The efficacy of a DNA vaccine encoding herpes simplex virus type 1 (HSV-1) glycoprotein D in decreasing ocular disease severity following corneal HSV-1 challenge.

Antiviral effects of a DNA vaccine against herpes simplex virus 1 (HSV-1) glycoprotein D (gD) were evaluated in eight week-old female BALB/c mice. The nuclease-insensitive construct (gD-ASOR) consisted of an HSV-1 gD encoding plasmid coupled to asialo orosomucoid (ASOR), targeting it to cells bearing ASOR receptors. Mice were immunized on day 0 and 7 with 10 microg doses of gD-ASOR or control substances. Fourteen days later, mice were infected by the corneal route with 10(5) pfu or 10(6) pfu HSV-1, strain 17syn+. Immunized mice showed a significant decrease in ocular disease severity over a 21-day observation period following infection compared to sham-immunized mice. Acute replication kinetic assays demonstrated a 100-fold decrease in viral titers on day 6 in trigeminal ganglia from immunized BALB/c mice compared to sham-immunized mice. Immunized mice showed a significant increase in numbers of CD4(+)T cells infiltrating the trigeminal ganglia at day 6 post infection compared to sham-immunized mice. Significant differences were not seen in latent viral reservoir between immunized and unimmunized mouse groups. Immunization with gD-ASOR decreased the severity of acute ocular HSV-1 infection, induced a CD4(+) T cell response, decreased the viral load in the trigeminal ganglia, but did not diminish viral latency.

CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus.

A subset of CD161 (NK1) T cells express an invariant Valpha14Jalpha281 TCR-alpha chain (Valpha(invt) T cells) and produce Th2 and Th1 cytokines rapidly in response to CD1d, but their physiological function(s) remain unclear. We have found that CD1d-reactive T cells mediate to resistance against the acute, cytopathic virus diabetogenic encephalomyocarditis virus (EMCV-D) in relatively Th1-biased, C57BL/6-based backgrounds. We show now that these results generalize to Th2-biased, hypersensitive BALB/c mice. CD1d-KO BALB/c mice were more susceptible to EMCV-D. Furthermore, alpha-galactosylceramide (alpha-GalCer), a CD1d-presented lipid antigen that specifically activates Valpha(invt) T cells, protected wild-type (WT) mice against EMCV-D-induced encephalitis, myocarditis, and diabetes. In contrast, neither CD1d-KO nor Jalpha281-KO mice were protected by alpha-GALCER: Finally, disease in Jalpha281-KO mice was comparable to WT, indicating for the first time equivalent roles for CD1d-reactive Valpha(invt) and noninvariant T cells in resistance to acute viral infection. A model for how CD1d-reactive T cells can initiate immune responses, which synthesizes current results, is presented.

Murine response to DNA encoding herpes simplex virus type-1 glycoprotein D targeted to the liver.

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.

Targeted delivery of DNA encoding herpes simplex virus type-1 glycoprotein D enhances the cellular response to primary viral challenge.

Intravenous injection of plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) complexed with asialoorosomucoid-poly-L-lysine (gD-ASOR) targets foreign DNA to the liver, leading to hepatic expression of gD-1. BALB/c mice were given two intravenous injections of gD-ASOR, pBK-ASOR (plasmid lacking the gD-1 gene but complexed with ASOR), or PBS. The skin was inoculated with 1 x 10(4) PFU of HSV-1 or sham-inoculated, and analyzed for infectious virus and cellular infiltration 1, 3, and 5 days after inoculation. Prior immunization with gD-ASOR led to significantly lower (P < 0.05) viral titers in the skin 5 days after inoculation compared with controls. Infiltration of the skin at the site of inoculation by polymorphonuclear neutrophils (PMNs), T cells, B cells, dendritic cells, and macrophages was monitored immunohistochemically. Significantly higher numbers (P < 0.05) of CD4+ and CD8+ T cells, dendritic cells, and macrophages responded to HSV-1 challenge in mice immunized with gD-ASOR than in mice immunized with pBK-ASOR or PBS. The response by PMNs and B cells was indistinguishable among the treatment groups. These results suggest that BALB/c mice sensitized to gD-1 following gD-ASOR immunization develop an enhanced T-cell response to primary HSV-1 infection.

