The A and B subunits of Shiga toxin were isolated by high performance liquid chromatography and their physicochemical properties were examined. The A subunit of Shiga toxin purified from culture supernatant was not nicked, but it could be nicked in vitro by trypsin. The isoelectric points of the A and B subunits were determined to be 8.2 and 5.8, respectively. Amino acid compositions of the two subunits were also determined. The isolated A and B subunits were reconstituted to form active holotoxin which showed lethal activity to mice which was similar to that of native Shiga toxin.

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Assuming that salivary immunity to indigenous microorganisms could develop, we assessed antibacterial reactivities of natural salivary antibodies in specific pathogen-free inbred mice. An ELISA was set up, using whole bacterial cells, to map reactivities of salivary IgA and IgG which accounted respectively for 91% and 8.7% of salivary Ig's in the BALB/c mouse. Representative strains of seven species from three genera (Lactobacilli, Staphylococci, and Streptococci), including major and minor components of the murine oral flora (38, 43, and 8%, respectively), were used to determine the presence and level of specific antibodies in individual saliva. It was verified that naturally occurring IgA antibodies can display diverse antibacterial reactivities. A characteristic profile emerged for salivary IgA where antibodies to Streptococcus faecalis predominate. Natural salivary IgG antibodies did not show the same reactivity pattern as IgA, anti-Lactobacilli and anti-Staphylococci reactivities being much less frequent in the salivary IgG repertoire. However, antibodies to S. faecalis occurred at the same high frequency for both isotypes (62-70% of the samples). Besides being species-specific, antibacterial reactivities were also found to be strain-specific. Broad variations in antibacterial titers were detected among individual mice under standardized experimental conditions. Present data thus suggest that the dynamics of salivary antibody production in the mouse reflect a differential natural sensitization of the secretory (IgA) versus the systemic (IgG) immune systems by distinct populations of indigenous bacteria.

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Murine peritoneal macrophages (Mφs) elicited by a single injection of zymosan A showed a higher responsiveness to the wheat germ agglutinin (WGA)-mediated triggering for H₂O₂ release than Mφs activated by double injections of the agent. On the contrary, the response to phorbol myristate acetate(PMA)-mediated triggering was higher in the latter Mφs than in the case of the former Mφs. Furthermore, PMA-triggered Mφ H₂O₂ release was found to be inhibited by sarcoma 180 tumor cell-derived proteinaceous factor in a much more marked fashion than the WGA-triggered H₂O₂ release. These results indicate some significant differences between the cellular mechanisms of the WGA- and PMA-triggering for Mφ oxidative burst. On the other hand, microfilament-inhibitors (cytochalasins B and E) and serine protease-inhibitors (tosyl-L-lysine-chloromethyl ketone and tosylamido-2-phenylethyl-chloromethyl ketone), but not microtubule-disrupting agents (colchicine, vinblastine, and vincristine), suppressed both the WGA- and PMA-triggerings for Mφ H₂O₂ release to a similar degree, thereby indicating that the WGA- and PMA-triggerings for Mφ oxidative burst have a common process which is dependent on microfilament and serine protease functions. In relation to this, the WGA- and PMA-triggerings for Mφ spreading were also depressed by microfilament- and serine protease-inhibitors but not by microtubule-inhibitors, indicating a participation of common membrane functions in the signal transduction in cases of Mφ oxidative burst, and cell spreading induced by the WGA- as well as PMA-triggering.

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A polyvalent radiovaccine of Salmonella was shown to induce protective immunity in mice. The results revealed that the immunized mice progressively eliminated the challenged organisms and no Salmonella could be isolated after a period of 21 days. In contrast, Salmonella grew in the control mice and reached levels of 10⁸ to 10⁹cfu/spleen resulting in the death of animals. Sera from both control and the immune mice were found to lack bactericidal activity. It was further observed that the vaccine induced delayed type of hypersensitivity and that antibody production as measured by bacterial agglutination and passive hemagglutination were low in response to the vaccine. However, the phagocytic activity of the reticuloendothelial system was considerably enhanced by the immunization. The results of experiments with immunosuppressed mice suggested the role of thymus derived (T) lymphocytes in the protection.

