Biomedical subjects

K T HayGlass

Publications and source records attributed to K T HayGlass.

At least 19 recordsLinked to original sources

PubMed2003

Improved mRNA quantitation in LightCycler RT-PCR.

BACKGROUND: Real-time polymerase chain reaction (PCR) utilizing the LightCycler and similar systems is an increasingly used technique for quantitative reverse transcription (RT)-PCR of mRNA levels from genes of immunologic interest. A commonly encountered limitation with these systems is that the fluorescence induced by SYBR Green (a fluorophore that binds double-stranded DNA) can result from primer dimers (PDs) as well as the PCR product of interest, thus interfering with the ability to reproducibly quantitate mRNA levels. METHODS: We use a modification of the LightCycler PCR strategy to overcome this problem by altering the PCR strategy to take advantage of the LightCycler's ability to measure fluorescence at a temperature greater than the melting point of PDs. The resulting measurements determine fluorescence of only the desired PCR product. RESULTS: We demonstrate that by using this modified PCR strategy, one can eliminate the fluorescence induced by PDs and obtain accurate product quantitation. CONCLUSIONS: This simple modification allows more precise quantitation of sample mRNA levels by eliminating the contaminating fluorescence induced by the formation of PCR PDs. This modification obviates the need to redesign PCR primers in RT-PCR experiments where this is impractical or impossible.

Animals↗

PubMed2002

Evaluation for synergistic suppression of T cell responses to minor histocompatibility antigens by chloroquine in combination with tacrolimus and a rapamycin derivative, SDZ-RAD.

The 4-aminoquinolines, chloroquine and hydroxychloroquine, can suppress chronic graft-versus-host disease (GVHD) following blood and marrow transplantation (BMT) in mice and humans, respectively. We hypothesized that chloroquine in combination with tacrolimus and the rapamycin derivative SDZ-RAD can synergistically suppress T cell responses and antigen-presenting cell (APC) function in vitro. We used the APC-dependent C57BL/6 anti-BALB.B T cell response and APC-independent anti-CD3epsilon antibody-induced response to evaluate the role of synergism between chloroquine and tacrolimus or SDZ-RAD on each component of a T cell response to minor histocompatibility antigens. We found that chloroquine with tacrolimus had a greater synergistic suppression of APC-dependent compared to the APC-independent T cell responses, with a combination index (CIx) for 50% inhibition by mean effect analysis of 0.16 and 0.50, respectively (a lower number indicates greater suppression). By contrast, chloroquine with SDZ-RAD had a similar CIx between the two responsed 0.50 vs0.45) suggesting only T cell suppression. Synergy between chloroquine and SDZ-RAD involved a direct effect on T cell cytokine production, whereas synergism between chloroquine and tacrolimus was due to an effect on both T cells and APCs. We conclude that the renal-sparing 4-aminoquinolines may be used syneristically with immunosuppressive drugs currently used for BMT.

Animals↗

PubMed2001

Comparison of chemiluminescent assays and colorimetric ELISAs for quantification of murine IL-12, human IL-4 and murine IL-4: chemiluminescent substrates provide markedly enhanced sensitivity.

Many available ELISAs lack the sensitivity required to reliably quantify levels of cytokines released in response to antigenic stimulation. In an effort to increase sensitivity of these assays, we compare the sensitivity of standard colorimetric ELISAs and corresponding chemiluminescent assays for three cytokines which are usually produced in very low quantities: mouse IL-12 p70, human IL-4 and mouse IL-4. Use of a chemiluminescent substrate enhanced the sensitivity of these assays 12-29-fold as compared to current colorimetric ELISAs. Accompanying this increase in sensitivity was an increase in dynamic range, a decrease in the time required to obtain maximum sensitivity and a decrease in the concentration of reagents required. These findings are of particular interest to those wanting to quantitate levels of any cytokine which is typically produced in low levels.

Animals↗

PubMed2001

In vivo stability of human chemokine and chemokine receptor expression.