A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D.

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.

Staphylococcal enterotoxin B primes cytokine secretion and lytic activity in response to native bacterial antigens.

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-gamma. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to "bystander" fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-gamma decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.

Cytokines produced early in picornavirus infection reflect resistance or susceptibility to disease.

Gender bias favoring female resistance to picornavirus disease is not seen in ICR Swiss mice following infection with the MM strain of encephalomyocarditis virus (EMCV) (causing encephalitis and death) as it is with D variant of EMCV (causing diabetes in males). To define this difference, an in vitro virus-infected splenocyte culture system was used to explore virus effects on lymphoid cells. Infected and sham-infected splenocyte cultures, prepared from both genders of mice and infected with either virus variant, were examined for immunoregulatory cytokines in the first 24 h of infection using ELISA or bioassays. Disease resistance was associated with increased levels of interferon-y (IFN-gamma) and undetectable levels of interleukin-10 (IL-10) by 12 h postinfection in splenocytes from ICR Swiss females infected with EMCV-D. Disease susceptibility was associated with high levels of IL-10 at 12 h after infection of spleen cells from ICR Swiss males infected with EMCV-D or from both genders infected with EMCV-MM. This information was used to protect susceptible mice against picornavirus disease (either diabetes or death) by giving them an inducer of IFN-alpha/beta, to induce natural killer (NK)-like cells to produce high levels of IFN-gamma and rat monoclonal anti-IL-10 to neutralize the effects of mouse IL-10.

Development of a novel in vitro co-culture system for studying host response to native bacterial antigens.

We have developed a novel co-culture system in which murine splenocytes are cultured with live bacteria in the presence of a bacteriostatic antibiotic. Superantigens, like staphylococcal enterotoxin B (SEB) are important factors in bacterial pathogenicity. Research has shown that superantigens affect numerous immune cell types, either directly or indirectly, yet their involvement in pathogenic mechanisms remains poorly defined. In these studies, we utilize the co-culture system to study how superantigen pretreatment affects interferon-gamma (IFN-gamma) production by splenocytes co-cultured with gram-positive bacteria. Streptococcus mutans, S. sanguis and Bacillus subtilis were tested for susceptibility to a panel of antibiotics. Spectinomycin was found to maintain a bacteriostatic state of approximately 10(5) bacteria ml(-1) at optimal concentrations for each bacterial strain. Co-culturing splenocytes with bacteria did not affect splenocyte viability and cultured splenocytes responded to mitogenic stimulation as expected. Two days after SEB pretreatment, isolated splenocytes cultured with either Streptococcus species produced 10-15 times more IFN-gamma than splenocytes from sham-injected controls; however, no differences in CD4+ or CD8+ T cell populations appeared in cultures with or without bacteria. Splenocytes isolated four days after SEB treatment did not produce significant amounts of IFN-gamma in co-culture. Co-cultures containing live bacteria produced four times more IFN-gamma than cultures containing heat-killed bacteria. Splenocytes depleted of natural killer (NK) cells prior to SEB treatment produced 25% less IFN-gamma after 20 h co-culturing with S. mutans. T lymphocytes were identified to be the major producer of IFN-gamma at this time point by intracellular cytokine staining. Apparently SEB exposure primes a response to live bacteria and the response is evident two days after initial exposure. The in vitro co-culture system allows us to observe host responses to bacteria in the context of the multicellular interdependent immune response. With this assay we can more closely 'mimic' in vivo events, particularly immune cell interactions in microfloral environments, to study how the pathogenic effects of superantigens alter this response.

Does the gender difference in interferon production seen in picornavirus-infected spleen cell cultures from ICR Swiss mice have any in vivo significance?

