Norbert A. Wolf

Vaginal-Associated Immunity in Women with Recurrent Vulvovaginal Candidiasis: Evidence for Vaginal Th1-Type Responses following Intravaginal Challenge with Candida Antigen

Studies from women with recurrent vulvovaginal candidiasis (RVVC) and from an animal model of experimental vaginitis suggest that deficiencies in immune function should be examined at the local rather than systemic level. Evidence of vaginal cell-mediated immunity (CMI) was evaluated for the first time in cervicovaginal lavage (CVL) fluid from RVVC patients. Results showed that although constitutive Th1- and Th2-type cytokine expression was detectable in CVL fluid from normal women, and differences in cytokines were observed in RVVC patients, limitations in experimental design of such de novo analyses urged caution in interpretation. Alternatively, attempts were made to establish conditions in control subjects whereby vaginal immunity could be detected after intravaginal challenge with Candida antigen. Preliminary results showed that Th1-type cytokines (interleukin-2 and -12, interferon-gamma) and histamine were increased 16-18 h after intravaginal introduction of Candida skin test antigen. Intravaginal antigenic challenge represents a novel approach for studying Candida-specific vaginal CMI.

Candida-Specific Systemic Cell-Mediated Immune Reactivities in Human Immunodeficiency Virus—Positive Persons with Mucosal Candidiasis

Oropharyngeal candidiasis (OPC), as opposed to vulvovaginal candidiasis (VVC), is a common opportunistic infection in human immunodeficiency virus (HIV)-positive persons that correlates with reduced CD4 T cell counts. Although cell-mediated immunity (CMI) by CD4 Th1-type cells is considered to be the predominant host defense against mucosal candidiasis, the immune factors associated with susceptibility to OPC in HIV-positive persons are not well understood. This study investigated Candida-specific systemic CMI in HIV-positive persons with OPC and/or VVC. Reductions in delayed skin test reactivity to Candida antigen were observed in HIV-positive persons with CD4 cell counts <200 cells/microL, irrespective of the presence of mucosal infection. Likewise, despite the correlate of OPC with reduced CD4 cell counts in HIV-positive persons, differences in Candida-specific peripheral blood mononuclear cell proliferation and Th1/Th2 cytokine production between HIV-positive and HIV-negative persons were not consistent in a manner to suggest that deficiencies in Candida-specific systemic CMI account solely for the susceptibility to OPC.

A Chlamydia pneumoniae-Specific Peptide Induces Experimental Autoimmune Encephalomyelitis in Rats

It has been reported recently that the bacterial respiratory pathogen Chlamydia pneumoniae is present in the cerebrospinal fluid of a subset of multiple sclerosis (MS) patients. However, it is not known whether this organism is a causative agent of MS, or merely an opportunistic pathogen that takes advantage of a disease process initiated by some other means. We report identification of a 20-mer peptide from a protein specific to C. pneumoniae which shares a 7-aa motif with a critical epitope of myelin basic protein, a major CNS Ag targeted by the autoimmune response in MS. This bacterial peptide induces a Th1 response accompanied by severe clinical and histological experimental autoimmune encephalomyelitis in Lewis rats, a condition closely reflective of many aspects of MS. Studies with peptide analogues suggest that different populations of encephalitogenic T cells are activated by the C. pneumoniae and myelin basic protein Ags. Mild experimental autoimmune encephalomyelitis was also observed when rats were immunized with sonicated C. pneumoniae in CFA.

Treatment With the Interleukin-1 Receptor Antagonist and Soluble Tumor Necrosis Factor Receptor Fc Fusion Protein Does Not Prevent Endotoxin-Induced Preterm Parturition in Mice

