An immunochemical staining technique for the spore coat proteins of Bacillus megaterium ATCC 12872 was developed using colloidal gold as a second antibody. For reducing the non-specific immunogold binding and increasing the specific binding, the affinity-purified IgG was used as a first antibody. In sporulating cells at t₁₀, gold particles were found not only in the spore coat but also in the mother cell cytoplasm, suggesting that some coat proteins were synthesized in the cytoplasm. Use of the specific affinity-purified antibody to 48K-protein demonstrated that this protein was one of the components of the outer coat.

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A colicin plasmid in Escherichia coli strain B177 isolated from a septicemic calf was characterized. The colicin type was identified as ColV by using reference ColV producers. The colicin plasmid was labeled with transposon Tn903 and subjected to conjugation. The transconjugants examined suggest that the colicin plasmid confers serum resistance. There was no difference in siderophore utilization ability between the transconjugants and host strain SF800. Bioassay for siderophore suggests that the colicin plasmid specifies the production of iron-chelating compounds available for the host strain.

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The genome of antigenic variant CV (CT3)-1 derived from Leptospira interrogans-serovar canicola was compared by cleavage with restriction endonucleases with the parent and serovar bafani, to which the variant was serologically most closely related. No differences were observed between the parent and variant in DNA restriction endonuclease patterns using eight restriction endonucleases. Serovar bafani was different in the patterns from the parent and antigenic variant CV(CT3)-1. The two antigenic variants derived from serovar hebdomadis, HV(H16)-1 and HV(H19)-1 which belonged serologically to serovars jules and hebdomadis, respectively, were compared by restriction endonuclease DNA analysis with the parent and serovar jules. No differences were observed between the parent and variants in DNA restriction endonuclease patterns using the same enzymes. But some differences were observed in DNA restriction endonuclease patterns between HV(H16)-1 and serovar jules. Thus, the antigenic variant selected from the parent by the anti-parent monoclonal antibody and serologically different from the parent, being identified either as a new serovar or as a known one, was found to be similar to the parent by the restriction endonuclease DNA analysis.

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Monospecific factor serum for identifying Candida tropicalis was obtained either from rabbit antiserum to heated cells of C. tropicalis M 1519 (S 96) or from antiserum to C. tropicalis IFO 1400, by adsorption with heated cells of Candida albicans serotype A, or C. albicans (A) and Candida krusei, respectively. We designated this adsorbed serum factor t serum. The monospecific factor serum reacted with 31 out of 32 strains of C. tropicalis, only when tested on heat-treated cell antigens, whereas it did not react with any of 72 strains of the six other medically important species of Candida. The morphological and physiological characteristics of the one strain of C. tropicalis that did not react with the factor t serum, designated the t^--strain, were shown to be similar to those of the type strain of C. tropicalis by most of the methods employed for identifying Candida. Therefore, cell wall mannan from the t^--strain was compared with that from several typical strains of C. tropicalis for its specificity by the precipitation reaction and also for its ¹H-nuclear magnetic resonance spectrum. The results showed that these mannans are similar to each other serologically and physicochemically, suggesting that the new antigen t is not mannan. Taxonomic characterization of the t^-- and several typical strains of C. tropicalis was carried out by determining the mol% G+C of their DNA and also their DNA homology. Although the mol% G+C values of four typical strains of C. tropicalis were fairly similar (35.2 to 36.2mol% by the Tm method and 35.5 to 36.4mol% by the HPLC method), the t^--strain had a G+C content of 44.1 (Tm) and 43.3 (HPLC) mol%. Furthermore, the DNAs of the t^--strain and the type strain of C. tropicalis showed only 18.2% relatedness. These results suggest that the antigen corresponding to serum factor t exists only in the cell wall of C. tropicalis strains, not in those of the other medically important Candida, and that the t^--strain should not be classified as C. tropicalis. In conclusion, the taxonomic value and usefulness of factor t serum is primarily for differentiating C. tropicalis from C. albicans serotype A serologically.

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The protease produced by Vibrio vulnifrcus enhances vascular permeability through histamine release from mast cells and activation of the plasma kallikrein-kinin system which generates bradykinin when injected into the dorsal skin. V. vulnificus living cells also enhanced vascular permeability within a few hours after the injection into the dorsal skin. The permeability-enhancing activity of living cells was greatly reduced by addition of soybean trypsin inhibitor, a specific inhibitor for plasma kallikrein-kinin system, or anti-protease IgG. Two protease-deficient mutants induced by nitrosoguanidine treatment had only one-tenth permeability-enhancing activity of a wild-type strain. These results indicate that V. vulnificus elaborates the protease in vivo and that the protease elaborated enhances vascular permeability through release of chemical mediators such as histamine and bradykinin and forms edema.