Cross-sectional analyses of human PBMC, plasma, and tissue have reported altered chemokine and/or chemokine receptor expression in several inflammatory diseases. Interpretation of such studies is difficult without data on the in vivo stability of such parameters. Using four color flow cytometry, we longitudinally followed CXCR3, CCR5 (Th1-associated), and CCR3 (Th2-associated) expression within CD4+/CD45RO+ and CD8+/CD45RO+ T cell populations in peripheral blood of healthy individuals over a 21 day period. In parallel, we quantified plasma levels of IP-10, Mig, eotaxin and TARC. Chemokine and receptor expression differed markedly between subjects but was highly stable, varying by <5% within individuals. Differences in chemokine receptor expression between subjects were markedly altered when quantified as absolute cell numbers rather than frequencies. Finally, CCR3 expression by CD4+/CD45RO+ T cells was positively correlated with plasma levels of its ligand, eotaxin, whereas strong negative correlations were evident between CXCR3 expression and IP-10 or Mig. These data demonstrate longitudinal stability of chemokine receptor and ligand expression among healthy individuals; reveal that both frequency and absolute cell count analysis is essential for accurate assessment of chemokine receptor expression; and identify inverse relationships between type 1 and type 2 immunity-associated receptors and their ligands in vivo.

Adult↗

PubMed2001

CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to down-regulate ongoing IgE responses in mice.

Antigen-specific IgE plays an important role in the pathogenesis of allergic disorders. Immunostimulatory CpG motifs (CpG) in bacterial DNA or synthesized oligodeoxynucleotides (ODN) are gaining recognition as potential immunomodulators for switching on protectiveT(h)1-mediated immunity and preventing or potentially inhibiting T(h)2-dependent allergic responses. To date, allergic models used in CpG ODN studies have been established by immunization of mice with allergen in the presence of adjuvant. This, in addition to failure to assess specific IgE production in most of the studies, has limited understanding of the role of CpG ODN vaccination in allergic responses. Here, we examine the effects of synthesized CpG ODN on both developing and ongoing IgE responses in mice sensitized using a recombinant mosquito salivary antigen (rAed a 2) without adjuvant. Pretreatment of mice with CpG ODN mixed with rAed a 2 successfully inhibited subsequent induction of serum rAed a 2-specific IgE (but not IgG1) and antigen-induced IL-4 and IL-5 production in spleen cells. This was associated with an increase of serum IgG2a and IL-12, and increased IFN-gamma and IL-12 production by spleen cells. In this model, however, co-administration of CpG ODN with rAed a 2 to presensitized mice failed to down-regulate ongoing IgE responses despite significant up-regulation of serum IL-12 and specific IgG2a. Strikingly, a transient skin delayed-type hypersensitivity reaction occurred in CpG ODN-treated mice. These observations provide a new insight into the potential therapeutic application of CpG ODN to allergic disorders.

Adjuvants, Immunologic↗

PubMed2001

Cytokine-based immunotherapy of allergic disease.

Human immediate hypersensitivity diseases are strongly associated with an excessive type 2 response to normally innocuous environmental antigens, and are a growing health care concern in developed nations. Commonly prescribed treatments provide effective symptomatic relief, but are unable to consistently ameliorate the underlying cause of allergic disease: the excessive generation of allergen-specific Th2 cells. IL-12 and IL-18 are potent inducers of type 1 immunity, and, as such, have been proposed as candidates for treatment of allergic diseases. This review critically assesses the potential of recombinant IL-12 and IL-18 immunotherapy to redirect both de novo and established allergic responses in animal models of human allergic disease to clinically protective immune responses.

Animals↗

PubMed2000

Experimental approaches to analysis of immune dysregulation in human allergic disease.

Over the last 40 years, much attention has been directed towards identification of the immunologic, genetic and environmental factors that predispose towards development of allergic disease. An implicit assumption in many such studies is that clinical tolerance reflects from immunologic tolerance. Here we critically review the conceptual background and experimental data arguing for the alternative hypothesis that failure to develop atopic disease reflects the success of type 1 dominated immunity that constitutively impedes development of type 2 responses to environmental antigens, hence, clinical immediate hypersensitivity. We report that endogenous production of type 1 chemokines such as IP-10 by non-atopic individuals may play a substantive role in maintaining this putatively protective type 1 bias in non-atopic subjects. Polyclonal activators (superantigen TSST-1, anti-CD3, PHA) were used to activate distinct intracellular signaling pathways, inducing quantitatively different IFNgamma:IL-4 ratios in primary culture of human PBMC. In parallel, physiologic stimuli such as grass pollen or cat antigen were used to evaluate the impact of IP-10 on CD4 T cell dependant, chloroquine-sensitive cytokine synthesis. IFNgamma responses by non-atopic subjects were markedly increased in the presence of nM concentrations of rhIP-10 while type 2 cytokine synthesis remained unaffected. Optimal rIP-10 concentrations for promoting expression and maintenance of type 1 cytokine synthesis in vitro (0.1 to 10 ng/ml) were at or well below those generally used for chemotaxis (5 to 100 ng/ml). Collectively, our findings suggest a potential role for this T cell focused chemokine in maintaining the default type 1 responses usually caused to environmental antigens in non-atopic subjects. These may play a role in determining the relative susceptibility of individuals to develop atopic disease. Taken together with recent reports of other roles played by chemokines in shaping the nature of immune responses, the data suggest that constitutive, endogenous type 1 chemokine synthesis may play a homeostatic role in inhibiting development of atopic disease.

