Search

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.

It is hypothesised that complex interactions between genetic and environmental factors give rise to allergy and asthma in childhood. The Canadian Healthy Infant Longitudinal Development (CHILD) study was designed to explore these factors.CHILD is a longitudinal, general population birth cohort study following infants from mid-pregnancy to age 5 years. Over this time period, biological samples, questionnaires, clinical measures and environmental data are collected.A total of 3624 families have been recruited, and many thousands of samples and questionnaires have been collected, annotated, and archived. This report outlines the rationale and methodology for collecting and storing diverse biological samples from parents and children in this study, and the mechanisms for their release for analyses.The CHILD sample and data repository is a tremendous current and future resource and will provide a wealth of information not only informing studies of asthma and allergy, but also potentially in many other aspects of health relevant for Canadian infants and children.

Cytokine patterns elicited by infection are critical in the regulation of the adaptive immune response and in the resolution of infection. Using a murine model of pneumonia induced by intranasal inoculation with the Chlamydia trachomatis mouse pneumonitis (MoPn) biovar, we found that the patterns of immune responses and cytokine production by spleen cells were correlated with quantitative growth of MoPn in the lungs of C57BL/6 and BALB/c mice. Specifically, BALB/c (H-2d) mice had a significantly higher mortality rate and a slower clearance of the organism from the lungs than did C57BL/6 mice (H-2b). BALB/c mice exhibited higher IL-10 production, higher IgG1 Ab responses, and less IFN-gamma production than C57BL/6 mice, which showed higher IFN-gamma production, stronger delayed-type hypersensitivity (DTH) responses, and significantly less IL-10 production. In vivo neutralization of IL-10 in BALB/c mice with an anti-IL-10 mAB during MoPn infection significantly increased the DTH response and enhanced clearance of MoPn. These findings support the hypothesis that excessive IL-10 production in BALB/c mice inhibits Th1-like responses, including IFN-gamma expression and the DTH response following chlamydial infection and consequently delay infection resolution.

Ag-induced, CD4 T cell-dependent synthesis of IL-4 and IFN-gamma plays a critical role in the induction, maintenance, and regulation of allergic responses. The requirement for IL-2 and IL-4 in the initial induction of IL-4 T cell responses is well established. However, it remains unclear whether IL-2 is required for priming of IL-4 responses, IL-4 gene expression, or both. In this report, we dissociate the IL-2 dependence of developing and established IL-4 responses. The capacity of spleen cells from naive and allergen-immunized mice to synthesize IL-4 after Ag-specific vs polyclonal restimulation is examined in overnight cultures. Although de novo induction of IL-4 and IFN-gamma production obtained after in vitro activation of cells from naive animals with immobilized anti-CD3 is IL-2 dependent, Ag-driven IL-4 synthesis by CD4 T cells from OVA (alum)- or ragweed extract (alum)-immunized mice is independent of IL-2. The data demonstrate the IL-2 independence of CD4 T cell-derived IL-4 synthesis by allergen-primed cells, distinguishing IL-2-dependent from IL-2-independent stages of IL-4 responses. In contrast to the finding that IL-2 is not required for IL-4 gene expression by primed T cells in allergen-driven responses, both induction and expression of IFN-gamma responses are strictly IL-2 dependent. This differential requirement for IL-2 in IL-4 and IFN-gamma gene expression may be significant in the maintenance of IL-4-dominated responses in allergic individuals.

Administration of high m.w. glutaraldehyde-polymerized OVA (termed OVA-POL) before OVA-[A1(OH)3] immunization of C57BL/6 mice markedly impairs their capacity to generate OVA-specific IgE responses, while simultaneously resulting in striking enhancement of Ag-specific IgG2a responses. We demonstrate here that treatment with this class of chemically modified allergen also results in pronounced inhibition of ongoing IgE responses in vivo. The abrogation of well established murine IgE responses that is elicited after treatment with OVA-POL (i) is potent (97%), (ii) is long lived, and (iii) reflects reciprocal regulation of Ag-specific IgE and IgG2a responses in vivo. Moreover, the capacity of OVA-POL-treated mice to generate secondary IgE responses remains strongly decreased for at least 260 days and six subsequent immunizations with native allergen, despite there being no further treatment with modified allergen. These changes in IgE and IgG2a responsiveness are Ag specific and T cell dependent.