Splenocyte cultures from female ICR Swiss mice produced greater interferon (IFN) levels, particularly IFN-gamma, than did cultures from males by 12 h post-infection (pi) with the D variant of encephalomyocarditis virus (EMCV-D). This early IFN-gamma is produced by natural killer (NK)-like cells and is dependent on plastic adherent cells and IFN-alpha/beta. In this study, we evaluated the significance of this observation on the innate resistance of ICR Swiss females to EMCV-D-mediated disease. Treatment of females with rabbit anti-mouse IFN-alpha/beta serum rendered them susceptible to the diabetogenicity of EMCV-D. Although sera from both sexes of ICR Swiss mice exhibited peak IFN levels day 3 pi, IFN-gamma was present in the sera of males at only 1 day pi and in the sera of females at days 1-3 pi. Females cleared virus from the circulation by day 2 pi, 1 day earlier than did males. Flow cytometric evaluations of lymphoid cell phenotypes in spleens and pancreata of infected mice revealed that percentages of L3T4+ cells were significantly decreased only in spleens from males at day 1 pi and were diminished along with Ly2+ cells in pancreata of males at 7 days pi, suggesting that T-cell responses were impaired in virus-infected males.

Enhanced capacity of females for early interferon-gamma production by natural killer-like cells following stimulation by staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) induced the production of interferon-gamma (IFN-gamma) by spleen cells from ICR Swiss mice during the first 24 h of culture. Splenocytes from females produced higher levels of IFN-gamma than did those from males at 8, 12, and 16 h. By 20 h after SEA stimulation, IFN-gamma production by spleen cells from males was similar to that of females. The cell types involved in IFN-gamma production in this SEA/spleen cell system were analyzed by depletion studies. Removal of Thy-1+ cells by panning prevented production of IFN-gamma in the 24 h after SEA stimulation. In vivo depletion of asialo GM1+ (AGM1+) cells prevented production of IFN-gamma through 16 h of culture with SEA, but permitted a modest IFN-gamma response at 20 h that was similar in magnitude in both sexes. Following removal of L3T4+ and Lyt-2+ cells by panning, IFN-gamma production was detected at 12 h after SEA stimulation and maintained through 24 h of culture with cells from females producing higher levels of IFN-gamma. These data suggest that male ICR Swiss mice are deficient in the activity of Thy-1+, AGM1+, L3T4-, and Lyt-2- cells in the early (8-16 h) production of IFN-gamma following SEA stimulation of spleen cells.

AGM1+ spleen cells contain gamma interferon (IFN-gamma) gene transcripts in the early, sex-dependent production of IFN-gamma after picornavirus infection.

Encephalomyocarditis D variant (EMCV-D)-infected spleen cell cultures prepared from diabetes-resistant ICR Swiss female mice produce more gamma interferon (IFN-gamma) activity over a 24-h period than do spleen cell cultures from diabetes-susceptible male mice of this strain. Pretreatment of mice with anti-asialo GM1 eliminates early in vitro IFN-gamma production from 4 to 16 h postinfection (p.i.) and reduces IFN-gamma production from 16 to 24 h p.i. In this study, depletion of spleen cells with anti-Thy-1 by panning greatly reduced IFN-gamma activity in EMCV-D-infected spleen cell cultures throughout a 24-h period. Populations of asialo GM1 (AGM1), L3T4, and Lyt-2-positive cells were isolated from cells harvested at 9 h p.i. from EMCV-D-infected spleen cell cultures by a modified panning technique on polystyrene microscope slides. By in situ hybridization with a [35S]dATP-labeled IFN-gamma cDNA probe, only the AGM1-bearing cells were found to contain detectable IFN-gamma gene transcripts. An AGM1+, Thy-1+ natural killer-like cell is the probable producer of the early, sex-dependent IFN-gamma activity in this system.

Sex hormone modulation of interferon (IFN) alpha/beta and gamma production by mouse spleen cell subsets following picornavirus infection.

