Nobuya Ohtomo

Mechanism of Antibody-Mediated Protection against Herpes Simplex Virus Infection in Athymic Nude Mice: Requirement of Fc Portion of Antibody

Experiments were performed to investigate the resistance of the host due to antibody‐mediated mechanisms to herpes simplex virus (HSV) infection. Transfer of hyperimmune anti‐HSV mouse serum inhibited the development of skin lesions and prolonged the survival of lethally HSV‐infected nude mice. Relatively high concentrations of antibody were required to achieve this protection. Antisera prepared in heterologous animals were also effective, while administration of anti‐cowpox virus serum or interferon provided no protection. This type of protection is therefore due to specific antibody and cannot be attributed to interferon. In order to delineate the requirement for antibody in antibody‐mediated protection, human gamma globulin preparations were transferred to lethally HSV‐infected nude mice. Transfer of intact human gamma globulin (GG) was effective in controlling infection. S‐sulfonation of GG did not diminish the protective ability. However, purified F (ab′)2 did not have any protective action even when it was administered frequently to maintain serum neutralizing antibody titer. GG was effective in C5‐deficient mice lethally infected with HSV. These results indicate that in vivo antibody‐mediated protection to HSV infection requires the Fc region of the intact IgG molecule and suggest that antibody‐dependent cell‐mediated cytotoxicity may be operative in vivo.

Pathogenesis of Vibrio parahaemolyticus: Intraperitoneal and Orogastric Challenge Experiments in Mice

It has been noted that V. parahaemolyticus isolated from patients with food poisoning are almost always thermostable direct hemolysin (TDH)‐positive, whereas, isolates from foods or environmental sources are usually TDH‐negative. The virulence of V. parahaemolyticus in mice was examined by using intraperitoneal and orogastric challenge models, where the strains used were those isolated from patients and foods with food poisoning and included TDH‐positive and ‐negative. The LD50 in mice was estimated to be approximately 107 in the former and 108 in the latter challenge model. In the mice killed by challenge, either intraperitoneal or orogastric, the following pathological changes were almost always observed: swelling, redness and fluid accumulation in the small intestine, particularly the upper part of it. Histologically, congestion, edema, and vacuolation were observed in mucosal and submucosal tissues; furthermore, ulceration at tips of some villi was noticed. The lethality and pathological finding in mice due to V. parahaemolyticus were demonstrated similarly in both challenge models and irrespective of the TDH phenotype of the strain used. It will be suggested that the findings in challenged mice may be. attributed to enteropathogenic factor(s) other than TDH of V. par ahaemolyticus.

Passive immunization of mice with monoclonal antibodies to glycoprotein gB of herpes simplex virus

To investigate the protective ability of monoclonal antibodies (MCAs) to viral glycoprotein in herpes simplex virus (HSV) infection, athymic nude mice were inoculated intracutaneously with HSV type‐1 (HSV‐1) in the midflank. Three hours after inoculation, one group of mice was passively immunized with one of a series of MCAs to glycoprotein gB of HSV‐1, and a control group of mice was given phosphate buffered saline alone. The control mice died within 16 days after infection, whereas the mice passively immunized with any of the MCA showed suppressed development of skin lesions. Three of six mice given MCA failed to develop any visible lesions and no HSV could be isolated from the lumbar dorsal root ganglia of these mice 60 days after the challenge. BALB/c mice were also protected from infection with HSV type 2 by passive immunization with MCA to HSV‐1 gB.

Entrance of Cholera Enterotoxin Subunits into Cells

Quantitative analysis of the staining of cholera enterotoxin on the surface of cells with specific antibodies against each subunit of cholera toxin, using a Fluorescence-Activated Cell Sorter, showed that not only subunit A but also subunit B penetrates the cell membrane. The detection of each subunit inside the cell was facilitated by the use of saponin, an agent which increases membrane permeability.

Increase in Intradermal Vascular Permeability Caused by Pertussis Toxin from Bordetella pertussis

Rabbits that were injected intradermally with pertussis toxin (PT), produced from Bordetella pertussis, showed slight edema and erythema at the injection sites, but not hemorrhage nor necrosis. The edema lesions were stained blue by the intravenous injection of Pontamine Sky Blue 6B dye, suggesting that PT caused increased vascular permeability, similarly to the permeability factor (PF) of cholera toxin. The reaction of the PF of PT could be determined by measuring the diameter of the blue area. The diameter of the blue area bore a good linear relationship to the logarithm of the dose of PT. The activity of the PF was neutralized by anti‐PT rabbit serum. Detoxification of PT with formalin did not increase the vascular permeability, but reverted pertussis toxoid showed a PF reaction in proportion to the reverted leukocytosis‐promoting and histamine‐sensitizing activities of PT. The supernate of a Bordetella pertussis culture also induced a PF reaction and the reaction could be made clear by heating the supernate at 56 C for 30 min, but the supernate of Bordetella bronchiseptica did not induce the reaction at all.

Experimental Cholera in Germ-free and Gnotobiotic Piglets

We have previously reported that two to five weeks old germ-free piglets developed cholera-like watery diarrhea consistently after orogastric inoculation with cholera toxin or Vibrio cholerae Inaba 569B.1) Because Inaba 569B is an extraordinarily hypertoxigenic strain of Vibrio cholerae, we have further evaluated the germ-free piglet model by using strains with lower toxigenicity as the challenge organism. Vibrio cholerae El Tor Inaba P6973, El Tor Inaba T348 and Ogawa NIH41, which produce toxin at the same level as current isolates, were used as challenge strains.2)

Further Evidence Showing that Subunit B of Cholera Toxin Enters the Cell

Lymphocyte has been known to bind cholera toxin (CT) on its surface (7, 13, 16). Since the subsequent accumulation of intracellular cyclic AMP is observed (9, 19), it is believed that the A subunit (SA) of CT penetrates the cell membrane after binding of CT to the receptor ganglioside with the B subunit (SB) to stimulate intracellular adenylate cyclase as suggested in other CT-cell system [reviews see 3, 6, 11, 18]. There has been a speculative model for the internalization of CT into cell (2), in which SA penetrates the cell membrane through the channel constructed by SB’s extending themselves into the bilayer of the membrane. In such a model, it is expected that SA disappears from the surface after absorption, although SB remains on the surface. We have started the present study with an intention to give a substantial basis for CT-cell interaction. Mouse thymus cells have been chosen for this study because these cells are easily obtainable in large numbers with minor contamination of other type of cells and suitable for the analysis with a fluorescence-activated cell sorter, which facilitates to determine the relative amount of specific antigen on the cell surface.