Protection Against Allergy-Study In Rural Environments Research Articles

Symptom trajectories in infancy for the prediction of subsequent wheeze and asthma in the BILD and PASTURE cohorts: a dynamic network analysis

Host and environment early-life risk factors are associated with progression of wheezing symptoms over time; however, their individual contribution is relatively small. We hypothesised that the dynamic interactions of these factors with an infant's developing respiratory system are the dominant factor for subsequent wheeze and asthma. In this dynamic network analysis we used data from term healthy infants from the Basel-Bern Infant Lung Development (BILD) cohort (435 neonates aged 0-4 weeks recruited in Switzerland between Jan 1, 1999, and Dec 31, 2012) and replicated the findings in the Protection Against Allergy Study in Rural Environments (PASTURE) cohort (498 infants aged 0-12 months recruited in Germany, Switzerland, Austria, France, and Finland between Jan 1, 2002, and Oct 31, 2006). BILD exclusion criteria for the current study were prematurity (<37 weeks), major birth defects, perinatal disease of the neonate, and incomplete follow-up period. PASTURE exclusion criteria were women younger than 18 years, a multiple pregnancy, the sibling of a child was already included in the study, the family intended to move away from the area where the study was conducted, and the family had no telephone connection. Outcome groups were subsequent wheeze, asthma, and healthy. The first outcome was defined as ever wheezed between the age of 2 years and 6 years. Week-by-week correlations of the determining factors with cumulative symptom scores (CSS) were calculated from weeks 2 to 52 (BILD) and weeks 8 to 52 (PASTURE). The complex dynamic interaction between the determining factors and the CSS was assessed via dynamic host-environment correlation network, quantified by a simple descriptor: trajectory function G(t). Wheeze outcomes at age 2-6 years were compared in 335 infants from BILD and 437 infants from PASTURE, and asthma outcomes were analysed at age 6 years in a merged cohort of 783 infants. CSS was significantly different for wheeze and asthma outcomes and became increasingly important during infancy in direct comparison with all determining factors. Weekly symptoms were tracked for groups of infants, showing a non-linear increase with time. Using logistic regression classification, G(t) distinguished between the healthy group and wheeze or asthma groups (area under the curve>0·97, p<0·0001; sensitivity analysis confirmed significant CSS association with wheeze [BILD p=0·0002 and PASTURE p=0·068]) and G(t) was also able to distinguish between the farming and non-farming exposure groups (p<0·0001). Similarly to other risk factors, CSS had weak sensitivity and specificity to identify risks at the individual level. At group level however, the dynamic host-environment correlation network properties (G(t)) showed excellent discriminative ability for identifying groups of infants with subsequent wheeze and asthma. Results from this study are consistent with the 2018 Lancet Commission on asthma, which emphasised the importance of dynamic interactions between risk factors during development and not the risk factors per se. The Swiss National Science Foundation, the Kühne Foundation, the EFRAIM study EU research grant, the FORALLVENT study EU research grant, and the Leibniz Prize.

Continuous Rather Than Solely Early Farm Exposure Protects From Hay Fever Development

An important window of opportunity for early-life exposures has been proposed for the development of atopic eczema and asthma. However, it is unknown whether hay fever with a peak incidence around late school age to adolescence is similarly determined very early in life. In the Protection against Allergy-Study in Rural Environments (PASTURE) birth cohort potentially relevant exposures such as farm milk consumption and exposure to animal sheds were assessed at multiple time points from infancy to age 10.5 years and classified by repeated measure latent class analyses (n= 769). Fecal samples at ages 2 and 12 months were sequenced by 16S rRNA. Hay fever was defined by parent-reported symptoms and/or physician's diagnosis of hay fever in the last 12 months using questionnaires at 10.5 years. Farm children had half the risk of hay fever at 10.5 years (adjusted odds ratio [aOR] 0.50; 95% CI 0.31-0.79) than that of nonfarm children. Whereas early life events such as gut microbiome richness at 12 months (aOR 0.66; 95% CI 0.46-0.96) and exposure to animal sheds in the first 3 years of life (aOR 0.26; 95% CI 0.06-1.15) were determinants of hay fever, the continuous consumption of farm milk from infancy up to school age was necessary to exert the protective effect (aOR 0.35; 95% CI 0.17-0.72). While early life events determine the risk of subsequent hay fever, continuous exposure is necessary to achieve protection. These findings argue against the notion that only early life exposures set long-lasting trajectories.

