Stability of pro- and anti-inflammatory immune biomarkers for human cohort studies.

BACKGROUND: Although discovery research has identified the importance of dozens of pro- and anti-inflammatory immune mediators in the pathogenesis, maintenance, exacerbation and resolution of inflammatory diseases, most human cohort studies have incorporated few or no immunological intermediate phenotypes in their analyses. Significant hindrances have been (1) the limited panel of biomarkers known to be readily detected in healthy human populations and (2) the stability, hence utility, of such biomarkers to repeated analysis. METHODS: The frequency and stability of 14 plasma biomarkers linked to in vivo immune regulation of allergic and autoimmune inflammatory disorders was determined in 140 healthy pediatric and adult participants. The impact of initial and multiple subsequent freeze/thaw cycles on pro-inflammatory (CCL2, CXCL10, IL-18, TNFα, IL-6), anti-inflammatory (IL-10, sTNF-RII, IL-1Ra), acute phase proteins (CRP, PTX3) and other biomarkers (sST2, IL-1RAcP) was subsequently quantified. RESULTS: Multiple biomarkers capable of providing an innate immune signature of inflammation were readily detected directly ex vivo in healthy individuals. These biomarker levels were unaffected when comparing paired data sets from freshly obtained, never frozen plasma or serum and matched aliquots despite extensive freeze/thaw cycles. Neither age nor sex affected stability. Similarly, no quantitative differences were found following repetitive analysis of inflammatory biomarkers in culture samples obtained following in vitro stimulation with TLR and RLR ligands. CONCLUSIONS: A broad panel of in vivo and ex vivo cytokine, chemokine and acute phase protein biomarkers that have been linked to human chronic inflammatory disorders are readily detected in vivo and remain stable for analysis despite multiple freeze thaw cycles. These data provide the foundation and confidence for large scale analyses of panels of inflammatory biomarkers to provide better understanding of immunological mechanisms underlying health versus disease.

Infant gut microbiota and food sensitization: associations in the first year of life.

BACKGROUND: The gut microbiota is established during infancy and plays a fundamental role in shaping host immunity. Colonization patterns may influence the development of atopic disease, but existing evidence is limited and conflicting. OBJECTIVE: To explore associations of infant gut microbiota and food sensitization. METHODS: Food sensitization at 1 year was determined by skin prick testing in 166 infants from the population-based Canadian Healthy Infant Longitudinal Development (CHILD) study. Faecal samples were collected at 3 and 12 months, and microbiota was characterized by Illumina 16S rRNA sequencing. RESULTS: Twelve infants (7.2%) were sensitized to ≥ 1 common food allergen at 1 year. Enterobacteriaceae were overrepresented and Bacteroidaceae were underrepresented in the gut microbiota of food-sensitized infants at 3 months and 1 year, whereas lower microbiota richness was evident only at 3 months. Each quartile increase in richness at 3 months was associated with a 55% reduction in risk for food sensitization by 1 year (adjusted odds ratio 0.45, 95% confidence interval 0.23-0.87). Independently, each quartile increase in Enterobacteriaceae/Bacteroidaceae ratio was associated with a twofold increase in risk (2.02, 1.07-3.80). These associations were upheld in a sensitivity analysis among infants who were vaginally delivered, exclusively breastfed and unexposed to antibiotics. At 1 year, the Enterobacteriaceae/Bacteroidaceae ratio remained elevated among sensitized infants, who also tended to have decreased abundance of Ruminococcaceae. CONCLUSIONS AND CLINICAL RELEVANCE: Low gut microbiota richness and an elevated Enterobacteriaceae/Bacteroidaceae ratio in early infancy are associated with subsequent food sensitization, suggesting that early gut colonization may contribute to the development of atopic disease, including food allergy.

The Canadian healthy infant longitudinal development birth cohort study: biological samples and biobanking.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Maternal distress in early life predicts the waist-to-hip ratio in schoolchildren.

