 and lasted for 7 days. It was the first case of a community outbreak of the disease to be recognized in Japan. The major symptoms observed in the patients consisted of diarrhea (88%), fever (82%), abdominal pain (39%), and vomiting (6.1%). The rate of isolation of the organism from the patients was 39%. Sera of four patients showed elevated agglutinin titers against the organism ranging from 1: 80 to 1: 320. Although it is suggested that the outbreak was caused by a communal lunch or between-meal snacks prepared by and provided at the nursery school, the incriminated food, source and route of contamination could not be pinpointed.

Hydrocephalus in Suckling Rats Infected Intracerebrally with Mouse Hepatitis Virus, MHV-A59

Abstract: After intracerebral inoculation of mouse hepatitis virus, MHV-A59 strain, into 3- to 5-day-old Wistar rats, some survivors at 14 days postinoculation (p.i.) were found to lack the cerebral cortex and to have an accumulation of considerable amount of cerebrospinal fluid. The virus titer in the brain increased exponentially after inoculation, reaching a maximum 4 to 6 days p.i. when immunofluorescence revealed virus-specific antigen within neurons in the cerebral cortex. A small amount of infectious virus was also detectable 14 days p.i. when the cerebral anomaly was evident. This brain malformation causing hydrocephalus was due to cerebral damage by viral infection.

Adjuvant action of capsular polysaccharide of Klebsiella pneumoniae on antibody response. IX. Its effect on the histology of the regional lymph node and other lymphoid organs.

Abstract: The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.

Polyclonal B Cell Activation by Cell Wall Preparations of Gram-Positive Bacteria

Abstract: The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram-positive bacteria were studied using the anti-sheep red blood cell (SRBC) or anti-trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS-responder), C3H/HeJ (LPS-non-responder), (CBA/N × Balb/c) F1 male with an X-linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA-ability of synthetic peptidoglycan, muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.

Purification and Some Properties of Tetanolysin

Abstract: Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050-fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate-gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ-toxin of Cl. perfringens.

Microbial adjuvant and autoimmunity. IV. Production of lesions in the exocrine pancreas of mice by repeated injection of syngeneic pancreatic extract together with the capsular polysaccharide of Klebsiella pneumoniae.

Abstract: Definite lesions in the exocrine pancreas were produced when SMA mice were immunized eight times at intervals of 30 days with a mixture of extract of pooled pancreas from syngeneic mice and the capsular polysaccharide of Klebsiella pneumoniae type I Kasuya strain (CPS-K), whereas no pancreatic lesions were produced in mice given CPS-K alone or pancreatic extract alone. The typical histological changes were characterized by infiltration with lymphocytes, plasma cells, and other mononuclear cells, degeneration and lysis of the acinar cells, destruction of the lobular architecture, and replacement of fatty tissue and fibrous connective tissue. The endocrine islets were well preserved. No specific histological changes were produced in the organs other than the pancreas in these mice. Most of mice immunized with pancreatic extract mixed with CPS-K produced serum precipitins to syngeneic pancreatic antigens. However, severe pancreatic lesions were also produced in mice showing no definite precipitin production.

Heat-Stable Enterotoxin Produced by Yersinia enterocolitica Isolated from Patients

Abstract: Twenty-three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15-fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 micrograms of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C X 20 min or 100 C X 60 min). These observations indicated that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat-stable enterotoxin of Escherichia coli. However its physiochemical properties seem to be different from those of E. coli.

Nocardia farcinica as a Pathogen of Lung Infection

Abstract: Strains of Nocardia asteroides and the type strain of Nocardia farcinica, ATCC 3318, have been considered to be homogeneous (7, 8), and Gordon and Mihm (8) proposed N. asteroides as the type species of the genus Nocardia, assigning strain ATCC 3318 to the species N. asteroides. In contrast, Mariat (13) and Cerbon (3) suggested the heterogeneity of N. asteroides. Magnusson and Mariat (12) differentiated strains received as N. farcinica from strains of N. asteroides by skin reactions in guinea pigs. Tsukamura (18) stated that strains received as N. asteroides could be divided into two distinct clusters by numerical classification, and designated the group containing the ATCC 3318 strain as N.