Maturation of the gut microbiome during the first year of life contributes to the protective farm effect on childhood asthma.

Growing up on a farm is associated with an asthma-protective effect, but the mechanisms underlying this effect are largely unknown. In the Protection against Allergy: Study in Rural Environments (PASTURE) birth cohort, we modeled maturation using 16S rRNA sequence data of the human gut microbiome in infants from 2 to 12 months of age. The estimated microbiome age (EMA) in 12-month-old infants was associated with previous farm exposure (β = 0.27 (0.12-0.43), P = 0.001, n = 618) and reduced risk of asthma at school age (odds ratio (OR) = 0.72 (0.56-0.93), P = 0.011). EMA mediated the protective farm effect by 19%. In a nested case-control sample (n = 138), we found inverse associations of asthma with the measured level of fecal butyrate (OR = 0.28 (0.09-0.91), P = 0.034), bacterial taxa that predict butyrate production (OR = 0.38 (0.17-0.84), P = 0.017) and the relative abundance of the gene encoding butyryl-coenzyme A (CoA):acetate-CoA-transferase, a major enzyme in butyrate metabolism (OR = 0.43 (0.19-0.97), P = 0.042). The gut microbiome may contribute to asthma protection through metabolites, supporting the concept of a gut-lung axis in humans.

Parents know it best: Prediction of asthma and lung function by parental perception of early wheezing episodes.

Childhood asthma is often preceded by early wheeze. Usually, wheezing episodes are recorded retrospectively, which may induce recall bias. The aim of this study was to investigate true-positive recall of parent-reported wheeze at 1year of age, its determinants, and its implications for asthma and lung function at 6years of age. The PASTURE (Protection Against Allergy-Study in Rural Environments) study followed 880 children from rural areas in 5 European countries from birth to age 6years. Wheeze symptoms in the first year were asked weekly. At age 6, parent-reported asthma diagnosis was ascertained and lung function measurements were conducted. Correct parental recall of wheeze episodes at the end of the first year was assessed for associations with lung function, asthma, and the asthma risk locus on chromosome 17q21. Parents correctly recalled wheeze after the first year in 54% of wheezers. This true-positive recall was determined by number of episodes, timing of the last wheeze episode, and parental asthma. Independently from these determinants, true-positive recall predicted asthma at age 6years (odds ratio 4.54, 95% confidence interval (CI) [1.75-14.16]) and impaired lung function (β=-0.62, 95% CI [-1.12; -0.13], P-value=.02). Associations were stronger in children with asthma risk SNPs on chromosome 17q21. Correct parental recall of wheezing episodes may reflect clinical relevance of early wheeze and its impact on subsequent asthma and lung function impairment. Questions tailored to parental perception of wheezing episodes may further enhance asthma prediction.

Phenotypes of Atopic Dermatitis Depending on the Timing of Onset and Progression in Childhood

C Roduit, R Frei, M Depner and the PASTURE study group. JAMA Pediatr. 2017;171(7):655–662 To use a symptom-based definition of atopic dermatitis to identify different phenotypes and to determine if certain subtypes are at higher risk to develop comorbid atopic disease. A total of 1038 European children from The Protection Against Allergy Study in Rural Environments (PASTURE) birth cohort (designed to assess impact of living on a farm) were recruited between August 2002 and March 2005. This study included participants …

Disagreement Between Skin Prick Tests and Specific IgE in Early Childhood

A Chauveau, ML Dalphin, V Kaulek. Int Arch Allergy Immunol. 2016;170(2):69–74 To evaluate for agreement between the results of skin prick tests (SPTs) and serum-specific immunoglobulin E (SSIgE) testing for sensitivity to environmental aeroallergens and food allergens in children. The authors of this study also assessed the associations between SPTs with positive results and the presence of atopic dermatitis in children from ages 1 to 6 years. Test results were available for 204 children from rural areas in France, who were recruited through the Protection against Allergy: Study in Rural Environments (PASTURE) project, which included a cohort of European children managed from birth to 10 years of age to determine the effect of farm exposure on the occurrence of allergies. SPTs, atopic dermatitis evaluations using Scoring Atopic Dermatitis Severity Scores, and SSIgE levels were completed at the …

Functional phenotypes determined by fluctuation-based clustering of lung function measurements in healthy and asthmatic cohort participants

RationaleAsthma is characterised by inflammation and reversible airway obstruction. However, these features are not always closely related. Fluctuations of daily lung function contain information on asthma phenotypes, exacerbation risk and...