Allergens↗

PubMed2000

gammadelta T-cell-deficient mice do not differ from normal controls in their induction or expression of type 2 dominant responses to exogenous antigen.

The roles that gammadelta T cells play in shaping initial CD4 T cell activation, and sensitivity to development of atopic diseases, remain controversial. Using a genetic knockout model of gammadelta T-cell deficiency, we investigated the role of these cells in initiation of exogenous antigen specific murine cytokine and antibody responses. Given that the most widely distributed and clinically prominent class of allergens are soluble protein antigens, we utilized OVA to examine the role played by gammadelta T cells in shaping the induction and expression of exogenous Ag specific immune responses. Focusing on immunization conditions that stimulate in vivo induction of type 2 dominant immunity, we report that gammadelta deficient and intact C57Bl6 mice exhibit similar OVA-specific responses as indicated by the (i) intensity of initial T-cell activation (ii) the type1 vs. type 2 balance of exogenous Ag specific cytokine synthesis and the (iii) intensity and the relative balance of the resulting IgE vs. IgG(2a) responses in vivo seen in these strains. Taken together, the data are consistent with the hypothesis that gammadelta T cells do not play an essential role in shaping induction of systemic immune responses to soluble exogenous antigen in type 2 dominated responses.

Animals↗

PubMed2000

Endogenous IL-12 synthesis is not required to prevent hyperexpression of type 2 cytokine and antibody responses.

Endogenous IL-12 production is hypothesized to play an essential role preventing spontaneous expression of type 2 responses, acting as a natural inhibitor limiting development of immediate hypersensitivity. Here, IL-12-deficient p35(- / -) and p40(- / -) mice were used to examine the role of endogenous IL-12 and p40 homodimer during in vivo development of exogenous antigen-driven responses. In the absence of deliberate immunization, IL-12-deficient mice exhibited greatly reduced serum IgG2a but IgG1 / IgE levels no higher than controls. Immunization to elicit polarized ovalbumin-specific type 1 or type 2 dominant responses, or using Trichinella spiralis extract in the absence of adjuvants, led to IFN-gamma production of approximately 10 % of C57BL / 6 controls yet the kinetics and intensity of primary and secondary type 2 cytokine (IL-4, IL-5, IL-13) and antibody (IgG1, IgE) responses, as well as functional IL-12 receptor expression, were consistently unaltered. Thus, while IL-12 provides an important positive signal for Th1 development, antigen exposure in its absence does not lead to generalized enhancement of type 2 cytokine or antibody responses. The data argue that endogenous IL-12 production is not required as a constitutive negative regulator limiting induction or expression of type 2 effector responses.

Animals↗

PubMed2000

Failure of rIL-12 administration to inhibit established IgE responses in vivo is associated with enhanced IL-4 synthesis by non-B/non-T cells.

Administration of rIL-12 offers a widely successful tactic for preferential induction of type 1 immune responses in vivo. Its use to modulate ongoing cytokine or effector responses has proven to be substantially more difficult. Immediate hypersensitivity is the most common human immunologic disease. Here, rIL-12 was administered to C57Bl/6 and outbred CD1 mice with ongoing ovalbumin (OVA)-specific IgE responses in an attempt to redirect established type 2 cytokine and antibody production. Despite use of a broad range of treatment protocols for >4 months following initial immunization, recall IgE responses were consistently unaffected. rIL-12-treated mice exhibited strong in vivo and in vitro IFN-gamma responses, increased approximately 40-fold relative to controls, but also markedly enhanced (15- to 20-fold) OVA-specific IL-4 production. CD4 T cell function was successfully transformed from a type 2- to a type 1-dominated pattern following long-term IL-12 administration in vivo, as measured by strongly reduced IL-4 and IL-10 responses in antigen-stimulated primary culture, and 5-fold reductions in the frequencies of IL-4- and IL-10-producing OVA-specific CD4 T cells. However, chronically rIL-12-treated mice exhibited increased numbers of non-B/non-T cells that when re-stimulated with specific allergen, produce IL-4 at levels 20-fold higher than did CD4 T cells while IL-13 responses are unaffected. Collectively, the data indicate that even effectively shifting CD4 T cell activation from a type 2- to a type 1-dominated response does not in itself lead to altered effector (IgE) responses upon antigen re-exposure.