Replication of the diabetogenic variant of encephalomyocarditis virus (EMCV-D) in spleen cells and its association with subpopulations of spleen cells (L3T4+, Lyt-2+, Mac 1+, 33D1+ and AGM1+ cells) from both sexes of ICR Swiss mice were examined. Virus replication was limited to less than 0.5 log in suspensions of whole spleen cells, nonadherent cells or a B cell subfraction from both sexes of ICR Swiss mice following infection with EMCV-D at an MOI of 10; no virus replication was seen in adherent spleen cells from either sex. After 1 hour adsorption of EMCV-D onto spleen cells at a multiplicity of infection (MOI) of either 10 or 0.1, virus-associated cells were isolated using a monoclonal murine anti-EMCV-D and anti-mouse IgG conjugated to magnetic beads. Using an MOI of 0.1, less than 1% of spleen cells bound virus particles after 1 hour adsorption at 4 degrees C. Among the virus-positive cells, relatively higher percentages of adherent cell populations (Mac 1+ and 33D1+ cells) of both sexes bound virus particles within the first hour post-infection (PI) than did the other spleen cell subpopulations. Interferon (IFN) alpha/beta production was detected as early as 4 hours PI in female spleen cell cultures infected with EMCV-D at an MOI of 0.1 while no IFN alpha/beta activity was found in comparably infected male spleen cell cultures. Inhibiting IFN alpha/beta activity in the virus-infected spleen cell cultures during the first 20 hours of infection using polyclonal rabbit anti-mouse IFN alpha/beta serum eliminated production of IFN gamma as well as IFN alpha/beta. Spleen cell cultures depleted of adherent cells were unable to produce IFN alpha/beta or IFN gamma in the first 24 hours PI. The capacity to produce IFN gamma at 12 hours after virus infection of spleen cells from both sexes of mice was restored to adherent cell-depleted cultures by addition of mouse IFN alpha/beta at the time of infection. These results suggest that IFN alpha/beta and adherent cells play critical roles in the early production of IFN gamma (less than 16 hours PI) characteristic of the infected spleen cell cultures of females. Production of IFN alpha/beta and IFN gamma by spleen cells from both sexes of ICR Swiss mice was enhanced by administrating estrone to donor mice during the week before harvesting spleen cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Sex-dependent, early cytokine production by NK-like spleen cells following infection with the D variant of encephalomyocarditis virus (EMCV-D).

Spleen cell cultures from diabetes-resistant ICR Swiss females exhibited an increase in expression of Ia antigens 24 hours post-infection (PI) with EMCV-D while comparable spleen cell cultures from diabetes-susceptible males of this strain did not exhibit this increase in Ia antigens expression. A monoclonal antibody specific for mouse interferon-gamma (IFN gamma) eliminated this increase in Ia antigens expression. Interferon-gamma (IFN gamma) and interleukin 2 (IL-2) production by EMCV-D-infected spleen cell cultures were monitored at 4-hour intervals for 24 hours. Female spleen cells produced IFN gamma earlier (less than 16 hours PI) and in greater amounts than did comparably treated male spleen cells. Addition of a monoclonal rat anti-mouse IL-2 to virus-infected cultures did not significantly affect the early (less than 16 hours PI) production of IFN gamma by spleen cells of females. Treatment of the spleen cell donors with rabbit anti-asialo GM1 (AAGM1) abolished early production of IFN gamma in virus-infected female spleen cell cultures and reduced the early IL-2 production by infected male and female cells. These results suggest that an NK-like cell is responsible for the early female IFN gamma production; this may be a factor in the resistance of female ICR Swiss mice to EMCV-D-induced diabetes.

Impaired cytokine response in male ICR Swiss mice after infection with D variant of encephalomyocarditis virus.

