In this review, we have emphasized: 1) bacterial lipopolysaccharide (LPS) involvement in IgA responses to orally administered thymic-dependent (TD) antigens; 2) characterization of Peyer's patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti-LPS immunity to infection.
Gut LPS, which interacts with PP lymphoreticular cells, is a major determinant for host responses to orally administered TD antigens. Bacteroides species are the principal microflora present in the gastrointestinal tract and our studies with phenol-water LPS extracts from Bacteroides fragilis indicate that both polysaccharide and lipid A activate lymphoreticular cells. The B. fragilis lipid A moiety, like that derived from E. coli and Salmonella LPS, induces B cell mitogenic responses in cultures from LPS responsive mice, but does not stimulate C3H/HeJ B cells. The inability of lipid A to stimulate gut-associated lymphoreticular tissue (GALT) cells of C3H/HeJ mice results in the induction of greater T helper cell activity in this tissue in response to orally administered TD antigens and ultimately results in an elevated IgA response pattern.
Murine PP contain accessory cells (-1% dendritic cells and 6-8% macrophages) and lymphocytes T (35-38%) and B (40-42%). Recent studies with antigen-specific T cell clones from C3H/HeJ PP have resulted in the isolation of IgA isotype-specific T helper cells (PP Th A cells). PP Th A cells are antigen-specific, bear Fcα receptors, and require H-2 histocompatibility with B cells for helper activity. PP Th A cells most effectively collaborate with surface IgA (sIgA)-bearing B cells (IgA committed B cells) for IgA isotype responses. Other studies have shown that PP dendritic cells and T cells form clusters when stimulated in vitro with sodium periodate and that these clusters promote polyclonal IgA responses in B cell cultures. Polyclonal IgA responses in cultures containing PP cell clusters from C3H/HeJ mice are considerably higher than those in identical cultures from LPS responsive mice, In other studies, the environmental influence on GALT B cells and their resultant commitment to IgA isotype is under investigation. CBA/N, X-linked immunodeficient (xid) mice possess an immature splenic B cell population which cannot respond to thymic-independent class-2 (TI-2) or certain TD antigens. However, GALT B cells of xid mice possess a mature Lyb-5⁺ B cell subpopulation capable of both TI-2 and TD responses. GALT T cells and environmental influences are responsible for the induction of mature B cells in PP of xid mice.
The gastrointestinal route represents both a mode for induction of IgA responses and the major site of infection by pathogenic E. coli and Salmonella species. We have compared immune responses to the three major LPS regions in LPS nonresponsive, high IgA responsive C3H/HeJ mice and in LPS responsive C3H/HeN animals. Oral immunization of C3H/HeJ mice with smooth Salmonella cells induced elevated splenic IgA anti-LPS PFC responses. In separate studies, we have produced monoclonal antibodies to antigenic determinants of Salmonella LPS. These monoclonal antibodies are being tested to determine the isotype and specificity of antibodies required for immunity to Salmonella typhimurium infection.

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One hundred and eight strains of Clostridium perfringens were collected from polluted waters and tested for their drug resistance and bacteriocinogeny. Thirty of the strains were tetracycline resistant and 36 were bacteriocinogenic. Only one strain possessed both tetracycline resistance and bacteriocinogen.
Transfer experiments on tetracycline resistance and bacteriocinogeny were carried out with several selected strains among these isolates. Both tetracycline resistance and bacteriocinogeny were shown to be transferable by conjugation-like procedures. Transfer experiments on these two properties revealed that there were two types of recipient strains among the isolates in regard to the acceptance of tetracycline resistance and bacteriocinogeny. In one type of the recipient strain, these two properties seemed to be incompatible although in the other, both tetracycline resistance and bacteriocinogeny were transferred by two-step mating. Transcipients of this two-step mating retransferred their tetracycline resistance but not bacteriocinogeny to another recipient strain. The existence of an incompatibility-like relationship between tetracycline resistance and bacteriocinogeny in one recipient strain, and the inability of these two properties to be co-transferred from the two-step transcipients, suggest a certain interrelationship between the factors responsible for tetracycline resistance and bacteriocinogeny.

