An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6μg/ml. One hundred and forty-nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.

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The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated-antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT-producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated-antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti-VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT-enzyme-linked immunosorbent assay (VT-ELISA). These results showed that serological assay particularly for antibodies against VT and unheated-antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.

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Human cancers express organ-specific neoantigens (OSNs) which elicit specific cellular immune responses in the cancer patient, as demonstrated by leukocyte adherence inhibition (LAI), an in vitro immune response assay. A purified protein of MW 40, 000 (p40) exhibiting OSN (colon specific) activity was cleaved into specific peptide fragments and their partial amino acid sequences determined. This information was used in the polymerase chain reaction (PCR) to obtain a 992bp cDNA clone (PCR-992) from a human colon adenocarcinoma cell line (LS-180). By comparison of the predicted amino acid sequence of PCR-992 with the known sequence of p40 peptides, PCR-992 was shown to correspond to almost the entire coding region of p40. Nucleotide sequence analysis suggested that the protein was mycoplasmal in origin due to its high A+T content (76%) and the presence of five in frame TGA termination codons; at least two of the latter are actually read as tryptophan, a known feature of mycoplasma translation. We have confirmed this origin by direct isolation of a contaminating mycoplasma species from the LS-180 cell line and demonstration that it could be hybridized with the PCR-992 probe. Northern and PCR analysis of RNA preparations from the contaminated LS-180 cell line showed that p40 was part of the high affinity transport system operon of Mycoplasma hyorhinis (Dudler et al, EMBO J., 7: 3963-3970, 1988). Total protein lysates of Mycoplasma hyorhinis cultivated without animal cells could elicit positive LAI responses when incubated with cancer patient leukocytes but not with normal patient leukocytes. The organ-specific nature of the response was, however, not observed indicating that host cell-mycoplasmal interactions may play a role in determining the organ-specific nature of p40 seen with the LAI. The significance of these findings will be discussed in the context of previous thinking regarding the origin of OSNs.

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The latex agglutination test was used to compare cryptococcal antigen titers before and after protease treatment in 19 patients with pulmonary cryptococcosis. Antigen was detected by the LA test in 13 of 33 serum samples before protease treatment, and in an additional 13 samples following treatment. Of 26 antigen-positive serum samples, 22 (84.6%) showed an increased antigen titer after protease treatment. Using the cell slide agglutination test, antibody was detected in 3 of 19 cases. In one of these 3, antigen could only be detected after protease treatment. Soluble immune complex was prepared in vitro using anti-C. neoformans rabbit antiserum and polysaccharide antigen of C. neoformans, and the effects of immune complexes on the LA test were examined. In this experimental model, soluble immune complexes seemed to be observed in antibody excess region, because the antigen titers were increased after the protease treatment. We concluded that C. neoformans capsular polysaccharide antigen and anti-C. neoformans antibody formed soluble immune complexes in patients' sera, which interfered with antigen detection by the latex agglutination test without protease treatment.

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In foregoing work, we identified at least 5 distinct epitopes on human type II collagen (CII), using 8 murine monoclonal antibodies (mAb) against human CII, and suggested that a species-nonspecific epitope on CII recognized by anti-CII mAb termed 1-5 is an arthritogenic epitope. We also found that antibody response against a selected epitope of human CII could be induced by immunization with rabbit anti-idiotypic (Id) antibody against anti-CII mAb. The author developed and characterized monoclonal anti-Id antibodies against 1-5mAb recognizing a putative arthritogenic epitope. The author also investigated whether the anti-Id mAb could regulate antibody response directed against a selected epitope recognized by 1-5mAb, and the induction of collagen-induced arthritis in DBA/1J mice. DBA/1J mice intravenously preinjected with anti-Id mAb to 1-5, did not produce anti-CII antibody expressing 1-5 Id upon immunization with human CII. Furthermore, as the development of collagen-induced arthritis (CIA) in DBA/1J mice pretreated with anti-Id mAb to 1-5 was significantly suppressed, anti-Id mAb will be a useful tool for studying the regulation of antibody response to a selected epitope. This study lends support to our hypothesis that the 1-5 epitope is an arthritogenic epitope.

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The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L-glycero-D-manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.

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We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze-substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti-O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron-dense mass.

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A chemotaxonomic study was carried out with a new serotyping scheme comprising 35 O-antigen groups of Vibrio fluvialis on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharide (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. The 35 O-antigen groups were divided into 21 chemotypes. It is noted that a rarely occurring component sugar of gram-negative bacterial LPS, D-glycero-D-manno-heptose, and two kinds of uronic acids, i.e., galacturonic acid of a weakly bound type and glucuronic acid of a strongly bound type, were found in common in all the 21 chemotypes. A characteristic sugar component of gram-negative bacterial LPS, 2-keto-3-deoxyoctonate (KDO), was not detectable in any of the 21 chemotypes. Instead, three kinds of “KDO-like substances” were found, one in each of three chemotypes (III, XI and XVII). They were strongly positive in Weissbach's periodate-thiobarbituric acid test for KDO, but definitely not identical to it in high-voltage paper electrophoresis (HVPE); the substance present in chemotype XI was indicated by HVPE to be 3-deoxy-D-threo-hexulosonic acid which is a sugar constituent of Vibrio parahaemolyticus O7 and O12 LPS.

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Two polypeptide antigens with molecular sizes of 34, 000 daltons (34kDa) and 38kDa were separated from heated cells of a human clinical treponeme strain G7201 and Treponema denticola ATCC 35404, respectively. The rabbit polyclonal antisera against these antigens were produced and examined for their immunological reactions with the two heated antigens or intact spirochetal cells. Immunoblot analysis showed that the 34-kDa protein was also detected in T. denticola ATCC 35404 and ATCC 33520, and the 38-kDa protein was detected only in the two ATCC strains. Immunoelectron microscopy using the two rabbit antisera and protein A-gold complexes demonstrated that the 38-kDa protein antigen was present on the axial flagella of two T. denticola strains, and that the 34-kDa protein was located in the axial flagella of the G7201 cell, but neither in axial flagella nor on outer envelopes of the two ATCC strains cells, suggesting that the native 34-kDa axial flagellar protein of the G7201 strain may be different from that of T. denticola in terms of immunological reactivity.

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This paper describes an attempt to effectively induce antibody-dependent cell-mediated cytotoxicity (ADCC) in nude mice. A monoclonal antibody against α-fetoprotein, 80G, coadministered with spleen cells from other nude mice bearing HuH-7N (xenograft of human hepatoma cell line, HuH-7) significantly suppressed the growth of HuH-7N as compared to treatment with 80G alone. 80G with spleen cells from normal nude mice also had some suppressive effect. In contrast, no effect was observed with each spleen cells alone as well as 80G alone. These results suggest that further supply of effector cells could enhance ADCC activity in nude mice.

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