SummaryChemically modified allergens have been extensively studied in an attempt to develop materials of increased efficacy and improved safety for use in the immunotherapy of allergic disease. Most of the strategics that have been developed yield products that strongly inhibit de novo IgE responses but have only marginal impact on ongoing IgE responses. We report the virtual abrogation of pre-established murine anti-ovalbumin IgE responses using a glularaldehyde-polymerized ovalbumin preparation (OA-POL) of Mr 3.5 × 107. Secondary IgE responses are inhibited by 97–99% over a period of at least 8 months following three i.p. courses of OA-POL treatment. Administration of five additional ovalbumin [Al(OH)3] booster immunizations over tbis period fails to alter this unresponsive state. The inhibition of antigen-specific IgE responses is isotype specific.

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.

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.

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.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.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.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.

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.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.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.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.

Ag-induced, CD4 T cell-dependent synthesis of IL-4 and IFN-gamma plays a critical role in the induction, maintenance, and regulation of allergic responses. The requirement for IL-2 and IL-4 in the initial induction of IL-4 T cell responses is well established. However, it remains unclear whether IL-2 is required for priming of IL-4 responses, IL-4 gene expression, or both. In this report, we dissociate the IL-2 dependence of developing and established IL-4 responses. The capacity of spleen cells from naive and allergen-immunized mice to synthesize IL-4 after Ag-specific vs polyclonal restimulation is examined in overnight cultures. Although de novo induction of IL-4 and IFN-gamma production obtained after in vitro activation of cells from naive animals with immobilized anti-CD3 is IL-2 dependent, Ag-driven IL-4 synthesis by CD4 T cells from OVA (alum)- or ragweed extract (alum)-immunized mice is independent of IL-2. The data demonstrate the IL-2 independence of CD4 T cell-derived IL-4 synthesis by allergen-primed cells, distinguishing IL-2-dependent from IL-2-independent stages of IL-4 responses. In contrast to the finding that IL-2 is not required for IL-4 gene expression by primed T cells in allergen-driven responses, both induction and expression of IFN-gamma responses are strictly IL-2 dependent. This differential requirement for IL-2 in IL-4 and IFN-gamma gene expression may be significant in the maintenance of IL-4-dominated responses in allergic individuals.

Administration of high m.w. glutaraldehyde-polymerized OVA (termed OVA-POL) before OVA-[A1(OH)3] immunization of C57BL/6 mice markedly impairs their capacity to generate OVA-specific IgE responses, while simultaneously resulting in striking enhancement of Ag-specific IgG2a responses. We demonstrate here that treatment with this class of chemically modified allergen also results in pronounced inhibition of ongoing IgE responses in vivo. The abrogation of well established murine IgE responses that is elicited after treatment with OVA-POL (i) is potent (97%), (ii) is long lived, and (iii) reflects reciprocal regulation of Ag-specific IgE and IgG2a responses in vivo. Moreover, the capacity of OVA-POL-treated mice to generate secondary IgE responses remains strongly decreased for at least 260 days and six subsequent immunizations with native allergen, despite there being no further treatment with modified allergen. These changes in IgE and IgG2a responsiveness are Ag specific and T cell dependent.

B cell-deficient, rabbit anti-mouse IgM-treated mice were compared with normal or normal rabbit immunoglobulin-treated controls in their ability to develop proliferative T cell responses, delayed hypersensitivity, and primary or secondary cytotoxic T cell responses. Immunization with hapten-coupled autologous spleen cells resulted in anti-mu-treated mice generating only marginal T cell responses. This decreased responsiveness was shown to be attributable not to an intrinsic T cell defect or to changes in the ability of macrophages from anti-mu-treated mice to present soluble antigen, but rather to the greatly diminished capacity of B cell-deficient spleen cells to present antigen. The results support the concept that B cells play a significant role in antigen presentation required for T cell activation.