We report on life course stress determinants of overweight in children, using data from the longitudinal follow-up of the nested case-control arm of the SAGE (study of asthma genes and the environment) birth cohort in Manitoba, Canada. Waist and hip measurements were obtained during a clinic visit at age 9-11 years. Multiple linear regression was conducted to determine the relationship between the waist-to-hip ratio and maternal smoking during pregnancy, postpartum maternal distress and stress reactivity in children (cortisol, cortisol-DHEA [dihydroepiandrostrenone] ratio quartiles) following a clinic stressor at age 8-10 years. We found waist-to-hip risk at age 9-11 years to be elevated among boys and girls whose mothers had experienced distress in the postnatal period. This association varied by gender and asthma status. In healthy girls, postpartum distress increased waist-to-hip ratio by a factor of 0.034 (P < 0.01), independent of the child's stage of puberty and adrenarche, cortisol-DHEA ratio and duration of exclusive breastfeeding. Among girls with asthma, maternal smoking during pregnancy was associated with an increased waist-to-hip ratio, if the mother also experienced distress in the postpartum period (0.072, P = 0.038). Among asthmatic boys, an association between maternal distress and waist-to-hip ratio was evident at the highest cortisol-DHEA ratios. Stress-induced changes to leptin and infant over-eating pathways were proposed to explain the postnatal maternal distress effects. Drawing on the theories of evolutionary biology, our findings underscore the significance of postnatal stress in disrupting hypothalamic-pituitary-adrenal axis function in infants and increasing risk for child overweight.

A novel study design to investigate the early-life origins of asthma in children (SAGE study).

This is a description of the Study of Asthma, Genes and the Environment (SAGE), a novel birth cohort created from provincial healthcare administrative records. It is a general population-based cohort, composed of children at high and low risk for asthma, living in urban and rural environments in Manitoba, Canada. The SAGE study captures the complete longitudinal healthcare records of children born in 1995 and contains detailed information on early-life exposures, such as antibiotic utilization and immunization, in relationship to the development of asthma. Nested within the birth cohort is a case-control study, which was created to collect information on home environmental exposures from detailed surveys and home dust sampling, to confirm asthma status in children and use this data to validate healthcare database measures of asthma, to determine differences in immune system responsiveness to innate and adaptive immune stimuli in asthma, to genotype children for genes likely associated with the development of asthma and to study the epigenetic regulation of pre-established protective vs allergic immune responses. The SAGE study is a multidisciplinary collaboration of researchers from pediatric allergy, population health, immunology, and genetic and environmental epidemiology. As such, it serves as a fertile, interdisciplinary training ground for graduate students, and postdoctoral and clinician fellows.

Novel cytokine peptide-based vaccines: an interleukin-4 vaccine suppresses airway allergic responses in mice.

BACKGROUND: Monoclonal antibodies or soluble receptors have been used to block over-produced endogenous cytokines. However, they have disadvantages of short half-lives, high costs, and possible adverse effects. Using interleukin (IL)-4 as a model target, we sought to develop a novel therapeutic strategy by constructing an IL-4 peptide-based vaccine for blocking IL-4 on a persistent basis, and to evaluate its efficacy in a mouse model of asthma. METHODS: A peptide was selected by antigenic prediction and structure analysis of IL-4/receptor complex. The vaccine was constructed by employing truncated hepatitis B core antigen as carrier with the peptide inserted using gene engineering methods. It was then expressed, purified and identified. Prior to intraperitoneal sensitization and intranasal challenge with ovalbumin, mice were subcutaneously immunized three times with the vaccine, or the carrier or saline as controls. Serum antibodies, inflammatory cells in bronchoalveolar lavage fluids (BALF), lung histology, and responsiveness to inhaled methacholine were analyzed. RESULTS: The vaccine presented as virus-like particles and reacted to polyclonal anti-IL-4 in Western blotting. Vaccinated mice produced high titers of IgG to IL-4. Serum ovalbumin-specific IgE, eosinophil accumulation in BALF, goblet cell hyperplasia, tissue inflammation and methacoline-induced respiratory responses were markedly suppressed in vaccinated mice with statistical significance, as compared with those in the control groups. CONCLUSIONS: Administration of this novel IL-4 vaccine led to an overall decrease in the development of airway allergic inflammatory responses. The results indicate that cytokine peptide-based vaccines hold potential for treatment of asthma and, by extension, other diseases where over-expressed cytokines play a pivotal role in pathogenesis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.