Objective: To determine whether the administration of anticytokine agents, the interleukin-1 receptor antagonist (IL-1ra) and a soluble tumor necrosis factor receptor Fc fusion protein (sTNFR-Fc), prevents endotoxin-induced preterm delivery in mice. Methods: C3H/HeN pregnant mice at 15 days of gestation (70% gestation) were randomized to receive phosphate-buffered saline (PBS) or lipopolysaccharide (LPS) (50 μg/mouse) intraperitoneally (i.p.). Randomly selected PBS- or LPS-treated mice were additionally treated intravenously (i.v.), i.p., or subcutaneously (s.c.) every 3 hours with IL-1ra (1-50 mg) or every 12 hours with sTNFR-Fc (200-400 μg) beginning 1 hour before LPS injection. Animals were observed for vaginal bleeding and preterm delivery. Results: Mice treated i.p. with 50 μg LPS (n = 13) had a shorter injection-to-delivery interval than mice treated similarly with PBS (n = 19) (median 13.5 hours, range 10-105 versus median 86.8 hours, range 53-120, respectively; P < .001) Saline-treated mice given 10 mg IL-1ra every 3 hours i.p. (n = 3) or 200 μg sTNFR-Fc every 12 hours i.v. (n = 4) had similar injection-to-delivery intervals as PBS-treated control mice (median 70 hours, range 70-76 versus median 58 hours, range 50-120, respectively). Similarly, LPS-treated mice given PBS every 3 hours (n = 20) had injection-to-delivery intervals comparable to LPS-treated mice (n = 13) (median 15.5 hours, range 9.8-92 versus median 13.5 hours, range 10-105, respectively). Lipopolysaccharide-treated mice given i.p. injections of 1 (n = 4), 10 (n = 31), or 50 (n = 15) mg of IL-1ra every 3 hours did not have longer injection-to-delivery intervals compared with LPS-treated mice (n = 13) (medians 11.6, 15, 14.5, and 13.5 hours; ranges 10.8-12, 8-95, 11-92, and 10-105, respectively). Lipopolysaccharide-treated mice given i.v. injections of 200 (n = 4) or 400 (n = 9) μg sTNFR-Fc every 12 hours did not have longer injection-to-delivery intervals compared with LPS-treated mice (n = 8) (medians 23.3, 22.5, and 21.9 hours; ranges 14.8-33, 15-95.5, and 15.5-44, respectively). The median injection-to-delivery interval of LPS-treated mice given both IL-1ra (10 mg) every 3 hours i.p. and sTNFR-Fc (200 μg) every 12 hours i.v. (n = 5) was not different from that of LPS-treated mice (median 26 hours, range 24.5-72 versus median 13.5 hours, range 10-105; respectively; P > .05). Conclusions: The anticytokine agents IL-1ra and sTNFR-Fc did not did not prevent preterm delivery or prolong pregnancy in endotoxin-induced preterm labor in mice.

NK Cells Inhibit T Cell Proliferation via p21-Mediated Cell Cycle Arrest

NK cells have been shown to influence immune responses via direct interaction with cells of the adaptive immune system, such as dendritic cells, B cells, and T cells. A role for NK cells in down-regulation of T cell responses has been implicated in several studies; however, the underlying mechanism of this suppression has remained elusive. In this study we show that dark Agouti rat NK cells inhibit syngeneic T cell proliferation via up-regulation of the cell cycle inhibitor, p21, resulting in a G0/G1 stage cell cycle arrest. The inhibition is cell-cell contact dependent, reversible, and Ag nonspecific. Interestingly, NK cells do not inhibit IL-2 secretion or IL-2R up-regulation and do not induce T cell death. Thus, our results show that NK cells do not affect early T cell activation events, but specifically inhibit T cell proliferation by direct interaction with T cells. Our findings suggest that NK cells may play an important role in maintaining immune homeostasis by directly regulating clonal expansion of activated T cells. This novel mechanism of T cell regulation by NK cells provides insight into NK cell-mediated regulation of adaptive immunity and provides a mechanistic link between NK cell function and suppression of T cell responses.

Synergistic interaction between Toll-like receptor agonists is required for induction of experimental autoimmune encephalomyelitis in Lewis rats

To investigate whether TLR agonists can replace mycobacteria in adjuvant to induce EAE in Lewis rats, we immunized rats with MBP peptide (MBP(68-86)) in IFA, supplemented with TLR agonists. Rats immunized with MBP(68-86) plus CpG-ODN or LPS in IFA did not develop EAE. In contrast, rats immunized with MBP(68-86) plus CpG-ODN and LPS in IFA developed clinical EAE. Spleen cells proliferated and secreted IFN-gamma in response to MBP(68-86), and secreted IL-6 and IL-12p40 in response to CpG-ODN and LPS. However, rats immunized with MBP(68-86) plus CpG-ODN and PolyI:C, a TLR3 agonist, did not develop EAE. We conclude that selected combinations of TLR agonists can facilitate the induction of EAE by MBP peptide via the innate immune system.