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Hemolysis by leptospiral hemolysin was strongly inhibited by bovine serum. The inhibitory activity was observed in the chloroform-methanol-soluble fraction of bovine serum. The inhibitor was eluted in a complex lipid fraction and was separated into two fractions (Fr. I and II) by silicic acid column chromatography. Fractions I and II inhibited approximately 75% and 95%, respectively, of hemolysis by leptospiral hemolysin. Fraction I was identified as phosphatidylethanolamine (PdE) by silica gel thin-layer chromatography (TLC). Two kinds of phospholipids (PLs) were detected in Fr. II by TLC. One was resistant to alkaline treatment and was identified as sphingomyelin (Spm), and the other was sensitive to such treatment and was identified as phosphatidylcholine (PdC). PLs, such as Spm, PdC, phosphatidylglycerol, PdE, phosphatidylserine and cardiolipin, inhibited hemolysis by leptospiral hemolysin, but phosphatidylinositol did not show any inhibitory activity. PLs lacking the amino group in the polar backbone of the molecules were more effective.
From experiments using erythrocytes of various kinds of animals, it was revealed that the hemolytic sensitivity of mammalian erythrocytes to leptospiral hemolysin depended on the Spm content in the erythrocyte membrane. On the other hand, phospholipase C (PLase C) activity with Spm and PdC as substrates was detected in the culture supernatant of Leptospira. Therefore, leptospiral hemolysin was presumed to be PLase C, perhaps sphingomyelinase. The inhibitors of leptospiral hemolysin present in bovine serum were identified as PLs. PLs in bovine serum were suggested to function as inhibitors of the interaction between leptospiral hemolysin and the surface of the erythrocyte membrane.

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Hemolysis mutants were produced by treating Mycoplasma pneumoniae FH-P24 strain with N-methyl-N-nitro-nitrosoguanidine and were classified into three different groups. The first group of mutants, strains P24-L1, L2, and L11, showed wide and clear hemolytic zones. Their attachment ability to erythrocytes of various animals and to hamster lung cells were the same as those of the parent strain. The second group, strain P24-S1, showed non-hemolysis and non-hemadsorption, but retained the attachment ability to lung cells, although not to erythrocytes. The third group, strain P24-S11, was non-hemolytic, had completely lost the attaching ability, and did not proliferate in vivo. Strains in the first group produced significant microscopic pneumonic lesions in hamsters while strain P24-S1 produced milder lung lesions. Strain P24-S11 did not cause any lung lesions, and organisms were not recovered from the lungs of hamsters. The attachment of M. pneumoniae to respiratory epithelium as a cause of infection and the existence of a relationship between the hemolytic abilities of the organisms and histopathogenicity in the hamster lung tissue were further supported by the present data.
It was also shown that the use of hemolysis mutants is useful for the elucidation of pathogenesis in mycoplasmal infections.

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The ecology of non-O 1 Vibrio cholerae and Vibrio mimicus as causes of cholera-like diarrhea or seafood-associated gastroenteritis has been investigated in Toyama Prefecture since 1980. The relationship between biological or serological characteristics of the isolates and their enteropathogenicity is discussed.
Overall isolation rates from river water, sea water, and fish were 24.0, 59.5, and 33.7%, respectively, the isolation frequency being, in general, extremely high in the summer season, although the organisms were detected all year around in the case of sea water. Most isolates from river water were unable to grow on plates of TCBS agar to which colistin was added at a concentration of 1μg/ml (CL-TCBS). These strains quickly fermented cellobiose. O-51 and O-70 were the two most frequently detected serogroups among them and they did not show enteropathogenicity in the rabbit ileal loop (RIL) test. On the other hand, almost all isolates from sea water and fish as well as those from human diarrhea cases were able to grow on CL-TCBS, but were unable to ferment cellobiose quickly. O-36, O-10, O-6, O-8, O-39, and O-26 were the dominant serogroups of these isolates, and some of them showed enteropathogenicity in the RIL test. Six out of 98 isolates from river water, 14 out of 116 from sea water, and 19 out of 112 from fish were classified as Vibrio mimicus. All of these strains were able to grow on CL-TCBS and quickly fermented mannose but not cellobiose. O-41 was the most common serogroup among them and some of these strains showed enteropathogenicity in the RIL test. Production of a cholera-like enterotoxin among the isolates in Toyama Prefecture, if any, seemed to be poor.