Exposure to nonmicrobial N-glycolylneuraminic acid protects farmers' children against airway inflammation and colitis

Childhood exposure to a farm environment has been shown to protect against the development of inflammatory diseases, such as allergy, asthma, and inflammatory bowel disease. We sought to investigate whether both exposure to microbes and exposure to structures of nonmicrobial origin, such as the sialic acid N-glycolylneuraminic acid (Neu5Gc), might play a significant role. Exposure to Neu5Gc was evaluated by quantifying anti-Neu5Gc antibody levels in sera of children enrolled in 2 farm studies: the Prevention of Allergy Risk factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle (PARSIFAL) study (n=299) and the Protection Against Allergy Study in Rural Environments (PASTURE) birth cohort (cord blood [n=836], 1year [n=734], 4.5years [n=700], and 6years [n=728]), and we associated them with asthma and wheeze. The effect of Neu5Gc was examined in murine airway inflammation and colitis models, and the role of Neu5Gc in regulating immune activation was assessed based on helper T-cell and regulatory T-cell activation in mice. In children anti-Neu5Gc IgG levels correlated positively with living on a farm and increased peripheral blood forkhead box protein 3 expression and correlated inversely with wheezing and asthma in nonatopic subjects. Exposure to Neu5Gc in mice resulted in reduced airway hyperresponsiveness and inflammatory cell recruitment to the lung. Furthermore, Neu5Gc administration to mice reduced the severity of a colitis model. Mechanistically, we found that Neu5Gc exposure reduced IL-17+ T-cell numbers and supported differentiation of regulatory T cells. In addition to microbial exposure, increased exposure to non-microbial-derived Neu5Gc might contribute to the protective effects associated with the farm environment.

Phenotypes of Atopic Dermatitis Depending on the Timing of Onset and Progression in Childhood

Atopic dermatitis is an inflammatory, pruritic skin disease that often occurs in early infancy with a chronic course. However, a specific description of subtypes of atopic dermatitis depending on the timing of onset and progression of the disease in childhood is lacking. To identify different phenotypes of atopic dermatitis using a definition based on symptoms before age 6 years and to determine whether some subtypes are more at risk for developing other allergic diseases. The Protection Against Allergy Study in Rural Environments (PASTURE) is a European birth cohort where pregnant women were recruited between August 2002 and March 2005 and divided in 2 groups dependent on whether they lived on a farm. Children from this cohort with data on atopic dermatitis from birth to 6 years of age were included. Atopic dermatitis, defined as an itchy rash on typical locations from birth to 6 years. The latent class analysis was used to identify subtypes of atopic dermatitis in childhood based on the course of symptoms. Multivariable logistic regressions were used to analyze the association between atopic dermatitis phenotypes and other allergic diseases. We included 1038 children; of these, 506 were girls. The latent class analysis model with the best fit to PASTURE data separated 4 phenotypes of atopic dermatitis in childhood: 2 early phenotypes with onset before age 2 years (early transient [n = 96; 9.2%] and early persistent [n = 67; 6.5%]), the late phenotype with onset at age 2 years or older (n = 50; 4.8%), and the never/infrequent phenotype (n = 825; 79.5%), defined as children with no atopic dermatitis. Children with both parents with history of allergies were 5 times more at risk to develop atopic dermatitis with an early-persistent phenotype compared with children with parents with no history of allergies. Both early phenotypes were strongly associated with food allergy. The risk of developing asthma was significantly increased among the early-persistent phenotype (adjusted odds ratio, 2.87; 95% CI, 1.31-6.31). The late phenotype was only positively associated with allergic rhinitis. Using latent class analysis, 4 phenotypes of atopic dermatitis were identified depending on the onset and course of the disease. The prevalence of asthma and food allergy by 6 years of age was strongly increased among children with early phenotypes (within age 2 years), especially with persistent symptoms. These findings are important for the development of strategies in allergy prevention.

