Biomedical subjects

G N Milligan

Publications and source records attributed to G N Milligan.

14 recordsLinked to original sources

PubMed2001

Role of polymorphonuclear leukocytes in resolution of HSV-2 infection of the mouse vagina.

A naturally occurring population of polymorphonuclear leukocytes (PMNs) was detected in the vaginal lumen of uninoculated mice. A large population of these cells also infiltrated the vaginal mucosa following intravaginal HSV-2 inoculation. We examined the role of PMNs in preventing infection of the vaginal mucosa, virus clearance, and limiting virus spread to the lumbosacral ganglia. Depletion of PMNs prior to HSV-2 inoculation did not increase the incidence of infection suggesting that the small population of resident PMNs was ineffective in preventing infection by a viral pathogen. Depletion of PMNs impacted virus clearance from the vagina over a range of HSV-2 doses resulting in significantly higher virus titers on days 4 through 6 after inoculation. Virus clearance was delayed in PMN-depleted immune mu MT mice suggesting that PMN involvement in HSV-2 clearance did not require specific antibody. PMN-depletion of non-immune mice increased virus spread to the sensory ganglia only in mice inoculated with high virus doses. Immunization of mice with an attenuated strain of HSV-2 protected the sensory ganglia against acute infection with a challenge strain. Although PMN depletion of immune mice significantly increased virus titers in the vagina, the incidence of acute virus replication in the sensory ganglia was not different than in control-treated immune mice suggesting that PMNs were not required for protection of the sensory ganglia in immune animals. Taken together, these results suggest that PMNs were involved in resolving genital HSV-2 infections, but played only a limited role in preventing HSV-2 spread to the sensory ganglia.

Animals↗

PubMed2000

Immune protection against HSV-2 in B-cell-deficient mice.

The role of antibody in protection of the vaginal mucosa and sensory ganglia against HSV-2 infection was examined using HSV- immune, B-cell-deficient muMT mice. Significantly higher virus titers were detected in the vaginal mucosae of immune muMT mice compared to immune C57BL/6J mice 24 h after HSV-2 rechallenge. However, virus was rapidly cleared in immune muMT mice, and the infection was resolved with only a 2-day delay. Passive transfer of immune serum to immune muMT mice prior to rechallenge resulted in HSV-specific vaginal IgG levels comparable to those of immune C57BL/6J mice. Although transferred antibody failed to prevent reinfection of the majority of recipients, vaginal virus titers at 24 h and clearance kinetics were similar to those of immune C57BL/6J controls. Following vaginal rechallenge, HSV-2 did not spread to the sensory ganglia of immune C57BL/6J mice nor was the rechallenge virus detected in the ganglia of the majority of immune muMT mice. However, protection was severely compromised by T-cell depletion of immune C57BL/6J mice. These results suggest that HSV-specific antibody limits, but does not prevent, infection of the genital epithelia. Further, prevention of virus spread to the sensory ganglia in immune animals requires vigorous T-cell immune responses.

Animals↗

PubMed1999

Neutrophils aid in protection of the vaginal mucosae of immune mice against challenge with herpes simplex virus type 2.

Large numbers of polymorphonuclear leukocytes (PMNs) infiltrated the murine vaginal mucosa within 24 h after intravaginal inoculation with an attenuated strain of herpes simplex virus type 2 (HSV-2). The role of these cells in resolution of a primary genital infection and in protection of HSV-immune animals against challenge with a fully virulent HSV-2 strain was investigated. Depletion of greater than 95% of the PMNs at the vaginal mucosal surface prior to intravaginal inoculation with an attenuated HSV-2 strain resulted in significantly higher virus titers on days 3 to 7 but only slightly delayed resolution of the primary genital infection. These results suggest that neutrophils helped control the infection but that other immune mechanisms ultimately cleared the virus. Interestingly, depletion of PMNs from HSV-immune mice prior to challenge with a fully virulent HSV-2 strain resulted in a rise in virus titers to levels comparable to those of nonimmune mice and a more pronounced diminution of virus clearance from the vaginal mucosa despite the presence of HSV-specific B and T cells. Levels of gamma interferon (IFN-gamma) and HSV-specific antibody were comparable in neutrophil-depleted and control-treated immune mice following HSV-2 challenge, suggesting that RB6-8C5 treatment did not impair T- and B-cell function. Therefore, these results suggest that neutrophils play a role in limiting and clearing HSV-2 vaginal infections and that they are, in association with HSV-specific B and T cells, an important component in immune protection of the vaginal mucosa.

Animals↗

PubMed1998

T lymphocytes are required for protection of the vaginal mucosae and sensory ganglia of immune mice against reinfection with herpes simplex virus type 2.