Animals↗

PubMed2000

Functional status of the immune system after chronic administration of 2'-deoxycoformycin in the BB rat.

Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic beta cells with the primary mechanism being cell mediated. The BB rat develops insulitis and IDDM with many features analogous to the disease in man. In previous studies we reported that weekly administration of 2'-deoxycoformycin (dCF) for four months reduces significantly the incidence of IDDM in the BB rat by 70%, and that the animals remain free of diabetes for a minimum of two months after drug withdrawal. Since the diabetes-prone BB rat is lymphopenic, with a reduction of both CD4 and CD8 cells, the continuous failure of dCF treated animals to develop diabetes may have been due to generalized immunosuppression. To test this possibility, the ability of dCF treated diabetes-free BB rats to mount an immune response after challenge with Ovalbumin was examined five months after drug withdrawal. The results showed that the post-immunization levels of total IgG and specific IgG in these animals did not differ from those observed in non-dCF treated controls nor those of control diabetes-resistant non-lymphopenic BB rats. Moreover, FACS analysis indicated no change in the percentages of total numbers of CD4+ or CD8+ cells between the two groups of animals. Histological assessment of the pancreata of the post-dCF treated animals showed varying degrees of mononuclear cell infiltrates in the islets. These data demonstrate that treatment by dCF is not permanent, and may require intermittent or continuous administration to prevent development of diabetes. Further studies are needed to determine the mechanism of action of dCF in this model of IDDM.

Animals↗

PubMed2000

T cell chemokine receptor expression in human Th1- and Th2-associated diseases.

The interaction between chemokines and their receptors is an important step in the control of leukocyte migration into sites of inflammation. Chemokines also mediate a variety of effects independent of chemotaxis, including induction and enhancement of Th1- and Th2-associated cytokine responses. Recent studies have shown that human Th1 and Th2 clones, activated under polarizing conditions with polyclonal stimuli in vitro, display distinct patterns of chemokine receptor expression: Th1 clones preferentially express CCR5 and CXCR3, while many Th2 clones express CCR4, CCR8 and, to a lesser extent, CCR3. These differential patterns of chemokine receptor expression suggest a mechanism for selective induction of migration and activation of Th1- and Th2-type cells during inflammation and, perhaps, normal immune homoeostasis. Studies have begun to examine T cell chemokine receptor expression in vivo to determine the relevance of these in vitro observations to human Th1- and Th2-associated diseases. In this review, we critically examine recent reports of T cell chemokine receptor expression in human autoimmune disorders (multiple sclerosis and rheumatoid arthritis) and atopic disorders (allergic rhinitis and asthma) which are believed to arise from inappropriate Th1- and Th2-dominated responses, respectively.

Arthritis, Rheumatoid↗

PubMed1998

Environmental antigen-induced IL-13 responses are elevated among subjects with allergic rhinitis, are independent of IL-4, and are inhibited by endogenous IFN-gamma synthesis.

Human immediate hypersensitivity diseases represent the most common example of chronic excessive Th2-like activation in developed nations. While IL-13 shares many functional properties with IL-4, the intensity and regulation of environmental Ag-stimulated IL-13 synthesis by allergic vs nonallergic individuals remain ill defined. Here, we examine the intensity of polyclonally and Ag-stimulated IL-13 production by PBMC of 20 subjects with seasonal allergic rhinitis and 20 healthy controls. Polyclonally driven IL-13 responses did not differ significantly (Mann-Whitney U test, p = 0.68). In contrast, the median CD4-dependent IL-13 response among atopics was markedly stronger than nonatopics in Ag-stimulated primary culture (p = 0.0031) and exhibited a strong correlation with IL-5 (r = 0.76, p = 0.0009), but not IL-4 (r = 0.14, p > 0.05), responses. IL-13 production was unaffected by blocking endogenous IL-4 or IL-5 activity or by addition of rIL-4 or rIL-5. In contrast, it was inhibited by low levels of rIFN-gamma and strongly enhanced upon addition of neutralizing anti-IFN-gamma mAb. Collectively, the data are consistent with a negative regulatory role for endogenous IFN-gamma synthesis in controlling the intensity of systemic IL-13 responses evoked in both atopic and nonatopic populations following exposure to common Ags. They also suggest that the elevated levels of IL-4 and IL-5 characteristic of type 2-dominated responses in vivo are without detectable impact on the maintenance of recall Ag-stimulated IL-13 production.