The basis for the resistance of the female and the susceptibility of the male ICR Swiss mouse to the diabetogenicity of the D variant of encephalomyocarditis virus (EMCV-D) is unknown. This pattern of disease resistance and susceptibility can be reversed if females are treated with testosterone and males are treated with estrogen before virus infection. As a possible explanation for this sex difference in disease development, differences in early antiviral host responses were explored. Cellular antiviral resistance mechanisms operative early in virus infection were evaluated in ICR Swiss mice of both sexes after intraperitoneal infection with virus. No differences were seen in splenic natural killer (NK) cell responses of male and female mice during the 1st wk of infection, during which only the males became diabetic. Depletion of NK cell activity with rabbit anti-asialo GM1 serum did not render the infected ICR Swiss female susceptible to virus-induced diabetes. Treatment of ICR Swiss mice with type I carrageenan to compromise macrophage function rendered the female susceptible to diabetes after infection with EMCV-D but made only the male susceptible to diabetes by the usually avirulent interferon-inducing EMCV-D. Concanavalin A and recombinant interleukin 2, inducers of immune interferon, which in turn primes macrophages for activation and induces their expression of la antigens, protected the ICR Swiss male against the diabetogenic effects of EMCV-D. Interleukin 2 enhanced the male's capacity to exhibit an increase in the expression of Ia antigen by peritoneal exudate cells 1 day after injection with EMCU-D to a level seen in disease-resistant females.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus-induced murine diabetes. Enhancement by immunosuppression.

Adult, male ICR Swiss mice are susceptible to the diabetogenic effects of the D-variant of encephalomyocarditis virus (EMC-D) in contrast to adult C3H/HeJ male mice, which are relatively resistant. To date, experimental evidence suggests that the immune system plays a role in the pathogenesis of this infection. We have investigated the potential involvement of the immune system in the pathogenesis of EMC-D-induced diabetes using cyclosporin-A (CyA), a potent immunosuppressive drug. The data show that treatment with CyA results in increased severity and incidence of diabetes in susceptible ICR Swiss mice and induction of diabetes in resistant C3H/HeJ mice. It is concluded that immune mediation probably is not involved in the early pathogenesis of EMC-D-induced diabetes in mice.

A murine model of insulin-dependent diabetes mellitus resulting from the cumulative effects of the nondiabetogenic strain of encephalomyocarditis virus and a single low dose of streptozocin.

The induction of insulin-dependent diabetes in outbred male and female mice was examined using a combination of the usually nondiabetogenic B-variant of encephalomyocarditis (EMC-B) virus and single low doses of streptozocin (STZ). Neither EMC-B virus nor low doses of STZ were overtly diabetogenic when administered alone; however, when these two insults occurred 1 day apart, diabetes resulted in male but not in female mice. The induction of diabetes was dependent on the time interval between these two insults, since EMC-B virus and STZ given 4 days apart did not induce diabetes. Unexpectedly, when the order of these two insults was reversed, diabetes occurred. The absence of diabetes when EMC-B virus was given before STZ suggested the possibility that virus-induced interferon blocked the cytotoxic effects of STZ. This suggestion was supported by the observation that an antiserum against beta interferon abrogated the virus-mediated protection against STZ-mediated cytotoxicity. Also, Poly I:C administered before a single diabetogenic dose of STZ delayed the onset of severe hyperglycemia.

Exacerbation of the pathogenesis of the diabetogenic variant of encephalomyocarditis virus in mice by interferon.

Adult male ICR Swiss mice develop insulin-dependent diabetes when infected with the D variant of encephalomyocarditis virus (EMC-D). In contrast, adult C57Bl/6 males are relatively resistant to the diabetogenic effects of this virus. We have been studying the role of interferon (IFN) in the pathogenesis of infection by EMC-D and development of virus-induced murine diabetes mellitus. The data show that when IFN beta or IFN gamma were administered four days after virus infection, the frequency and severity of diabetes were exacerbated in ICR Swiss mice, and the diabetic state was induced in the resistant C57Bl/6 strain. In addition, animals treated with either of the IFNs or the IFN-inducer poly I:C, developed symptoms of severe encephalomyocarditis. Analysis of ICR Swiss mouse tissues revealed that IFN-treatment resulted in virus replication in the heart and brain and the reappearance of the virus in the pancreas. It is concluded that under certain conditions, the diabetic state is exacerbated and the normal course of (EMC-D)-infection in mice is altered by IFN.

New screening method for occult gastrointestinal bleeding: immunologic and guaiac slide tests.