Intravaginal inoculation of mice with an attenuated strain of herpes simplex virus type 2 (HSV-2) resulted in vigorous HSV-specific immune responses that protected against subsequent challenge with fully virulent HSV-2 strains. Even in the presence of high titers of HSV-specific Ab, T cell-dependent mechanisms were required for protection of the vaginal mucosae of HSV-immune mice and could be detected by 24 h after intravaginal reinoculation. Depletion of specific T cell subsets from HSV-immune mice before HSV-2 reinoculation demonstrated that CD4+ T cells were primarily responsible for this protection. Similarly, optimal protection of the sensory ganglia against reinfection with HSV-2 was dependent on the presence of T cells. Infectious HSV-2 was not detected in the sensory ganglia or spinal cord of HSV-immune mice depleted of only CD4+ or CD8+ T cells, suggesting that the T cell-mediated protection could be provided by either subset. Similarly, neutralization of IFN-gamma during challenge of HSV-immune mice resulted in diminished protection of the vaginal mucosa, but not of the sensory ganglia. These results suggest that the ability to induce vigorous HSV-specific T cell responses is an important consideration in the design of vaccines to protect both the vaginal mucosa and sensory ganglia against HSV-2.

Animals↗

PubMed1997

Interferon-gamma enhances resolution of herpes simplex virus type 2 infection of the murine genital tract.

The requirement for interferon-gamma (IFN gamma) in resolution of an HSV-2 vaginal infection and the cellular sources of this cytokine in the vaginal mucosa were assessed. IFN gamma levels in vaginal secretions peaked on Days 2 and 5 following HSV-2 inoculation. Natural killer (NK) cell depletion greatly diminished the early production of IFN gamma but had no significant effect on the rate of virus clearance. CD4+ T cells were primarily responsible for the second peak of IFN gamma levels and neutralization of this IFN gamma beginning 3 days after virus inoculation delayed, but did not prevent, virus clearance from the vagina. HSV-2 persisted in mice depleted of both CD4+ and CD8+ T cells while clearance was delayed in CD4+, but not CD8+ T cell-depleted mice, demonstrating the T cell dependence and predominant role of CD4+ T cells in resolution of the infection. Together, these data suggest that IFN gamma is not essential for virus clearance but plays an important role in enhancing T cell-mediated clearance mechanisms. The implication of these results is that IFN gamma produced locally in the genital tract enhances virus clearance and may ultimately be important for reducing the amount of virus available to infect sensory ganglia.

Animals↗

PubMed1996

DNA immunization confers protective immunity on mice challenged intravaginally with herpes simplex virus type 2.

The immunogenicity and protective efficacy of a nucleic acid vaccine encoding the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene under the control of the CMV immediate early gene promoter was examined. Mice immunized three times by intramuscular injection with the vaccine developed an HSV specific IgG but not IgA antibody response detectable in both serum and vaginal secretions. In addition, antigen-specific cellular immune responses were detected in splenic lymphocytes isolated from DNA immunized animals. Immunization reduced virus replication in the genital tract following a lethal intravaginal HSV-2 challenge. Furthermore, symptomatic genital HSV disease was reduced in immunized mice and the animals were completely protected from death. We conclude that a nucleic acid vaccine expressing HSV-1 gD induced both humoral and cell mediated immune responses in mice which proved highly protective against disease following virus challenge.

Animals↗

PubMed1995

Analysis of herpes simplex virus-specific T cells in the murine female genital tract following genital infection with herpes simplex virus type 2.

A murine model of genital infection with a thymidine kinase-deficient (tk-) strain of herpes simplex virus type 2 (HSV-2) was utilized to examine the development of the local T cell response in the genital mucosa and draining genital lymph nodes (gLN). HSV-specific cytokine-secreting T cells were detected in the gLN 4 days postintravaginal inoculation but not in the urogenital tract or spleen until 5 days postinoculation, suggesting the cellular immune response originates in the gLN. More CD4+ than CD8+ gLN T cells were detected by flow cytometric analysis following primary vaginal inoculation and the majority of HSV-specific gLN T cells detected by ELISPOT were CD4+ and Th1-like based on secretion of IFN gamma and not IL-4 or IL-5. A similar population of HSV-specific memory T cells persisted in the genital tract 2 months following HSV-2 tk- genital inoculation. These data suggest that the urogenital cellular immune response elicited in mice following genital inoculation with HSV-2 tk- is predominantly CD4+ and Th1-like, resembling that observed in humans. The results of this study are important for the rational design of vaccines capable of inducing protective immunity in the genital tract.

Animals↗

PubMed1995

Generation of humoral immune responses against herpes simplex virus type 2 in the murine female genital tract.

