The Journal of Allergy and Clinical Immunology
Volume 120, Issue 1 , Pages 200-206, July 2007

Protein kinase Cζ: A novel protective neonatal T-cell marker that can be upregulated by allergy prevention strategies

  • Susan L. Prescott, MBBS, PhD, FRACP

      Affiliations

    • From the School of Pediatrics and Child Health, University of Western Australia, Perth
    • Corresponding Author InformationReprint requests: Susan L. Prescott, MBBS, PhD, FRACP, School of Pediatrics and Child Health, University of Western Australia, PO Box D184, Princess Margaret Hospital, Perth WA 6001, Australia.
    • ,
  • James Irvine, BSc(Hons)

      Affiliations

    • Department of Immunopathology, Women and Children's Hospital, Adelaide, and the Department of Pediatrics, University of Adelaide, Australia
    • ,
  • Janet A. Dunstan, BAppSc, PhD

      Affiliations

    • From the School of Pediatrics and Child Health, University of Western Australia, Perth
    • ,
  • Charles Hii, BSc(Hons), PhD

      Affiliations

    • Department of Immunopathology, Women and Children's Hospital, Adelaide, and the Department of Pediatrics, University of Adelaide, Australia
    • ,
  • Antonio Ferrante, PhD, FRCPath

      Affiliations

    • Department of Immunopathology, Women and Children's Hospital, Adelaide, and the Department of Pediatrics, University of Adelaide, Australia

Received 15 December 2006; received in revised form 9 March 2007; accepted 13 March 2007. published online 04 June 2007.

Perth and Adelaide, Australia

Article Outline

Background

Variations in neonatal T-cell function have been associated with allergic disease.

Objectives

To examine the relationship between neonatal T-cell protein kinase (PKC) expression and subsequent allergic disease.

Methods

T cells were purified from cord blood samples (n = 74) obtained from a cohort of mothers who received either 4 g/d fish oil or a placebo from 20 weeks of gestation. PKC expression was examined in relationship to supplementation, fatty acid levels, cytokine production, and allergic outcomes at 1 year and 2.5 years of age.

Results

Neonatal T-cell PKCζ expression was lower in children who had evidence of allergic disease at 1 year (P = .001) and 2.5 years (P = .052) of age. It was also lower in children with sensitization (positive skin prick test) at each age (P = .02 and P = .072, respectively). PKCζ expression was inversely correlated to PKCα (r = −0.28; P = .025), which was strongly related to IL-5 responses to allergens (ovalbumin, r = 0.59; P = .003; dust mite, r = 0.52; P = .011) at 1 year of age. Fish oil supplementation was associated with significantly higher PKCζ expression (P = .014), whereas most other isozymes were reduced by fish oil supplementation.

Conclusion

This is the first study to show that allergic disease is associated with altered expression of T-cell PKC isozymes in the neonatal period. It has also demonstrated that fish oil can modulate expression of PKC isozymes in a potentially favorable direction.

Clinical implications

Protein kinase Cζ should be explored further as an early marker and potential target for disease prevention.

Key words: Protein kinase C, neonate, cytokines, fish oil, polyunsaturated fatty acids, atopic dermatitis, sensitization, allergy prevention, predictive markers, cord blood, allergy

Abbreviations used: DHA, Docosahexaenoic acid, EPA, Eicosapentaenoic acid, MC, Mononuclear cell, ω-3 LCPUFA, ω-3 Long chain polyunsaturated fatty acid, OR, Odds ratio, PKC, Protein kinase C, SEB, Staphylococcus aureus enterotoxin B, SPT, Skin prick test

 

The development of allergic disease is frequently preceded by immunologic differences that are most evident in the neonatal period.1, 2, 3, 4, 5, 6 To date, none of these measures has been of any predictive value.7 Preliminary studies also suggest that maternal environmental exposures (such as infection,8 maternal diet,9, 10 and smoking10, 11) can modify neonatal T-cell function, although the mechanisms are not clear. With rising disease rates, there is a continuing urgency to identify the pathways involved and to explore the effects of early interventions that could favorably influence the functional development of T-cell responses and prevent allergic disease.

Maternal dietary supplementation with ω-3 long-chain polyunsaturated fatty acids (ω-3 LCPUFA) in pregnancy modifies neonatal immune function.9 The predominant effects were seen on T-cell responses, as evidenced by reduced neonatal T-cell cytokine production in vivo12 and in vitro.9 Interestingly, we have previously demonstrated in neonatal T lymphocytes that reduced ability to activate mitogen-activated protein kinases (which are crucial for cytokine production) is associated with reduced expression of several protein kinase C (PKC) isozymes.13 It has also been reported that mitogen-activated protein kinases differentially regulate TH1 from TH2 cytokine production.14, 15, 16 Furthermore, our observations (Hii and Ferrante, unpublished data, January 2005) revealed that normalization of isozymes expression precedes acquisition of functional responses by neonatal T lymphocytes in culture. There is also large individual variation in neonatal T-cell expression in PKC isozymes.