Skin prick tests and specific IgE in 10-year-old children: Agreement and association with allergic diseases.

Accurate assessment of atopic sensitization is pivotal to clinical practice and research. Skin prick test (SPT) and specific IgE (sIgE) are often used interchangeably. Some studies have suggested a disagreement between these two methods, and little is known about their association with allergic diseases. The aims of our study were to evaluate agreement between SPT and sIgE, and to compare their association with allergic diseases in 10-year-old children. Skin prick test, sIgE measurements, and assessment of allergic diseases were performed in children aged 10years in the Protection against Allergy: STUdy in Rural Environments (PASTURE) cohort. The agreement between SPT and sIgE was assessed by Cohen's kappa coefficient with different cutoff values. Skin prick tests and sIgE were performed in 529 children. The highest agreement (κ=.44) was found with a cutoff value of 3 and 5mm for SPT, and 3.5IU/mL for sIgE. The area under the curve (AUC) obtained with SPT was not significantly different from that obtained with sIgE. For asthma and hay fever, SPT (cutoff value at 3mm) had a significantly higher specificity (P<.0001) than sIgE (cutoff value at 0.35IU/mL) and the specificity was not different between both tests (P=.1088). Skin prick test and sIgE display moderate agreement, but have a similar AUC for allergic diseases. At the cutoff value of 3mm for SPT and 0.35IU/mL for sIgE, SPT has a higher specificity for asthma and hay fever than sIgE without difference for sensitivity.

Latent class analysis reveals clinically relevant atopy phenotypes in 2 birth cohorts

Phenotypes of childhood-onset asthma are characterized by distinct trajectories and functional features. For atopy, definition of phenotypes during childhood is less clear. We sought to define phenotypes of atopic sensitization over the first 6years of life using a latent class analysis (LCA) integrating 3 dimensions of atopy: allergen specificity, time course, and levels of specific IgE (sIgE). Phenotypes were defined by means of LCA in 680 children of the Multizentrische Allergiestudie (MAS) and 766 children of the Protection against allergy: Study in Rural Environments (PASTURE) birth cohorts and compared with classical nondisjunctive definitions of seasonal, perennial, and food sensitization with respect to atopic diseases and lung function. Cytokine levels were measured in the PASTURE cohort. The LCA classified predominantly by type and multiplicity of sensitization (food vs inhalant), allergen combinations, and sIgE levels. Latent classes were related to atopic disease manifestations with higher sensitivity and specificity than the classical definitions. LCA detected consistently in both cohorts a distinct group of children with severe atopy characterized by high seasonal sIgE levels and a strong propensity for asthma; hay fever; eczema; and impaired lung function, also in children without an established asthma diagnosis. Severe atopy was associated with an increased IL-5/IFN-γ ratio. Apath analysis among sensitized children revealed that among all features of severe atopy, only excessive sIgE production early in life affected asthma risk. LCA revealed a set of benign, symptomatic, and severe atopy phenotypes. The severe phenotype emerged as a latent condition with signs of a dysbalanced immune response. It determined high asthma risk through excessive sIgE production and directly affected impaired lung function.

ω-3 fatty acids contribute to the asthma-protective effect of unprocessed cow's milk

Living on a farm has repeatedly been shown to protect children from asthma and allergies. Amajor factor involved in this effect is consumption of unprocessed cow's milk obtained directly from a farm. However, this phenomenon has never been shown in a longitudinal design, and the responsible milk components are still unknown. We sought to assess the asthma-protective effect of unprocessed cow's milk consumption in a birth cohort and to determine whether the differences in the fatty acid (FA) composition of unprocessed farm milk and industrially processed milk contributed to this effect. The Protection Against Allergy-Study in Rural Environments (PASTURE) study followed 1133 children living in rural areas in 5 European countries from birth to age 6years. In 934 children milk consumption was assessed by using yearly questionnaires, and samples of the "usually" consumed milk and serum samples of the children were collected at age 4years. Doctor-diagnosed asthma was parent reported at age 6years. In a nested case-control study of 35 asthmatic and 49 nonasthmatic children, 42 FAs were quantified in milk samples. The risk of asthma at 6years of age was reduced by previous consumption of unprocessed farm milk compared with shop milk (adjusted odds ratio for consumption at 4years, 0.26; 95% CI, 0.10-0.67). Part of the effect was explained by the higher fat content of farm milk, particularly the higher levels of ω-3 polyunsaturated FAs (adjusted odds ratio, 0.29; 95% CI, 0.11-0.81). Continuous farm milk consumption in childhood protects against asthma at school age partially by means of higher intake of ω-3 polyunsaturated FAs, which are precursors of anti-inflammatory mediators.