Effect of Oxidizing Agents and Sulfhydryl Group Reagents on Beta Toxin from Clostridium perfringens Type C

Abstract: Purified beta toxin from Clostridium perfringens type C was inactivated by the oxidizing agents o-iodosobenzoate (OIBA), oxidized glutathione, and ferricyanide, and by the sulfhydryl group reagents 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, iodoacetamide, and iodoacetic acid, causing loss of activity in various degrees depending on the concentration used. The activity of the toxin was not influenced by exposure to 1.0 mm of p-chloromercuribenzoate. The toxin treated by OIBA or DTNB was reactivated by incubation with 2-mercaptoethanol and dithiothreitol. The data suggest that beta toxin contains thiol groups which are essential for the activity. Beta toxin of Clostridium perfringens is produced only by types B and C. It is necrotizing and lethal, but not hemolytic (6). It is thought to be a main factor in necrotic enteritis of animals and man caused by C. perfringens type C. We have purified the toxin extensively (11), and reported that the toxin is a heat labile protein with a molecular weight of approximately 30,000 (12). To study the role played by the toxin in necrotic enteritis of C. perfringens type C, it is important to know the relationship between the functional groups and the activity of the toxin. However, there have been no previous reports on this. We previously found that the toxin is completely inactivated by bubbling of air ( J. Sakurai, Y. Fujii, and M. Matsuura, unpublished data). In this paper, we report that beta toxin is inactivated by some oxidizing agents and sulfhydryl group reagents and that the toxin is reversibly inactivated by mild oxidation.

A Mouse Model for the Pathogenesis and Postexposure Prophylaxis of Rabies

Abstract: A mouse model for the study of postexposure prophylaxis of rabies was established. Mice injected intramuscularly with a street strain of rabies virus were significantly protected from death by five daily 0.2-ml doses of inactivated rabies vaccine of chick embryo cell culture origin initiated immediately or 3 hr after infection. In these mice, a large amount of circulating interferon was induced as early as 1 hr after the first dose of vaccine and lasted until at least 12 hr but no such amount of interferon was induced by additional doses of vaccine. Serum antibody was first detected in the mice on day 6. It was noted that some of the surviving mice manifested an ataxia or paralysis of the legs. Increasing mortality rates were shown in mice treated with decreasing doses of the vaccine. Passive protection tests using concentrated IgG and IgM antibodies with equivalent neutralization titers showed that IgG antibody gave total protection when given 24 hr before the infection, while it was almost totally ineffective in reducing the mortality when given 2 days or more after infection. IgM antibody did not protect the mice even when given 24 hr before infection. These results suggest that interferon production is more important than antibody production in the initial stages of protection by postexposure vaccination. However, the mechanisms of postexposure prophylaxis in this model could not be explained only by the interferon produced by the vaccine and the possible contributions of additional mechanisms were suggested.

Electron microscopy of the spherical body of oral spirochetes in vitro. Further studies.

Abstract: The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB Spirochetal cells adhered firmly to the surface of the resultant body The membrane of the SB, ie the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and the cylinder Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils These findings suggest that the oral spirochete produces an SB by terminal expansion of the outer envelope in the presence of high concentrations of sucrose The outer envelope of the SB, which consists of two electron-dense layers, has the property of binding spirochetal cells to its outer layer and the protoplasmic cylinder and axial fibrils to the inner layer Some protoplasmic cylinders were also observed to be swollen in the presence of high sucrose concentrations