It was therefore logical to surmise that these variations in PKC isozyme expression lead to corresponding differences in maturation characteristics, patterns of cytokine response, and disease susceptibility. Our preliminary evidence that young children exposed to high-dose fish oil in pregnancy may subsequently have less allergic sensitization and less severe atopic dermatitis12 further suggests that that effects on T-cell function could be clinically significant and effected through these lipid pathways. Specifically, the aims of this study were to assess (1) whether variations in patterns of neonatal PKC expression are related to the subsequent risk of allergic immune responses and/or symptoms; (2) whether variations in PKC expression were associated with immune development; and (3) whether fish oil supplementation was associated with differences in PKC isozyme expression.

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Methods 

Study design 

This study entailed a more detailed analysis of mononuclear cell (MC) samples collected as part of a previously described randomized controlled trial that examined the effects of maternal fish oil supplementation from 20 weeks of gestation on neonatal immune function.12

Population 

The study population is discussed in detail elsewhere.12 Briefly, the study group was composed of 98 pregnant nonsmoking women with documented allergic disease. Those randomized to the fish oil group received 4 × 1 g/d fish oil capsules (Ocean Nutrition, Halifax, Nova Scotia, Canada), for a total of 3.7 g ω-3 LCPUFA with 56.0% as docosahexaenoic acid (DHA) and 27.7% as eicosapentaenoic acid (EPA). The control group received 4 × 1 g/d capsules of olive oil (containing 66.6% ω-9 oleic acid and less than 1% n-3 LCPUFA; Pan Laboratories, Moorebank, Australia).

Blood samples 

This study used cord blood MCs that were previously collected and cryopreserved from this cohort by using standardized techniques.9 Samples were available from 67 infants (n = 36 in the placebo group and n = 31 in the fish oil group). At 12 months of age, MCs were also available from a subgroup of children (22 infants; equal number in each study group), and these were used for cytokine studies.

Fatty acid analyses 

Phospholipid fatty acid analyses were performed as previously described.17, 18 Briefly, total lipids were extracted from red cell membranes with methanol and chloroform. The phospholipid fraction was obtained from total lipid extracts by thin-layer chromatography, and the fatty acid methyl esters were analyzed by gas liquid chromatography.

Cytokine responses 

T-cell cytokine (IL-5, IL-13, and IFN-γ) responses were assessed at birth and 12 months of age as previously described.12 Briefly, MC were cultured (2 × 106 MC/mL in duplicate 250 μL aliquots) in AIM V (Gibco, Life Technology, Paisley, United Kingdom [UK]) serum-free medium for 48 hours with or without house dust mite extract (10 μg/mL; CSL, Parkville, Australia), ovalbumin (100 μg/mL; Sigma, Castle Hill, Australia), cat hair extract (30 μg/mL; ALK-Abelló, Hørsholm, Denmark), tetanus toxoid 0.5 Lf, tuberculin purified protein derivative 10 μg/mL, phytohemagglutinin mitogen (1 μg/mL, HA16; Murex, Biotech Ltd, Dartford, UK), and superantigen Staphylococcus aureus enterotoxin B (SEB, 200 ng/mL; Sigma). For phytohemagglutinin stimulation (1 μg/mL), 109 MC/L was used. Supernatants were analyzed for cytokine using an in-house ELISA (IL-5) or time-resolved fluorometry techniques (IL-13 and IFN-γ) as previously described.9, 19 The limit of detection was 3 pg/mL. Cytokines were expressed as the difference between the stimulated culture and the control (pg/mL).

Source of adult peripheral blood 

Adult blood was obtained by venipuncture from healthy volunteers within the Women's and Children's Hospital according to the institution's protocol on human ethics.

T-cell preparation and determination of PKC expression 

Cord and adult blood T cells were prepared from the MC fraction as described.13 The cells were at least 95% pure and 99% viable. After sonication in buffer containing 2% Triton X-100 (AppliChem, Cheshire, Conn), PKC expression was investigated by Western blot analysis using isozyme-specific anti-PKC antibodies.13 Each gel contained at least 1 lane loaded with adult sample, pooled from 5 volunteers. The blots were stripped (Western blot recycling kit; Alpha Diagnostic International, San Antonio, Texas) and reprobed with anti–β-actin antibody (Santa Cruz Biotech, Santa Cruz, Calif). The immunoreactive bands were subjected to densitometric analysis (ImageQuant scanner; Molecular Dynamics, Krefeld, Germany), and the level of PKC expression was corrected against the level of β-actin within each sample. Results are expressed as percentage of the value found in adult samples.