Adolescent↗

PubMed1998

Depletion of natural killer cells from the graft reduces interferon-gamma levels and lipopolysaccharide-induced tumor necrosis factor-alpha release in F1 hybrid mice with acute graft-versus-host disease.

BACKGROUND: We wished to determine whether removal of NK1.1+ cells from the graft provides protection against acute graft-versus-host disease (GVHD) by obviating the Th1 immune response that underlies the development of this disease. METHODS: Graft-versus-host (GVH) reactions were induced in two groups of (C57BL/6 x DBA/2)F1 hybrid mice. The first received grafts harvested from polyinosinic:polycytidylic acid-stimulated, C57BL/6 donors and depleted in vitro of NK1.1+ cells. This treatment provides protection against GVHD-associated mortality and cachexia. The second received unmodified grafts. We compared interferon-gamma and interleukin-10 production as well as the levels of engraftment in these two groups. Lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) release was also compared since TNF-alpha levels in GVH mice following injection of a sublethal dose of endotoxin provide an index of macrophage priming by Th1 cytokines. RESULTS: Interferon-gamma production was absent in recipients of NK1.1-depleted grafts at the time when high levels were seen in recipients of unmodified grafts. Following lipopolysaccharide injection, high levels of TNF-alpha were observed in recipients of unmodified grafts, whereas negligible amounts were present in recipients of NK1.1-depleted grafts. The use of NK1.1-depleted grafts did not result in a reduced level of engraftment of CD4+ or CD8+ cells. CONCLUSIONS: These results suggest that NK1.1 depletion of the graft confers protection against mortality by interfering with an immunoregulatory mechanism that results in the development of a Th1 response in GVH mice, and does not result in abortion of the graft. Because macrophage priming is prevented, recipients are also protected from the exaggerated sensitivity to endotoxin seen in mice with acute GVHD.

Animals↗

PubMed1998

Murine graft-versus-host disease in an F1-hybrid model using IFN-gamma gene knockout donors.

These experiments were performed to determine whether the absence of donor-derived IFN-gamma would influence the outcome of acute graft-vs-host disease (GVHD). Graft-vs-host reactions were induced in B6D2F1 hybrids using grafts from either IFN-gamma gene knockout (gko) or wild-type, C57BL/6J, parental strain donors. GVHD was equally lethal in both groups, but IFN-gamma gko graft recipients developed a more protracted form of the disease. These mice developed early wasting that persisted until death. IFN-gamma was present in spleen cell cultures from wild-type graft recipients, but was absent in cultures from IFN-gamma gko graft recipients. Both recipient groups showed macrophage priming for LPS-induced TNF-alpha release. Engraftment of donor-derived CD4+ and CD8+ cells was greater in IFN-gamma gko graft recipients. Pathologic changes in IFN-gamma gko graft recipients were different from those typically seen in acute GVHD. The syndrome developing in IFN-gamma gko recipients consisted of patchy alopecia, corneal dryness and clouding, and lymphocytic infiltration of the liver, pancreas, salivary gland, lung, and kidney. Lymphocytic infiltrates were also present in the epidermis and the epithelium of both bile and salivary gland ducts. Some of the lesions closely resembled those seen in the "sicca"/Sjogren's-like syndrome associated with chronic GVHD; however, there was no evidence of immune complex deposition in the kidney. These results indicate that GVHD in IFN-gamma gko graft recipients shares many features with acute GVHD, but both the duration of the disease and its pathologic manifestations are different. Our results suggest that IFN-gamma plays a significant role in the pathogenesis of acute GVHD by increasing the rate at which mortality develops.

Animals↗

PubMed1998

Natural killer cell depletion fails to influence initial CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses.