Location of Elements in Ashed Spores of Bacillus megaterium

Abstract: The structure of the skeleton of spores of Bacillus megaterium was examined after ashing in a plasma asher and the elemental composition of the ashed whole spores was determined with an analytical electron microscope. All spores were ashed in situ although they shrank by about 15%. Even P and S, in addition to metals, were recovered well from ashed samples. Ash was rich in the core and the coat, and poor in the cortex. Ca, P, S, and Mg were detected in the core and coat of the spore of B. megaterium QM B1551. Ca in the core was markedly decreased by germination or autoclaving. In the spore of B. megaterium ATCC 19213, almost all of the ash was detected in the core and its elemental composition was similar to that of the core of the strain QM B1551 spore. These results suggest strongly that the core is the site of Ca associated with dipicolinic acid.

Effect of Synthetic Muramyl Dipeptide (MDP) on Differentiation of Myeloid Leukemic Cells

Abstract: It has been reported that the M1 cell line, established from a myeloid leukemia of an SL strain mouse (9), can be induced to differentiate into mature macrophages and granulocytes by exposure to various factors (7-9, 12, 14, 17, 21, 24-26). As differentiation markers of Ml cells, various characters, such as Fc and C3 receptors, phagocytosis of latex particles, motility, lysosomal enzymes, cell morphology, and the capacity to cooperate with lymphocytes to produce antibody-forming cells (27), have been employed. Synthetic N-acetyl muramyl dipeptide (MDP) is known to be the minimal structure required for adjuvant activity of bacterial cell wall peptidoglycan for increasing both humoral and cell-mediated immune responses (1, 4, 5, 10, 11, 22). For the mode of action of MDP, it has been suggested that the dipeptide potentiates immune responsiveness by mediation of T cells (13) or of macrophages which release factors acting on B cells through T-cell mediation (6). Moreover, adherent peritoneal macrophages can be activated in vitro by MDP and acquire cytostatic activity against tumor cells (10, 23). Recently, it was also shown that MDP augments the cytolytic activity of macrophages of cultured cell lines without the participation of other cell types (22). In the present paper, we report the effect of MDP on the differentiation of M1 cells into mature macrophages. The clone of Ml cells, M1/436-7, established by Dr. Y. Ichikawa (Chest Disease Research Institute, Kyoto University) was provided through Dr. M. Saito (Central Research Laboratory, Morinaga Milk Industry Co., Ltd., Tokyo). This cell line can be induced to differentiate into macrophages but not into neutrophil granulocytes (17). J774.1, a macrophage cell line established by Dr. Peter Ralph (Sloan-Kettering Institute for Cancer Reserch, Rye, N.Y., U.S.A.) (20), was provided by Drs. T. Kishimoto and T. Igarashi (Osaka University Medical School). Cells were suspended in RPMI 1640 medium supplemented with 10% newborn calf serum inactivated by heating at 56 C for 30 min, and incubated at 37 C in a humidified atmosphere containing 5% CO2 in air. Differentiation of M1 cells was judged by the appearance of Fc receptors and phagocytosis of latex particles. The assay for Fc receptors was performed by counting EA (sheep erythrocytes sensitized with the 7S fraction of rabbit antisheep erythrocyte antiserum) rosette-forming cells by the method of Bianco et al (3). The percentage of cells with at least 10 attached erythrocytes was deter-

Transformation of mouse peritoneal macrophages and bone marrow cells by simian virus 40.

Abstract: A fraction of cultured mouse peritoneal macrophages and bone marrow cells acquired the ability to divide after infection by simian virus 40 (SV40). Two types of transformed lines were obtained. Most transformants isolated 40–60 days after infection did not display macrophage specific properties such as ingestion of opsonized red blood cells, possession of Fc receptors and complement receptors, and acid phosphatase activity throughout the whole culture phase. Cells of the transformed lines isolated by trypsin selection 2–6 months after infection displayed these properties when the cell density became high and cell growth was arrested. In the cells of the latter type of transformed lines, SV40 T-antigen was intensely demonstrated by immunofluorescence in the growing phase, but weakly or not at all in the stationary phase. It is suggested that the reversion to the phenotype of the progenitor (expression of macrophage specific functions) depends on the physiological state of the culture; however, it is uncertain whether the development of the macrophage functions is directly related to the SV40 T-antigen.