Clinical outcomes 

Infants were clinically evaluated at 12 months and 2.5 years of age, which included a detailed history and examination by the same pediatric allergist (S.L.P.). The main clinical outcome measures were (1) any physician-diagnosed allergic disease present in the preceding 12 months, (2) sensitization (ever), (3) IgE-mediated symptomatic food allergy (ever), (4) atopic eczema (with positive skin prick test [SPT]) ever, (5) SPT− eczema (ever), and (6) recurrent wheeze (with or without a diagnosis of asthma) in the preceding 12 months. The diagnostic criteria conformed to the recently published clinical guidelines.20

Allergen SPT 

Allergic sensitization was assessed by SPT at 12 months and 2.5 years of age. This was performed using common allergen extracts (milk, peanut, house dust mite, cat, grass, mold; Hollister-Stier Laboratories, Spokane, Wash) and whole egg, as well as histamine as a positive control and glycerine as a negative control. A wheal diameter of ≥2 mm was considered positive.

Statistical analysis 

Where possible, data that were not normally distributed (determined by Kolmogorov-Smirnov test and probability plot) were log-transformed to obtain a log-normal distribution. Pearson correlation coefficients were calculated where the data were normally distributed, and Spearman coefficients where the data remained nonparametric. Groups of normalized continuous variables were compared by using the Student t test and expressed as means and SEMs. Logistic regression was used to determine the weighted effects of neonatal variables (such as PKC expression or IFN-γ production) on subsequent clinical outcomes. All statistical analyses were performed using Macintosh SPSS software (version 11.0 for Mac OS X, SPSS Inc., Chicago, Ill). For all between group analyses, a P value < .05 was considered statistically significant. For correlation studies, because of the exploratory nature of this study, we did not wish to exclude any important relationships by using stringent correction factors for multiple analyses. However, we recognize the potential for type 1 error, and data have been interpreted conservatively in this context.

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Results 

Characteristics of PKC expression in neonatal period 

The pattern of neonatal T-cell PKC isozymes is shown in Fig 1. Although PKCα and PKCθ were expressed around (or even greater than) adult levels, other isozymes (particularly PKCδ, PKCζ, and PKCε) were substantially lower.

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  • Fig 1. 

    Neonatal PKC isozyme expression is shown relative to adult expression (as % of adult control samples). The levels are shown as the means and SEMs, and significance levels denote levels that are significantly different from the adult control (∗P < .05; ∗∗∗P < .001).

There were strong positive correlations between PKCα and PKCβ1 (Pearson correlation, r = 0.51; P < .001) and β2 (r = 0.45; P < .001), and similarly strong relationships between PKCβ1 and β2 (r = 0.78; P < .001). In contrast, there were inverse relationships between PKCα and PKCζ (r = −0.28; P = .025). PKCβI and PKCβII were also inversely related to PKCζ (not statistically significant), but there were no other consistent relationships.

Associations between PKC expression and immune function 

Next, we assessed the relationship between PKC expression and the capacity and patterns of cytokine production.

Cytokine production at birth 

We initially examined the relationship between PKC expression and capacity for TH1 responses, which have been previously associated (inversely) with atopic risk.1, 2, 3, 4, 5, 6 PKCζ expression was positively correlated (Spearman correlations) with polyclonal IFN-γ responses to SEB (r = 0.265; P = .03). There were no significant relationships with the other isozymes. IFN-γ responses to specific stimuli were below detection in most babies at birth.

We then examined PKC expression in relation to neonatal TH2 responses. Although PKCε was expressed at the lowest levels relative to other isozymes (Fig 1), this was consistently related to neonatal TH2 cytokine production, particularly IL-13, which was more readily detected than IL-5 at this age. Specifically, both IL-13 (r = 0.36; P = .001) and IL-5 (r = 0.37; P = .001) responses to cat allergen were positively related to PKCε expression. Similar relationships were seen for IL-13 responses to ovalbumin (r = 0.27; P = .019) and purified protein derivative (r = 0.29; P = .011). There were no consistent relationships between neonatal cytokine production and expression of the other isozymes.

