Humoral antibody responses to the encapsulated Smith diffuse strain ofStaphylococcus aureus were examined in cows immunized with the killed vaccine via different systemic routes. The sequential appearance of the antibody within different immunoglobulin classes in the sera during the course of immunization was followed by passive hemagglutination (PHA) and precipitation (PC) reactions and the mouse passive protection test. Repeated intravenous injections with the killed vaccine suspended in buffered saline stimulated production of IgM antibody exclusively during the whole period of immunization. On the contrary, following intramuscular administration with the vaccine incorporated in Freund's incomplete adjuvant, the antibodies appeared predominantly in IgG fractions of the sera.
Specific antibody to the homologous strain used for vaccination was prepared from bovine immune sera by an absorption and elution process. The mouse passive protective activity of the antibody preparation was removed by absorption with the capsular polysaccharide antigen as well as by the whole cell adsorbent of the Smith diffuse strain, but not by the Smith compact and Cowan I strains of S. aureus. IgM, IgG₁ and IgG₂ proteins were isolated from the purified antibody and were compared, on a weight basis, with respect to their biological activities. Slightly higher activity of the IgG over the IgM antibody was demonstrated both in the mouse passive protection test and PC reaction, whereas in the PHA reaction, IgM antibody was shown to possess a significantly higher activity than IgG antibody.
These studies suggest that IgG as well as IgM antibody might play an important role in protection against infection with encapsulated strains of S. aureus in cows.

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Heat-labile virus inhibitor (HLI) in normal sera of various mammalian species capable of neutralizing variola (VRV) and Newcastle disease viruses (NDV) was studied immunologically. After sucrose density gradient centrifugation of guinea pig serum, the HLI activity against VRV and that against NDV were both demonstrated in the same region sedimenting fastor than IgM. Absorption with partially purified VRV or NDV removed the HLI activity on the homologous virus but not that on the other. Prior saturation of virions with specific antibody blocked the absorption of HLI, suggesting a specific competition for binding site (s) between specific antibody and HLI.
The HLI level against variola virus was checked in connection with immunization with vaccinia virus. In human primary vaccination, the HLI level rose sharply within 4 weeks after vaccination, turning to decline gradually to settle at a level higher than that of the conventional neutralizing antibody (NA). In cases of human revaccination, a sharp rise of HLI started 4 days after vaccination and reached the highest level within 7 days, preceding the rise of conventional NA level which occurred about 3 days later. Three rabbits with negative HLI activity prior to vaccinia immunization obtained an HLI activity within 2 weeks, which showed a sharp rise up to 6-8 weeks. One rabbit with a positive prior HLI activity also showed a sharp rise of the HLI activity after immunization. In all rabbits the final HLI level was identical with that of conventional NA.
Groups of guinea pigs were immunized with either VRV or NDV. Rises of the HLI level after immunization were observed in all animals, the activity being restricted to the homologous virus used in the immunization. Complement-requiring NA was detected during the course of immunization but its behavior was different from that of HLI.
The above observations were interpreted to suggest a ubiquitous presence of HLI as a specific reactive agent and its role at an earliest stage of immune response.

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Incorporation of ³H-TdR into EL4 leukemic cells in vitro was inhibited by peritoneal exudate cells (PEC) harvested from syngeneic C57BL/6J mice given an intraperitoneal (i.p.) injection of 1 × 10⁷ viable Mycobacterium smegmatis ATCC 607 (Smeg) 4 days before. This phenomenon was also observed in the following five systems of PEC from animals and syngeneic tumor cells : C57BL/6J mice and B16 melanoma; DBA/2 mice and P815 mastocytoma ; SWM/Ms mice and K5 fibrosarcoma ; BALB/c, nu/nu mice and KKN-1 fibrosarcoma ; and strain 2 guinea pigs and line-10 hepatoma. The in vitro cytotoxicity of the PEC activated by viable Smeg was much higher than those activated by dead-Smeg, viable BCG or proteose peptone. The activity of the adherent fraction of the PEC was stronger than that of the nonadherent one, and not influenced by either anti-θ or anti-mouse lymphocyte rabbit sera. The PEC induced with Smeg 4 days before contained a large population of mononuclear cells (88.9%) and a significant level of polymorphonuclear cells (PMN) (3.2%), and showed a much higher cytotoxicity than the PEC induced with Smeg 3 hr before, which contained a much larger population of PMN (71.9%), suggesting that PMN were not the effector cells in this system. In vitro and in vivo treatment with macrophage-inhibitors such as carrageenan, trypan blue and cytochalacin B, reduced the activity of the PEC. All of these facts suggested macrophages as the effector. Viable macrophages were required for the growth inhibition of EL4 in vitro : gamma-ray irradiated or freeze-thawed macrophages were ineffective. Kinetic studies revealed that inhibition of ³H-TdR incorporation into EL4 cells started within 3 hr of incubation together with the activated macrophages at an effector to target (E/T) ratio of 5, and the incorporation decreased gradually with the lapse of incubation time. On the other hand, ⁵¹Cr release from labelled EL4 was undetected when the E/T ratio was 5 but detected at on E/T of 10 or more. Even at the higher E/T ratio, at least 10 hr were needed until the release of ⁵¹Cr, suggesting that the activated macrophages produced growth inhibition of tumor cells followed by cell destruction.

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