Interstrain variability of autoimmune encephalomyelitis in rats: multiple encephalitogenic myelin basic protein epitopes for DA rats

We investigated T cell epitopes of guinea pig myelin basic protein (MBP) that induce experimental autoimmune encephalomyelitis (EAE) in DA rats, using synthetic peptides that correspond to regions of the guinea pig MBP molecule that are homologous to rat MBP. Four peptides were encephalitogenic when tested in DA rats. MBP63-81, which partially overlaps the dominant encephalitogenic MBP epitope for Lewis (LEW) rats, caused severe EAE in the DA strain but did not elicit EAE in LEW rats. MBP66-81 and MBP63-76 were also encephalitogenic for DA but not LEW rats. MBP79-99 also induced EAE in DA rats, although MBP87-99, the minor encephalitogenic LEW epitope, was inactive. This indicates that part of the 79-86 sequence is necessary for encephalitogenic activity in the DA strain. MBP101-120, and MBP142-167 were also encephalitogenic for DA rats. T cells from DA rats immunized with intact MBP proliferated in response to the whole protein and to MBP79-99, but were not stimulated to a significant extent by the other encephalitogenic peptides, suggesting that these may represent cryptic or subdominant epitopes. However, MBP63-81-specific T cell lines could be isolated by repeated restimulation with peptide, indicating that the peptide-specific T cells were present in DA rats at low frequency.

Characterization of a subset of bone marrow-derived natural killer cells that regulates T cell activation in rats

We report that bone marrow‐derived natural killer (BMNK) cells from DA or F344 rats inhibit PMA/ionomycin‐induced T cell proliferation. These NK‐regulatory cells are NKR‐P1Adim, whereas a minor subpopulation is NKR‐P1Abright. Only the NKR‐P1Adim BMNK cells inhibit T cell proliferation. If activated with rat Con A supernatant, the NKR‐P1Adim cells become NKR‐P1Abright and lose the ability to inhibit T cell proliferation. In contrast to BMNK cells, all DA and F344 rat NK cells isolated from the blood, spleen, cervical, or mesenteric lymph nodes or Peyer’s patches are NKR‐P1Abright and lack the ability to inhibit T cell proliferation. Inhibition of T cell proliferation correlates with significant down‐regulation of CD3, suggesting that this may be the mechanism through which the NKR‐P1Adim cells mediate suppression. The nitric oxide synthase inhibitor NG‐monomethyl‐arginine acetate‐abrogated NKR‐P1Adim cell inhibition of T cell proliferation. We conclude that rat bone marrow NKR‐P1Adim cells represent a unique population that may play a role in maintaining immune homeostasis by regulating the clonal expansion of activated T cells.

DA rat NK(+)CD3(-) cells inhibit autoreactive T-cell responses.

We isolated natural killer (NK) cells from DA rat bone marrow (BM) by staining with PE anti-NKR-P1A and FACS sorting (>98% NK(+)). The purified NK cells inhibit T-cell proliferation in a dosage-dependent fashion and suppressed production of the proinflammatory Th1 cytokine, IFN-gamma. When activated in vitro with Con A supernatant (CAS), the purified NK cells secrete the beta-chemokine monocyte chemoattractant protein-1 (MCP-1) and upregulate MCP-1 mRNA. The activated NK cells also express IFN-gamma mRNA. Sorted NK(+)CD3(-) cells, from which NKT cells have been excluded, also inhibit autoreactive T-cell responses to myelin basic protein (MBP). These findings are consistent with a role for conventional NK cells in maintaining immune homeostasis, by eliminating autoreactive T cells that have inadvertently become activated.

Potential effects of midcycle cervical mucus on mediators of immune reactivity

OBJECTIVE To examine the effect of cervical mucus (CM) on immune mediators in cervicovaginal lavage fluid. DESIGN The stability of cytokines and immunoglobulins (Ig) within cervicovaginal lavage fluid was determined during 18-hour contact with unsolubilized midcycle CM in vitro. SETTING In vitro experiment. PATIENT(S) Healthy female volunteers from whom cervicovaginal lavage fluid and periovulatory CM were obtained. INTERVENTION(S) Changes in cytokine and Ig concentration in cervicovaginal lavage fluid after incubation with CM. MAIN OUTCOME MEASURE(S) Interleukin (IL)-4, IgA, and IgG concentrations were determined by ELISA. Interleukin-2 concentration was determined by bioassay using the IL-2-dependent cytotoxic T-lymphocyte cell line and ELISA. Results were expressed as percent antibody or cytokine recovery remaining after mucus contact, with differences over time evaluated by analysis of variance. RESULT(S) During 18-hour mucus contact, significant changes were detected for IL-4, IgA, and IgG concentration. Interleukin-2 was stable in mucus-associated cervicovaginal lavage fluid as determined by functional bioassay and ELISA. CONCLUSION(S) Immunoglobulins and cytokines differ in their recovery from aqueous media after CM contact, suggesting that midcycle CM could alter immune reactivity within the reproductive tract by modifying the availability or function of immunomodulatory substances.