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The production from murine resident peritoneal macrophages (Mφ) of a soluble factor, which was capable of enhancing the antigen-presenting (AP) function of dendritic cells (DC), was examined. The supernatants of peritoneal Mφ (Mφ sup) were prepared by culturing peritoneal Mφ with particles, i.e., zymosan A, latex, and sheep red blood cells (SRBC), or antigen-antibody (Ag-Ab) complexes such as keyhole limpet hemocyanin (KLH)-anti-KLH, ovalbumin (OVA)-anti-OVA, and SRBC-anti-SRBC complexes. When exposed to Mφ sup during antigen pulsing DC induced a marked antigen-specific T cell proliferation, relative to DC treated with the supernatants from Mφ cultured without stimuli (control sup). On the other hand, Mφ sup-treated splenic Mφ stimulated antigen-specific T cell activation to almost the same extent as did splenic Mφ treated with control sup. These results indicated that peritoneal Mφ elaborated a soluble factor which preferentially enhanced the AP capacity of DC when stimulated with particles or Ag-Ab complexes. Analytical gel filtration of Mφ sup revealed that the factor had an apparent molecular weight of 27, 000 daltons which was distinct from interleukin 1.

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Ribosomal preparations from Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Streptococcus pneumoniae were investigated with respect to their activating capacity towards murine lymphoid cells. The proliferation of BALB/c spleen cells was induced in a dose-dependent fashion (from 1 to 100μg/ml) by ribosomes of K. pneumoniae, H. influenzae, and S. pyogenes with a peak activity at 48 or 72hr of culture. The majority of the blast cells induced by these ribosomal preparations were positive for surface-immunoglobulin (S-Ig) and negative for Thy 1.2. Furthermore, K. pneumoniae, H. influenzae, and S. pyogenes ribosomes induced the synthesis of IgM and some IgA. Cell proliferation and induction of IgM production were also demonstrated with the 3 ribosomal preparations using spleen cells from athymic nude (nu⁺/nu⁺) mice, Lyb-5-defective CBA/N spleen cells, B cell-enriched and T cell-depleted BALB/c spleen cell suspensions, as well as spleen cells from the Ips gene-deficient C3H/HeJ strain. Cell culture supernatants contained specific anti-ribosome IgM antibodies. Antibodies of other specificities (anti-sheep erythrocytes) were also demonstrated in supernatants from K. pneumoniae-stimulated cultures. Evidence against a possible role of contamination of K. pneumoniae and H. influenzae ribosomes by lipopolysaccharide- or lipid A-associated proteins in this effect is discussed. Ribosomes from S. pneumoniae did not induce ³H-thymidinc incorporation nor Ig production. None of the 4 ribosomal preparations was found to stimulate T cell blastogenesis or to induce interleukin-2 production by naive BALB/c spleen cells. Finally, ribosomes from H. influenzae, S. pyogenes, S. pneumoniae but not those of K. pneumoniae stimulated interleukin-1 production by adherent spleen cells, from BALB/c mice.

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A mouse macrophage (Mφ) hybridoma which produces a soluble factor responsible for the cooperation between Mφ and spleen dendritic cells (DC) was constructed. The antigen-presenting activity and the stimulator cell activity in the allogeneic or syngeneic mixed leukocyte reaction of DC were significantly augmented when DC were incubated with the culture supernatant of the hybridoma treated with various stimulants including latex beads. The monokine present in the culture supernatant of the hybridoma, called dendritic cell-activating factor (DCAF), augmented the production of lymphocyte-activating factor by DC while Ia expression of DCAF-treated DC was not altered. DCAF had no effect on the antigen-presenting activity of peritoneal resident Mφ or B cell blasts while the antigen-presenting activity of spleen Mφ was enhanced, but the degree of the enhancement was much less than that of spleen DC. DCAF was found to have the following properties: its pI value is between pH 4 and 5; it is stable at pH 2 to 10; and it loses its activity on incubation at 75C for 30min. When the culture supernatant of the hybridoma stimulated with latex beads was subjected to gel filtration, the DCAF activity was detected in the 20Kd to 25Kd, 30Kd to 40Kd, and 50Kd to 60Kd molecular weight regions. The 30Kd to 40Kd fraction, which is the major peak fraction, was further purified by ion-exchange chromatography followed by gelfiltration chromatography. When each fraction was subjected to SDS-PAGE, a 30Kd band corresponding to the DCAF activity was observed and DCAF was purified to about 90% purity.

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We report here the strategy to isolate the DNA fragment of any species origin which encodes cell surface antigen by using cosmid library transfection and cell sortings with a monoclonal antibody. We took the mouse melanoma antigen defined by monoclonal antibody as a model system and rescued the genomic DNA by in vitro packaging, showing the feasibility of this procedure.

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Staphylococcal exfoliative toxin A (ETA) had a splitting effect at the granular layer of skin in humans and neonatal mice, but not in rabbits, guinea pigs, golden hamsters, or rats. Besides its splitting effect, ETA could stimulate productions of neutralizing antibody to ETA in rabbits, rats and B10D2 mice, but not in golden hamsters, guinea pigs, or ICR, HRS/J, and C57BL/10 mice. In our epidemiological investigation of human sera, the percentage of antibody to ETA in sera obtained from patients with impetigo (8%) was lower than those in sera of healthy males (23%) and females (29%). The relationship between susceptibility and immune response to ETA in these mammalians could be divided into three groups: the possession of resistant skin and high production of antibody to ETA; the possession of resistant skin and low production of antibody to ETA; the possession of sensitive skin and various titers of antibody to ETA.

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