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We succeeded in establishing a mouse-human (M-H) heterohybridoma clone which provides parental cells useful for human monoclonal antibody (hMoAb) production. Electron micrographs show that the M-H hybridoma cells retain characteristics of murine origin with regard to chromatin patterns, small granules, granular endoplasmic reticulum and Golgi apparatus. The human DNA incorporated into the M-H hybridoma is estimated to be about 1% of the total human chromosomal DNA. Mouse-human-human (M-H-H) hybridomas obtained by hybridization of the M-H hybridoma cells with Epstein-Barr virus(EBV)-transfected human B cells secrete immunoglobulin (Ig) in amounts comparable to those of murine hybridomas. Also the M-H-H hybridomas grow in nude mice and are capable of producing ascites containing large quantities of Ig. The Ig class switching takes place in the M-H-H hybridomas at a much higher frequency than in the original EBV transformant and the M-H hybridoma. Cells secreting specific monoclonal antibody of different Ig classes could be separated and concentrated by the use of fluorescence activated cell sorter (FACS).

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Alveolar macrophages (AM) from normal rats were separated into 4 different density fractions by centrifugation on a discontinuous Percoll gradient. These fractionated (I-IV) AM, as well as unfractionated (UF) AM, were then tested for their capacities to regulate mitogen-induced T cell proliferation. Concanavalin A (Con A)-induced response of nylon wool-passed non-adherent splenic T lymphocytes was suppressed by addition of UF or higher density (III and IV) AM, while an intermediate density (II) AM fraction could enhance T cell response in a dose-dependent manner. Similar effects of UF or fractionated (I-IV) AM on T cell responses were noted when the cultures were exposed in vitro to inert, non-fibrogenic titanium dioxide (TiO₂) particles. On the contrary, T cell response was sustained by addition of UF or higher density (III and IV) AM, and was also more prominently enhanced by an intermediate density (II) AM after the in vitro exposures to fibrogenic dust particles, like silica and asbestos. Higher interleukin 1 (IL-1) activity was detected from these silica- or asbestos-exposed cultures of UF and fractionated (II, III, and IV) AM. The IL-1 activity was also highly detectable from the cultures of an intermediate density (II) AM fraction when cultures were unexposed or exposed in vitro to TiO₂ particles. The Ia antigen expression on the surface of UF or fractionated (II, III, and IV) AM was elevated in the Con A-pulsed co-cultures, but not significantly different whether or not they were exposed in vitro to dust particles. These results may indicate the presence of heterogeneity in accessory cell functions and IL-1 production among rat AM.

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The culture supernatants of unstimulated T cells (TCS) from asthmatic patients with elevated serum IgE were tested for IgE-binding factors (IgE-BFs) displaying the IgE-potentiating activity. The IgE-BFs were detected by their ability to inhibit the rosetting of RPMI 8866 cells with ox erythrocytes coupled with mouse monoclonal antibody (E-Mab) specific to Fc receptors for IgE (Fc_εR). TCS showing the rosette-inhibiting activity significantly enhanced the spontaneous IgE synthesis by B cells of allergic individuals. Interestingly, rosette-inhibiting factors could be removed by absorption with IgE-Sepharose from which they were subsequently eluated with acid buffer, indicating that the rosette inhibition was indeed mediated by IgE-BFs. In addition, such IgE-BFs had affinity for concanavalin A and lost their IgE-potentiating activity after treatment with trypsin and neuraminidase. In contrast, T cells treated with tunicamycin released IgE-suppressing factors capable of inhibiting the IgE-potentiating activity of TCS derived from untreated T cells. On the other hand, the culture supernatants from subpopulations depleted of Fe_εR⁺ T cells but not of Fc_γR⁺ T cells contained neither rosette-inhibiting factors nor IgE-potentiating factors, suggesting that IgE-BFs were released by in vivo pre-activated Fc_εR⁺ T cells. With regard to circulating Fc_εR⁺ T cells determined by E-Mab, they were significantly higher in asthmatic patients with elevated serum IgE (0.77±0.15%) than in normal subjects (0.17±0.07%) in spite of a very small proportion of T cells bearing Fc_εR.

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Immunostimulatory effects of respirable mineral dust particles on alveolar macrophages (AM) and T lymphocytes were tested in vitro. When rat AM were incubated with fibrogenic silica and asbestos particles, a significant interleukin 1 (IL-1) activity was generated into the culture supernatants, whereas neither AM alone nor AM incubated with non-fibrogenic titanium dioxide (TiO₂) particles, however, produced a detectable amount of IL-1. Interleukin 2 (IL-2) activity, as tested with IL-2-dependent CTLL-2 assays, was not detectable from all of these cultures. It was also revealed that concanavalin A-induced proliferation of T lymphocytes was enhanced in autologous AM and nylon wool-passed spleen cell co-cultures incubated only with fibrogenic particles, but not with non-fibrogenic particles. Higher IL-1 activity was detected only from co-cultures exposed to fibrogenic particles, whereas IL-2 activity was almost similar among these co-cultures. These results indicate the differences in immunostimulatory effects on pulmonary macrophages and T lymphocytes among a variety of mineral dust particles.

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