Adherence of Yersinia enterocolitica to mammalian epithelial cell lines.

Abstract: Yersinia enterocolitica RIMD 2501003 grown at 25 C avidly adhered to various kinds of cultured epithelial cell lines (HeLa, FL, Y-1 adrenal, human intestine, human conjunctiva) but the bacteria grown at 37 C did not adhere. This phenomenon paralleled the temperature-dependent motility of the bacteria. To clarify the adherence mechanism, we obtained two kinds of mutants, an immobile mutant and a nonadherent mutant, by treatment with A-methyl-A-nitro-A-nitrosoguanidine. The immobile mutant did not move on soft agar but retained the capacity to adhere to cultured epithelial cells when grown at 25 C. The nonadherent mutant did not adhere to cultured epithelial cells but retained the ability to move on soft agar when grown at 25 C. When the bacteria were killed by heat, ultraviolet light irradiation or formaldehyde they lost their capacity to adhere to the cultured epithelial cells. Antiserum against Y. enterocolitica RIMD 2501003 grown at 25 C was absorbed with the bacteria grown at 37 C, with the bacteria grown at 25 C, with the nonadherent mutant grown at 25 C and with the bacteria killed by various means. Only the antiserum absorbed with bacteria grown at 37 C inhibited the adherence of bacteria. These data indicate that motility does not correlate with adherence of Y. enterocolitica. It appears that the adherence factor involves both a temperature-dependent surface factor and a factor synthesized de novo during the interaction of susceptible cells with the bacteria.

The Induction of Viral Inhibitor (s) in Mice Treated with Biological and Synthetic Immunopotentiators

Abstract: protective effect afforded by these agents has been explained as the result of potentiation of the host's defense mechanism rather than a direct inhibition of the tumor cells and viral growth. However, the precise mechanism is not yet clear. Thus, in an attempt to elucidate the mechanism of this enhanced resistance, we studied the interferon-inducing capacity of these agents in mice. Inbred DDI mice, 6 to 8 weeks old, were obtained from the Experimental Animal Center of Tohoku University. The nature and source of all immuno-

Intestinal microflora and bile acids. Effect of bile acids on the distribution of microflora and bile acid in the digestive tract of the rat.

Abstract: The population levels of intestinal microflora and bile acid composition in the digestive tract were examined in rats fed bile acids to determine the relationships between gastrointestinal microflora and the host. The population level of Bacteroides was increased in the ceca of rats fed cholic acid or deoxycholic acid. In the ileum, the concentration of conjugated bile acid in rats fed cholesterol, cholic acid, hyodeoxycholic acid or lithocholic acid was higher than that in control rats, and was very low in ceca and feces of all the rats. The concentration of total free bile acid was much higher in the ceca than in the ilea of rats fed hyodeoxycholic acid or lithocholic acid. Cholic acid and deoxycholic acid were found in the ilea, ceca and feces of the cholic acid-fed rats. In the deoxycholic acid-fed rats, cholic acid was localized in the ileum. 7-Ketodeoxycholic acid was also found in the ceca of the cholic acid-fed rats. 12-Ketolithocholic acid was found in the feces of rats fed cholic acid or deoxycholic acid. 3-Ketocholanic acid was found in some samples from the lithocholic acid-fed rats. Therefore, some kinds of bile acids influence the population levels of gastrointestinal microflora and bile acid composition in the intestine.