A murine model of genital infection with a thymidine kinase-deficient (tk-) strain of herpes simplex virus type 2 (HSV-2) was utilized to examine the local urogenital antibody response to HSV-2. Vaginal inoculation with HSV-2 tk- protected against a subsequent genital challenge with a lethal dose of virulent HSV-2. After primary vaginal infection, predominantly HSV-specific IgG antibodies were detected in serum and vaginal secretions. HSV-specific IgG antibody-secreting cells (ASC) were detected first and in greatest numbers in the genital lymph nodes (gLN) after primary HSV-2 tk- inoculation. HSV-specific IgG or IgA ASC were not detected in the urogenital mucosa after primary HSV-2 vaginal infection. Vaginal HSV-2 challenge of HSV-immune mice resulted in increased HSV-specific serum IgG antibody and vaginal IgA antibody titers. HSV-specific IgG ASC were detected by 4 days postchallenge in gLN and by Days 6 and 7 postchallenge in the spleen and genital mucosa. These results suggest that urogenital humoral responses originate in the gLN following HSV genital infection and that gLN may serve as the primary source of the HSV-specific IgG- and IgA-secreting cells present in the urogenital mucosa after vaginal challenge.

Animals↗

PubMed1994

Requirement for B cell-derived immunoglobulin for Th activation by the type 2 antigen polyvinylpyrrolidone.

Th specific for the type 2 antigen polyvinylpyrrolidone (PVP) could not be activated in vivo or in vitro in mice rendered B cell deficient by treatment from birth with anti-IgM. The requirement for B cells was apparently not due to a requirement for B cells to function as antigen presenting cells for Th activation since T cells also were not activated when anti-IgM treated mice were reconstituted with B cells prior to priming with PVP. In addition, removal of B cells from spleens of adult of adult mice had no effect on the ability of PVP to activate Th in vitro. Potential non-specific effects of chronic anti-IgM treatment on T cell function are unlikely since T cells from anti-IgM treated mice provided help to B cells for antibody responses to horse red blood cells responded normally to T cell mitogens and had a CD4:CD8 ratio like that of untreated mice; PVP primed T cells from anti-IgM treated mice also did not actively suppress the helper activity of PVP primed T cells from normal mice. In addition, PVP-specific Th were able to be activated in mice given multiple injections of anti-IgM beginning 2 weeks after birth and in mice given MOPC 104E IgM together with anti-IgM; under these conditions B cell depletion by the anti-IgM did not occur. Thus activation of PVP-specific Th requires that B cells be present during the first few weeks after birth. Experiments in which mice were given injections of normal mice Ig together with the anti-IgM indicate that the function of B cells for Th activation in this system can be replaced by B cell-derived Ig. Thus PVP-specific Th could be activated in mice treated from birth with anti-IgM plus normal Ig despite the absence of significant numbers of B cells in the spleens of these mice.

Animals↗

PubMed1991

Nonconventional (TL-encoded) major histocompatibility complex molecules present processed viral antigen to cytotoxic T lymphocytes.

A large number of class I-like genes are located distal to the K and D regions of the murine major histocompatibility complex (MHC) within the Q and TL region. The function of the molecules encoded within this region is obscure since unlike conventional MHC gene products, these molecules have not been reported to present processed environmental antigens to T cells. In the present report, we demonstrate that a peptide corresponding to processed influenza virus hemagglutinin can be recognized by CD8+ T cell receptor alpha/beta-positive cytotoxic T lymphocytes (CTL) in association with a MHC class I-like product encoded within the TL region. Thus, nonconventional class I MHC molecules can bind and present processed environmental antigens, and TCR-alpha/beta CTL directed to such peptide MHC complexes are represented in the mature T cell pool. Our data imply that Q/TL region products may be charged by peptides generated through an antigen processing and presentation pathway distinct from the pathway used by conventional MHC molecules and not normally available to environmental antigens.

Amino Acid Sequence↗

PubMed1990

The recognition of a viral antigenic moiety by class I MHC-restricted cytolytic T lymphocytes is limited by the availability of the endogenously processed antigen.

The transmembrane hydrophobic domain of the type A influenza A/JAPAN/305/57 (H2N2) hemagglutinin (HA) contains an immunodominant site encompassing amino acids 523-545 (J523-545) recognized by class I MHC-restricted cytolytic T lymphocytes (CTL). Class I CTL of two fine specificity subsets map to this transmembrane (TM) site. One of these CTL subpopulations is subtype specific. These T lymphocytes recognize the site generated during infection of target cells with A/JAPAN/305/57 virus (H2N2) but not target cells expressing the comparable TM site of the influenza A/PR/8/34 virus (H1N1) hemagglutinin (P527-549) after infection with this virus. The other CTL subpopulation is cross-reactive and recognizes the TM site of the A/JAPAN/305/57 HA and the A/PR/8/34 HA with similar efficiency. Analyses of the critical amino acids in the TM site necessary for CTL recognition with the use of synthetic peptides unexpectedly revealed reactivity for the A/PR/8 HA TM site by subtype-specific CTL. This reactivity was only observed with truncated peptides corresponding to a limited portion of the A/PR/8 HA TM site but also required peptide concentrations greater than 10(-7) M. These results suggested either that the endogenously processed A/PR/8 HA TM site generated during infection was larger than the site defined by the truncated cross-reactive peptides or that the concentration of endogenously processed TM site produced during infection was limiting. To distinguish between these possibilities, we expressed in target cells synthetic minigenes encoding only the portion of the A/PR/8 HA transmembrane sites defined by the synthetic peptides. Unlike the peptides, the "preprocessed" endogenous minigene products were not recognized by subtype-specific CTL. These data suggest that the level of available endogenously processed Ag rather than selectivity in the site of fragmentation of newly synthesized Ag may play a critical role in determining whether the complex of the antigenic moiety and class I MHC is efficiently presented to and recognized by class I CTL.