A valid mass screening method for occult, bleeding gastrointestinal pathology including colorectal cancer should be monospecific for human hemoglobin, sensitive for approximately 3 mg of human blood per 1 g of stool, capable of differentiating upper and lower gastrointestinal bleeding, cost effective, uncomplicated, and acceptable to patients. Hemoccult II, a guaiac peroxidase detection test, is nonspecific for human blood and cannot differentiate between upper and lower gastrointestinal bleeding. A radial immunodiffusion slide test for detecting human hemoglobin was compared with a guaiac test over a four-year period in 211 patients. in gastrointestinal problems diagnosed by endoscopy, roentgenographic rays, and other procedures, the Hemoccult II was positive in 9 of 41 cases of upper gastrointestinal tract origin (21 percent detection rate), whereas the radial immunodiffusion method, expected to be negative as a result of action of gastrointestinal proteases, was positive in only 3 and negative in 38 of the 41 samples (92 percent accuracy). The two tests were equally effective in detecting lower gastrointestinal bleeding (14 of 37 samples, 37 percent accuracy). The findings of this study indicate that the immunologic test may remedy the deficiencies of the guaiac test. The concomitant use of the immunologic and appropriately sensitive guaiac test appears to fulfill screening test requisites.

Replication of diabetogenic and nondiabetogenic variants of encephalomyocarditis (EMC) virus in ICR Swiss mice.

The replication of the diabetogenic D (EMC-D) and nondiabetogenic B (EMC-B) variants of encephalomyocarditis virus in various tissues of the murine host was determined. Pancreatic insulin levels were also measured. EMC-D replicated in the spleen, pancreas, heart, lung, and intestines, while EMC-B was limited to the spleen and pancreas. EMC-B interfered with the replication of EMC-D in each of the tissues examined. Insulin levels were initially increased by both viruses. By 4 days postinfection, insulin levels were either normal or undetectable in (EMC-D)-infected animals, but were dramatically elevated in those infected with EMC-B.

Antigenic modification, rosette-forming cells, and Salmonella typhimurium resistance in outbred and inbred mice.

To assess the separate contributions of host T cells and the physical state of the antigen in the development of effective. Salmonella resistance, glutaraldehyde-treated and untreated protein- and ribonucleic acid-rich extracts (E-RNA extracts) of virulent Salmonella typhimurium SR-11 or attenuated S. typhimurium RIA were used to immunize Salmonella-resistant Salmonella-susceptible strains of mice for the purpose of determining whether antigen-specific T-cell or B-cell responses were formed and, if so, which responses predominated. The resistance imparted to each mouse strain after vaccination with S. typhimurium RIA was used as the standard for comparison. The inbred mouse strains C57BL/6 and DBA/2 and their F(1) hybrid (strain BDF(1)), outbred ICR Swiss mice, and endotoxin-resistant C3H/HeJ mice were examined for the capacity to develop resistance to lethal Salmonella infections, as well as the ability to generate antigen-reactive T cells. Only the BDF(1), C3H/HeJ, and ICR Swiss mice were able to develop resistance to challenge infections mediated by the virulent SR-11 strain of S. typhimurium after vaccination with the living, attenuated RIA strain of S. typhimurium or immunization with E-RNA extracts. We developed an assay to identify the antigen-reactive rosette-forming lymphocytes present in lymph nodes and spleens of immunized mice. Levels of 0.2% or higher of theta antigen-bearing, antigen-reactive rosette-forming cells were found in the lymph nodes or spleens or both of only the BDF(1), C3H/HeJ, and ICR Swiss mice (i.e., in the "Salmonella responder" strains). Mouse strains C57BL/6 and DBA/2, which failed to develop resistance to lethal infections after immunization with the S. typhimurium RIA vaccine or with the E-RNA extracts, lacked effective numbers of antitheta antigen-sensitive rosette-forming cells. Modification of the effective E-RNA extracts by polymerization with glutaraldehyde resulted in a marked diminution in their abilities to induce resistance to salmonellosis in the two responder mouse strains tested (BDF(1) and ICR Swiss), even though detectable levels of antibody were induced.