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Phage Vf33, a filamentous phage about 1, 400nm long and 7nm wide, specific for Vibrio parahaemolyticus, was isolated and characterized. The buoyant density of Vf33 in CsCl was 1.292g/cm³. As with other filamentous phages, the lytic activity of Vf33 was resistant to heating below 80C and to treatment with diethylether, acetone or methanol but sensitive to chloroform. The nucleic acid of this phage is single-stranded circular DNA 8.4kb in size. The viral genome was converted to a double-stranded replicative form in the host cell. Among the strains tested, only V. parahaemolyticus strains possessing K38 antigen was sensitive to the phage.

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Two types of glycoproteins were isolated from the membrane fraction of adenovirus type 12 (Ad12) hamster tumor cells by recovering detergent-solubilized glycoproteins using concanavalin A-affinity chromatography and gel filtration. One of the glycoproteins consisted of a polypeptide of 130, 000 daltons (130K) with a pI value of 4.7-5.1, and the other consisted of a polypeptide of 18, 500 daltons (18.5K) with a pI value of 6.3-6.6. The glycoproteins were immunologically different. The 18.5K glycoprotein induced in vivo resistance to tumor growth and anti-tumor cytotoxic T cells, while the 130K glycoprotein induced in vivo suppressor T cells which inhibited the activity of anti-tumor cytotoxic T cells.

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An inhibitor of mouse factor B was purified from lysate of L cells by a human factor B-affinity column. The purified inhibitor was found to be homogeneous with a molecular weight of about 25, 000 and identical to the 25K protein isolated from L cell-lysate by the coprecipitation method with anti mouse factor B. Hemolytic titration of the activity and PAGE-analysis of the inhibitor-factor B complex showed that 1mol of inhibitor reacted with 1mol of mouse factor B and inactivated its hemolytic activity.

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Monoclonal antibodies against etiological agents of Weil's disease were produced by cell fusion technology. Twenty hybridomas were produced through the fusion of P3X63Ag8.653 cells with spleen cells from BALB/c mice immunized against Leptospira interrogans serovar icterohaemorrhagiae RGA strain and serovar copenhageni Shiromizu and M20 strains. Reactivities of the antibodies produced by the hybridomas were determined by the microscopic agglutination test. Among the five hybridoma antibodies to the RGA strain, two reacted specifically to serovar icterohaemorrhagiae, two reacted to serovar icterohaemorrhagiae at a high titer and serovar copenhageni at a low titer, and one reacted to serovars icterohaemorrhagiae, copenhageni, pyrogenes, and canicola. Of the ten hybridoma antibodies to the Shiromizu strain, one reacted specifically to serovar copenhageni, seven reacted to both serovars copenhageni and icterohaemorrhagiae at almost the same titer, and two exhibited intermediate properties. Of the five hybridoma antibodies to the M20 strain, three reacted to both serovars copenhageni and icterohaemorrhagiae at almost the same titer, one reacted to serovar copenhageni at a low titer and serovar icterohaemorrhagiae at a high titer, and one reacted to serovars copenhageni, icterohaemorrhagiae, and pyrogenes. The results revealed that each serovar has its own antigen(s) and their common antigens. In addition, 20 strains of leptospires were recently isolated and tested with three monoclonal antibodies characterized by different reactivities. Twenty strains were clearly identified by their antibodies, i.e., 16 strains were identified as serovar icterohaemorrhagiae and three strains were identified as serovar copenhageni. The remaining strain, which was not agglutinated by three antibodies, was identified as serovar autumnalis by an agglutination test with immune rabbit sera.

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It has been suggested that some polysaccharides play important roles in immune responses. Therefore, we used various types of polysaccharides for analysis of macrophage-mediated tumor cell killing.
We report here that fucoidan blocked macrophage activation occurs in an inductive phase but enhanced macrophage activation appears in an effector phase.

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