Amino Acid Sequence↗

PubMed1990

Type 6 and 19 pneumococcal polysaccharides coupled to erythrocytes elicit pneumococcal cell wall-specific primary IgM responses and capsular polysaccharide-specific secondary IgG responses.

Previous results have shown that the primary murine antibody responses to vaccine preparations of type 6 (S6; Danish type 6A) or type 19 (S19; Danish type 19F) pneumococcal capsular polysaccharides consist entirely of IgM antipneumococcal cell wall carbohydrate (PnC)-specific antibodies. No capsular polysaccharide-specific IgM antibodies were detectable by plaque-forming cell or enzyme-linked immunosorbent assay techniques. In this report, antibodies specific for S6 and S19 capsular polysaccharides were induced in secondary responses to chicken erythrocyte (CRBC) conjugates of S6 and S19. Essentially all detectable IgG produced in the secondary response was capsular polysaccharide specific and included all subclasses of IgG. In contrast, all detectable IgM produced in the primary response to S6-CRBC and S19-CRBC, and the IgM produced in the secondary response to S6-CRBC was not capsular polysaccharide specific since it reacted with PnC. Thus, there is a major change in the specificity of the primary IgM response compared to the secondary IgG response of mice immunized with S6-CRBC or S19-CRBC. Injection of PnC or any PnC-containing polysaccharide prior to immunization with S6-CRBC or S19-CRBC resulted in suppression of the primary IgM response. In contrast, only the capsular polysaccharide used in the immunizing polysaccharide-erythrocyte conjugate suppressed induction of the capsular polysaccharide-specific secondary IgG response. These results suggest that S6 and S19 capsular polysaccharide-specific IgG-producing memory B cells derive from capsular polysaccharide-specific precursors which do not produce detectable antibody after primary immunization.

Animals↗

PubMed1989

Regulation of IgG responses by helper and suppressor T cells activated by pneumococcal capsular polysaccharides.

Type 2 antigens are usually unable to prime the helper T cells (TH) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate TH. Low doses of S19 or S3 (subimmunogenic for a primary IgM response) activated TH capable of providing help to S19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses of these antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (TS). Removal of these TS prior to transfer of T cells to recipient mice resulted in expression of TH function. Therefore, the preferential activation of TH versus TS was dependent on the dose of antigen used for priming. TH activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if TS precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific TH.

Animals↗

PubMed1989

Production and characterization of monoclonal antibodies specific for types 6 and 19 pneumococcal capsular polysaccharides.

The primary IgM and the secondary IgG antibody responses to pneumococcal capsular polysaccharides type 19 (S19) and type 6 (S6) coupled to sheep erythrocytes (S19-SRBC or S6-SRBC) differ in specificity. Although the primary IgM response appears to be totally specific for the pneumococcal cell wall carbohydrate (PnC) which is present in these polysaccharide preparations, the secondary IgG response appears to be completely specific for the immunizing capsular polysaccharide. A library of B cell hybridomas from fusions of splenocytes undergoing a primary response to an S19 preparation consisted entirely of PnC-specific hybrids. Thus, no evidence was obtained for the presence of capsular polysaccharide-specific IgM secreting B cells. IgM and IgG antibody secreting hybridomas were obtained, from fusions of splenocytes undergoing secondary S6- or S19-SRBC responses, to examine the antigen specificity of secondary antibody response of B cells at the clonal level. Many of the secondary IgM hybridomas secreted PnC-specific antibody; however, several S6-specific IgM secreting hybrids were also obtained, demonstrating a previously undetected population of B cells. All IgG secreting hybridomas obtained from S19- or S6-SRBC secondary response fusions secreted capsular polysaccharide-specific antibody, thus confirming the apparent absence of PnC-specific IgG secreting B cells in these responses. This method of immunization and challenge of mice with capsular polysaccharide coupled to erythrocytes, which results in the production of capsular polysaccharide-specific IgG responses, offers a relatively straightforward means to generate monoclonal antibodies specific for pneumococcal capsular polysaccharides.

Animals↗