Toxigenicity of Clostridium difficile Isolates from Patients and Healthy Adults

Abstract: pseudomembranous colitis (PMC) of man (1, 2, 5, 9). Also in Japn, a clinical report on this organism has been issued recently (8). Toxigenic strains of this organism have also been isolated from stools of healthy neonates (9) and healthy adults (4, 8). George et al (6) devised a selective medium to facilitate the isolation of C. difficile from faecal specimens and isolated C. difficile from subjects without PMC who were given antibiotics. Since the relationship between toxigenic potentiality of C. dijficile isolates and PMC has not been mentioned as yet, we, in this study, examined C. difficile isolated from healthy subjects and antibioticadministered patients for the potentiality of toxigenicity. All of the 28 C. difficile strains tested in this study were recovered from adult stools by using CCFA medium devised for the isolation of this organism (6), and were identified by the method outlined in the V.P.I. Anaerobe Laboratory Manual (7). One strain was established from each of the healthy adults and patients. A total of 18 strains were isolated from healthy adults, i.e., from five out of 40 students and 13 out of 215 aged adults, while a total of 10 strains were recovered from clinical cases, i.e., from four patients who were given antibiotics but showed no induced diarrhea, another three patients with antibiotic-associated diarrhea without documented PMC and the other three patients with antibiotic-associated PMC. The test for cytotoxicity of culture supernatant fluid was performed as follows: chopped-meat-glucose medium (7) was inoculated with each strain, incubated at 37 C for 7 days, and then centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a membrane filter with 0.45-ym pore size ( JapanMillipore, Tokyo), and used for cytoxicity assay using HeLa cells cultured in tissue culture

Establishment of Autoantibody‐Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Abstract: Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.

Purification and polypeptides of respiratory syncytial virus.

Abstract: Polypeptides of partially purified respiratory syncytial (RS) virus have been reported by two groups (1, 3, 5, 8). However, there are discrepancies between their results. These discrepancies possibly arose from the insufficient purity of the virus obtained by sucrose or metrizamide density gradient centrifugation. We found that a sucrose-containing buffer is useful for overcoming the instability of the virus and that a gel filtration method that has recently been successfully applied to several viruses (2, 4, 9) is also efficient for the purification of RS virus. This report describes first a purification method for intact RS virus by a combination of gel filtration and discontinuous and linear sucrose density gradient centrifugation, and second the polypeptide pattern of the purified virions obtained by this method. To overcome the lability of RS virus, i.e. a marked loss of its infectivity encountered during the purification procedures, we used NTE buffer solution, which was composed of 0.15 M NaCl, 0.05 M Tris-HCl and 1 mm EDTA (pH 7.5), containing 20% sucrose for suspending the virus throughout the procedure. Monolayer cultures of HES cells, derived from human embryonic skin and established in our laboratory, were grown in Eagle's minimum essential medium with 3% fetal calf serum in roller bottles and were infected with the Long strain of RS virus at a multiplicity of approximately one plaque-forming unit per cell. The culture fluids were harvested after 36-hr incubation at 33 C (6). A virus preparation, concentrated by precipitation from infectious tissue culture fluids with polyethylene glycol 6000 and centrifuged through 30% sucrose

Complement proteins and macrophages. II. The secretion of factor B by lipopolysaccharide-stimulated macrophages.

Abstract: The secretion of factor B by mouse peritoneal macrophages was found to be enhanced following in vivo or in vitro stimulation with lipopolysaccharide (LPS). The intravenous administration of LPS to mice of various strains caused an increased release of factor B but not the release of acid phosphatase by the peritoneal macrophages obtained from the stimulated mice. In vitro stimulation of cultured macrophages with LPS resulted in an enhanced secretion of both factor B and acid phosphatase. The dose-dependent augmentation of factor B secretion by LPS was found in the macrophages from LPS-responsive C3H/HeN mice, whereas the macrophages from LPS-unresponsive C3H/HeJ mice did not respond to either phenol-extracted LPS or butanol-extracted LPS. The ability of LPS to cause the enhancement of factor B secretion by macrophages was abolished by alkali or acid treatment of LPS, indicating that its lipid A part was responsible for the observed effect.