The role played by NK- and NK1.1-expressing T cells in CD4 T cell activation and induction of immune responses in vivo is controversial. These effector cells of the innate immune response are hypothesized to play a pivotal role in shaping initial T cell activation, with some groups reporting that classical NK cells are required for optimal Th1-like T cell activation, and others supporting a role for NK1.1+ alphabeta T cells in Th2 generation. Here, we examine the impact of in vivo NK cell depletion on the development of exogenous Ag-specific cytokine and Ab responses using a murine model of human immediate hypersensitivity. OVA-specific immune responses were induced in 1) C57Bl/6 bg/bg and bg/+ mice, 2) BALB/c mice pretreated with anti-asialoGM1 or control Ab, and 3) C57Bl/6 mice depleted of NK1.1-expressing cells by in vivo administration of anti-NK1.1 mAb PK136. Depletion efficacy was assessed by functional assays and flow cytometric analysis. Each of these approaches indicated that depletion of NK cells and NK1.1+ CD4+ T cells fails to alter the Th1:Th2 balance of Ag-driven cytokine synthesis, as indicated by OVA-stimulated cytokine synthesis in primary bulk culture. Similarly, the kinetics and intensity of effector responses such as OVA-specific IgG2a and IgE synthesis were neither increased nor decreased in any of the three models examined. The results argue that NK cells and peripheral NK1.1+ T cells do not play an essential role in shaping the induction of Ag-specific immune responses to soluble exogenous Ags, the most common class of inhalant allergen.

Animals↗

PubMed1997

Immune suppression by lysosomotropic amines and cyclosporine on T-cell responses to minor and major histocompatibility antigens: does synergy exist?

BACKGROUND: Using murine models, we have shown that the lysosomotropic amine, chloroquine, is effective in the prevention of graft-versus-host disease (GVHD) mediated by donor T cells reactive with recipient minor histocompatibility antigens (MiHCs). Because lysosomotropic amines can suppress major histocompatibility complex (MHC) class II antigen presentation, their mechanism of action is potentially different from current immune suppressant drugs used to control GVHD such as cyclosporine. METHODS: We investigated the use of cyclosporine and the lysosomotropic amines chloroquine and hydroxychloroquine in combination for additive or synergistic immunosuppression on T-cell responses in vitro to MiHC and MHC in mice. RESULTS: We found that similar concentrations of chloroquine and hydroxychloroquine suppress the T-cell response to MiHC in mice (C57BL/6 anti-BALB.B) and that lysosomotropic amines in combination with cyclosporine result in synergistic suppression of a proliferative response to MiHC. Similar suppression and synergy appear to be present in an alloreactive response (C57BL/6 anti-BALB/c). Direct inhibition by chloroquine of T-cell proliferative responses induced by anti-CD3epsilon in the absence of antigen-presenting cells is present at higher concentrations than that required to suppress responses to MiHC or MHC. Chloroquine appears to induce decreased T-cell viability at high concentrations. This effect does not appear to be due to decreased T-cell production of interleukin-2 or interferon-gamma. At lower concentrations (<25 microg/ml), chloroquine can also decrease the ability of antigen-presenting cells to stimulate an a C57BL/6 anti-BALB/c T-cell response and can inhibit MHC class II expression after activation with lipopolysaccharide. CONCLUSIONS: Lysosomotropic amines in combination with cyclosporine appear to be synergistic in the suppression of T-cell proliferation to MiHC and MHC. Use of chloroquine in combination with cyclosporine may result in improved control of GVHD.

Animals↗

PubMed1997

In vivo IL-12 administration induces profound but transient commitment to T helper cell type 1-associated patterns of cytokine and antibody production.

There is much interest in the utility of exogenous IL-12 as a biologic adjuvant in immediate hypersensitivity and infectious or parasitic diseases where the induction of Th1 responses is strongly associated with protective immunity. Using an immediate hypersensitivity model in which C57Bl/6 mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of rIL-12 to direct and maintain OVA-specific cytokine and Ab responses in a Th1 direction. Exogenous IL-12 administered coincident with OVA immunization stimulated elevated serum IFN-gamma levels, enhanced IFN-gamma synthesis, and inhibited IL-4 synthesis in bulk culture; 80 to 99% inhibited primary Ag-specific serum IgE and IgG1 responses; and 15- to 20-fold enhanced IgG2a synthesis. However, each of these effects was highly transient, as exogenous IL-12, given at levels up to those associated with serious toxicity, failed to have a lasting impact on the OVA-specific T or B cell response. This transience was evident in primary bulk culture cytokine synthesis; in limiting dilution analysis of the frequency of OVA-specific IFN-gamma-, IL-4-, or IL-10-producing CD4 T cells; and, most importantly, in vivo effector responses such as IgE production to secondary and tertiary OVA immunization. The finding that the intense Th1-like phenomena seen following in vivo administration of rIL-12 with this exogenous Ag are highly transient and are not associated with alterations in the allergen-specific CD4 T cell repertoire to Th1-like patterns suggests a need for caution in the enthusiasm for the use of this cytokine as a biologic adjuvant.

Adjuvants, Immunologic↗