O07 ‐ Phenotypes of atopic dermatitis depending on the timing of onset and the evolution in childhood

Method 1045 children who participated in the birth cohort study, Protection Against Allergy-Study in Rural Environments (PASTURE), were included in the current study. Symptoms of atopic dermatitis were reported by parents from birth to 6 years of age by yearly questionnaires and defined as an intermittent or persistent itchy rash on typical locations. We used longitudinal latent class analysis (LCA) to identify different phenotypes of atopic dermatitis symptoms in childhood based on the first 6 years of life.

Farm exposure and time trends in early childhood may influence DNA methylation in genes related to asthma and allergy

Genetic susceptibility and environmental influences are important contributors to the development of asthma and atopic diseases. Epigenetic mechanisms may facilitate gene by environment interactions in these diseases. We studied the rural birth cohort PASTURE (Protection against allergy: study in rural environments) to investigate (a) whether epigenetic patterns in asthma candidate genes are influenced by farm exposure in general, (b) change over the first years of life, and (c) whether these changes may contribute to the development of asthma. DNA was extracted from cord blood and whole blood collected at the age of 4.5 years in 46 samples per time point. DNA methylation in 23 regions in ten candidate genes (ORMDL1, ORMDL2, ORMDL3, CHI3L1, RAD50, IL13, IL4, STAT6, FOXP3, and RUNX3) was assessed by pyrosequencing, and differences between strata were analyzed by nonparametric Wilcoxon-Mann-Whitney tests. In cord blood, regions in ORMDL1 and STAT6 were hypomethylated in DNA from farmers' as compared to nonfarmers' children, while regions in RAD50 and IL13 were hypermethylated (lowest P-value (STAT6) = 0.001). Changes in methylation over time occurred in 15 gene regions (lowest P-value (IL13) = 1.57*10(-8)). Interestingly, these differences clustered in the genes highly associated with asthma (ORMDL family) and IgE regulation (RAD50, IL13, and IL4), but not in the T-regulatory genes (FOXP3, RUNX3). In this first pilot study, DNA methylation patterns change significantly in early childhood in specific asthma- and allergy-related genes in peripheral blood cells, and early exposure to farm environment seems to influence methylation patterns in distinct genes.

Atopic sensitization in the first year of life

There is conflicting evidence on whether allergen-specific memory is primed prenatally, whether this priming affects persistent immunologic effects, and whether it is modulated by the first environmental exposures in infancy. We sought to explore the course of atopic sensitization between birth and 12 months of age. Specific IgE levels for 6 food and 13 common inhalant allergens were assessed in cord blood and 1-year blood samples in the Protection against Allergy-Study in Rural Environments (PASTURE) birth cohort including 793 children from rural regions of 5 European countries. Detailed information on children's health, nutrition, and farm-related exposures was gathered by using a pregnancy questionnaire, 2 questionnaires at 2 and 12 months of age, and a diary covering the time in between. Sensitization was more common at 12 months of age than at birth for almost all specificities. On an individual level, persistent sensitization to the same allergens was rare (1%), whereas transient (only at birth, 11%) and incident (only at 12 months, 34%) sensitization was seen in substantial proportions of children. Associations of transient sensitization with maternal sensitization differed with the allergen specificities, with the strongest associations for food allergens (odds ratio [OR], 10.6; 95% CI, 6.0-18.6) and the weakest associations for seasonal allergens (OR, 1.64; 95% CI, 0.94-2.86). Associations of maternal sensitization with incident sensitization were also seen. Incident sensitization was related to distinct prenatal and postnatal environmental exposures of mother and child, such as consumption of cereals for incident sensitization to seasonal allergens (OR, 0.66; 95% CI, 0.50-0.88). IgE sensitization patterns change between birth and 12 months and are related to maternal and environmental influences.

