The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6×1.2μm in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0×3.0μm and stained deep blue. Strain GIFU 10007 was internalized into 23%, of the HeLa cells within 10min after inoculation. About 90%, of the HeLa cells were infected after 24hr incubation in kanamycin (KM)-containing medium, Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24hr incubation in KM-containing medium by both light-microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40-fold within 24hr after inoculation, whereas in the four other less or non-infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.

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An electron microscopic study revealed that, within 30min after inoculation into the ligated ileal loop of anesthetized mice. cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyer's patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.

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In vitro effects of the Bordetella HLT on the isolated perfused lung and some other tissue preparations from guinea pigs were examined. When HLT (30 to 300MNDs/ml) was administered, an increase of the perfusion pressure was induced in the perfused lungs, indicating vasoconstriction. When 100 or 300MNDs/ml of HLT was given, the pressure increase appeared after a lag period of 3.5 to 4min, reached a maximum within 8 to 13min, and then slowly decreased by 60 to 80% 25min after exposure. In calcium-free medium, the pressure increase dur to HLT did not occur, but these HLT-treated lungs manifested an increase without any lag period immediately after the calcium-free medium was replaced by normal medium containing calcium. No difference in the response of the perfused lungs to histamine was observed before and after exposure to HLT. The arterial strip did not respond to HLT, but after predigestion with a collagenase and elastase solution the contractive response to 100MNDs/ml of HLT appeared with a lag period of 1min. HLT had no effect on the pharmacological responses of the isolated atria, deferent canal or intestinal preparations, or on the ciliary movement of cultured tracheal rings.

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Starting from the group II RNA phage GA which has an amber mutation in the maturation protein cistron, a spontaneous mutant of group II phage GA, whose serological and electrophoretic properties became similar to those of group I phage MS2, was isolated and analyzed. The mutant has now become sensitive to anti-MS2 serum and resistant to anti-GA serum. Analysis of the nucleotide sequence of the coat protein gene revealed that G↔A transition was the main change. The deduced amino acid sequence showed that five amino acids were substituted in the mutant, and three of the five became identical to MS2, resulting in increased molecular weight of the coat protein. However, it did not complement MS2. These results suggested that the serological change from group II phage GA type to group I phage MS2 type is induced spontaneously at high frequency by minor nucleotide changes in coat protein gene, and confirmed the previous results at the RNA level that MS2 and GA were related although the closeness between them seems somewhat remoter than that of groups III and IV (18, Inokuehi et al, unpublished data for the nucleotide sequence of group IV phage SP).

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The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV-1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the -GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated -GCr variant. When the attenuated variant was preinoculated i.p. 24hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV-1 was shown to be age-dependent.

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Two monoclonal antibodies against human IFN-α-one against natural leukocyte IFN-α and the other against recombinant human IFN-α 2 produced in E. coli-were prepared, and designated as HT-1, and 104-5-f respectively. These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN-α5s produced by E. coli and by mouse cells. The HT-1 antibody could bind and neutralize recombinant human IFN-α5 synthesized in mouse cells, but not recombinant human IFN-α5 synthesized in E. coli. On the other hand, the 104-5-f antibody could bind and neutralize recombinant human IFN-α5 synthesized in E. coli but not recombinant human IFN-α5 synthesized in mouse cells. Then these monoclonal antibodies or sheep polyclonal antibody against human IFN-α were used to immunoprecipitate the radioactively labeled recombinant human IFN-α5 synthesized either in E. coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO⁴. The labeled recombinant human IFN-α5 produced by mouse cells could be immunoprecipitated with the HT-1 monoclonal antibody or sheep anti-(human IFN-α) polyclonal antibody but not with the 104-5-f monoclonal antibody and showed a band of Mr. 17, 500 on polyacrylamide gel electrophoresis in the presence of NaDodSO⁴. On the other hand, the labeled recombinant human IFN-α5 produced by E. coli could be immunoprecipitated with the 104-5-f monoclonal antibody but not with the HT-1 monoclonal antibody and showed a band of similar Mr. on polyacrylamide gel electrophoresis. These results indicate that the two different preparations of recombinant human IFN-α5, one produced by mouse cells and the other in E. coli, have different epitopes on their molecules which are supposed to have the same primary amino acid sequence. Although the reason why these different epitopes are expressed on the two molecules is unclear, the results agree with our previous observation that recombinant human IFN-α5 produced by mouse cells but not by E. coli has a slight cross reactivity on mouse cells [Fukunaga, Sokawa, and Nagata, 1984. Proc. Natl. Acad. Sci. U.S.A. 81: 5086-5096]. And these results may suggest the different tertiary structure between recombinant human IFN-α5 produced in E. coli and mammalian cells.

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Tissue sites for synthesis of the fifth component of complement (C5) in vivo have been investigated by using allogeneic bone marrow chimeras and bone marrow chimeras which were transplanted in addition with hepatocytes. Our prior studies have demonstrated that bone marrow chimeras which had been prepared by transplanting marrow cells from C5-sufficient donor mice into irradiated C5-deficient recipients lacked detectable levels of C5 in the sera. However, when such potentially C5-deficient [C5(+)→C5(-)] chimeras were introduced into their spleens by means of injections of fully dispersed single cell suspensions of hepatocytes isolated from the C5-sufficient donor strain, they accepted the transplantation of hepatoeytes for prolonged periods and developed a measurable amount of C5 in the sera. These results indicate that C5 protein in sera is not synthesized in significant amount by cells that are descendants of bone marrow cells but rather that this complement component is synthesized and delivered to the blood in vivo by somatic cells including liver cells that are not derivatives of the bone marrow.

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Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.

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The relative role of thymus-derived (T-) lymphocytes and bone marrow-derived (B-) cells in acquired immunity to salmonellosis was examined in mice. The results demonstrate that the protective capacity of the donor immunized mice could be passively transferred to the recipient mice by spleen cells but not with peritoneal exudate cells or sera. A high cell number of spleen cells (2×10⁸/mouse) were required before passive transfer of immunity could be obtained. Of the T-lymphocytes and B-cell populations of spleen cells, T-cells from immune mice were effective in conferring protection to the recipient mice.

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To explore a possible correlation between susceptibility to Toxoplasma and interferon (IFN)-generating capacity in mice, we compared the levels of serum IFN induced by stimulation with Toxoplasma lysate antigen (TLA) in different strains of Toxoplasma-infected and uninfected mice. Injection of TLA into five strains of mice with chronic Toxoplasma infection resulted in the release of considerable amounts of IFN into the circulation. Most of these IFN activities were acid labile and not neutralized by sheep antiserum against mouse IFN-α/β, indicating that IFN-γ was the dominant form produced in this system. In contrast, the majority of IFN induced in uninfected mice was characterized as IFN-α/β by their acid stability and antigenicity. The response of IFN production in Toxoplasma-infected and uninfected mice varied quantitatively depending on the mouse strains examined. C57BL/6 mice were found to be the best producers of both IFN-α/β and IFN-γ, while BALB/c mice were consistently poor producers of both IFN populations. A/J, DBA/2, and C3H/He mice could be roughly classified as intermediate producers of both IFN populations. C57BL/6 and C3H/He mice showed a significant prolongation of mean survival time following primary or secondary infection with Toxoplasma compared to that of BALB/c mice. However, there was no direct correlation between the susceptibility to Toxoplasma and the levels of serum IFN.

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