Prenatal and early-life exposures alter expression of innate immunity genes: The PASTURE cohort study

There is evidence that gene expression of innate immunity receptors is upregulated by farming-related exposures. We sought to determine environmental and nutritional exposures associated with the gene expression of innate immunity receptors during pregnancy and the first year of a child's life. For the Protection Against Allergy: Study in Rural Environments (PASTURE) birth cohort study, 1133 pregnant women were recruited in rural areas of Austria, Finland, France, Germany, and Switzerland. mRNA expression of the Toll-like receptor (TLR) 1 through TLR9 and CD14 was assessed in blood samples at birth (n= 938) and year 1 (n= 752). Environmental exposures, as assessed by using questionnaires and a diary kept during year 1, and polymorphisms in innate receptor genes were related to gene expression of innate immunity receptors by using ANOVA and multivariate regression analysis. Gene expression of innate immunity receptors in cord blood was overall higher in neonates of farmers (P for multifactorial multivariate ANOVA= .041), significantly so for TLR7 (adjusted geometric means ratio [aGMR], 1.15; 95% CI, 1.02-1.30) and TLR8 (aGMR, 1.15; 95% CI, 1.04-1.26). Unboiled farm milk consumption during the first year of life showed the strongest association with mRNA expression at year 1, taking the diversity of other foods introduced during that period into account: TLR4 (aGMR, 1.22; 95% CI, 1.03-1.45), TLR5 (aGMR, 1.19; 95% CI, 1.01-1.41), and TLR6 (aGMR, 1.20; 95% CI, 1.04-1.38). A previously described modification of the association between farm milk consumption and CD14 gene expression by the single nucleotide polymorphism CD14/C-1721T was not found. Farming-related exposures, such as raw farm milk consumption, that were previously reported to decrease the risk for allergic outcomes were associated with a change in gene expression of innate immunity receptors in early life.

Wird die Protektion gegen Allergien bereits im Mutterleib angelegt?

Background Traditional farming represents a unique model situation to investigate the relationship of early-life farm-related exposure and allergy protection. The aim of PASTURE (Protection against Allergy-Study in Rural Environments) was to investigate associations between maternal farm exposures and cytokine production in cord blood (CB) mononuclear cells in a prospective multinational birth cohort of 299 farm and 326 nonfarm children and their families.

Cord Blood Allergen-Specific IgE Is Associated With Reduced IFN-γ Production by Cord Blood Cells: The Protection Against Allergy–Study in Rural Environments (PASTURE) Study

Pfefferle PI, Sel S, Ege MJ, et al; PASTURE Study Group. J Allergy Clin Immunol . 2008;122(4):711–716 PURPOSE OF THE STUDY. To investigate the relationship between allergen-specific cord blood (CB) immunoglobulin E (IgE) levels, parental allergen sensitization, CB cytokine production, and environmental influences. STUDY POPULATION. This study included families recruited for the multicenter Protection Against Allergy–Study in Rural Environments (PASTURE) study, an ongoing longitudinal birth cohort study designed to evaluate risk factors and preventative factors for atopic disease. Pregnant women in their third trimester were recruited for participation from Finland, Germany, France, Switzerland, and Austria. METHODS. CB samples were collected from umbilical cord veins, and parental blood …

Cord blood allergen-specific IgE is associated with reduced IFN-γ production by cord blood cells: The Protection against Allergy—Study in Rural Environments (PASTURE) study

It is currently discussed whether allergic sensitization may start in utero under the influence of the maternal immune system and environmental determinants. To investigate the relationship between allergen-specific cord blood (CB) IgE levels, parental sensitization, CB cytokine production, and environmental influences. As part of an ongoing multicenter birth cohort study, allergen-specific IgE antibodies against 20 common seasonal, perennial, and food allergens were measured in blood samples from 922 neonates, 922 mothers, and 835 fathers. Supernatants from stimulated CB cells were assessed for the production of IL-5, IFN-gamma, IL-10, and TNF-alpha. Allergen-specific IgE antibodies were detectable in 23.9% of newborns. Contamination with maternal serum was excluded by several means of analyses, including the absence of IgA antibodies. Clear correlation between maternal and fetal IgE was found only for hen's egg, cow's milk, and soybean allergen. Fetal IgE correlated negatively with the level of IFN-gamma production, but not with IL-5 and IL-10. Allergen-specific IgE antibodies most probably of fetal origin are detectable in CB and correlate with a lowered CB IFN-gamma production.