Cytokine production at 1 year of age 

Children who developed sensitization (most commonly to egg) tended to have higher production of TH2 IL-5 and IL-13 cytokines in response to this allergen, as shown in Fig 2, although this was not statistically significant. Twenty children (20/64; 31.25%) were SPT positive to egg at either 1 or 2 years of age. These children also had stronger TH2 IL-5 (P = .018) and IL-13 (P = .03) at birth. There were consistent strong relationships between PKCα expression at birth and the subsequent production of TH2 IL-5 at 1 year of age (Table I) in response to allergens, vaccines, and polyclonal stimuli. Similar trends were also seen for PKCβI and βII, although these were less consistent (Table I). At this age, there were no detectable differences in the cytokine responses between the fish oil and control groups (data not shown).

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  • Fig 2. 

    T-cell cytokine responses are shown at birth and 1 year of age comparing children with no egg allergen sensitization (open bars, 61 with cord blood samples and 18 with samples at 1 year) with egg-sensitized children (shaded bars, 9 with cord blood samples and 4 with samples at 1 year) at 2.5 year of age. Medians, interquartile ranges, and 95% CIs are shown.

Table I. Relationship (Spearman correlation) between neonatal PKC isozyme and IL-5 responses at 1 year of age (n = 23)

Variations in neonatal PKCζ activation were related to subsequent allergic symptoms 

Because early symptoms of allergic disease can be difficult to interpret, we examined a number of allergic outcomes including (1) physician-diagnosed disease (such as food allergy and atopic eczema), (2) reported symptoms (such as wheeze and urticarial reactions), and (3) objective evidence of sensitization (SPT reactivity). The detailed characteristics of this population are also discussed in detail elsewhere.12

The expression of PKCζ at birth was associated with atopic outcomes at both 1 year and 2.5 years of age (Fig 3, Fig 4). This relationship was evident for both allergic disease (at 1 year, P = .001; and 2.5 years, P = .052) as well as a history of allergen sensitization (at 1 year, P = .02; and 2.5 years, P = .072). None of the other isozymes showed any significant relationships with these clinical outcomes. PKCζ appeared best at predicting food allergy (odds ratio [OR], 0.02; 95% CI, 0.002-0.35; P = .007) and sensitization, with significantly lower levels in neonates who developed a positive SPT to egg (OR, 0.127; 95% CI, 0.02-0.69; P = .017) or any food allergen (OR, 0.13; 95% CI, 0.02-0.68; P = .01) at 1 or more of the subsequent clinical assessments (data not shown). The relationships with other specific allergy outcomes are shown in Fig 3, Fig 4. Only SPT+ (and not SPT−) dermatitis was associated with PKCζ expression (Figs 3, D, and 4, D). Recurrent wheeze in the first year of life was not associated with PKC expression, but there was relationship in children with recurrent wheeze during their third year of life (Fig 4, F).

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  • Fig 3. 

    Neonatal PKCζ expression is shown in relation to specific allergic outcomes (see Methods for definitions) measured at 1 year of age, comparing expression in affected (dark bars) and unaffected (light bars) children by using the Student t test (means and SEs). For variables based on SPT data. For variables based on SPT data, subjects who did not have SPT performed were excluded from the analyses. Significance levels are shown (∗P < .05; ∗∗P < .01).

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  • Fig 4. 

    Neonatal PKCζ expression is shown in relation to specific allergic outcomes (see Methods for definitions) measured at 2.5 years of age, comparing expression in affected (dark bars) and unaffected (light bars) children by using the Student t test (means and SEs). For variables based on SPT data, subjects who did not have SPT performed were excluded from that analyses. Significance levels are shown (∗∗P < .01).

Protein kinase C was the strongest independent predictor of allergic disease in this population. This effect was stronger than any predictive effect of neonatal polyclonal IFN-γ responses (to SEB), which were also lower in children with allergic disease by 1 year (P = .041; data not shown). Specifically, using multivariate logistic regression with these parameters as predictor variables, only PKCζ was significantly associated with allergic outcomes (OR, 0.161; 95% CI, 0.04-0.59; P = .006); and neonatal IFN-γ responses (OR, 0.964; 95% CI, 0.749-1.241; P = .77) did not. These relationships did not alter when controlled for fish oil supplementation.

Fish oil supplementation was associated with differences in PKC isozyme expression 

Next, we examined whether the supplementation with fish oil was associated with differences in PKC isozyme expression compared with the control group. Notably, PKCζ was expressed at significantly higher levels in the fish oil group (P = .014) as shown on Fig 5. All other PKC isozyme tended to be expressed at lower levels in the children whose mothers had received fish oil during pregnancy. Children in the fish oil group had significantly lower expression of these other isozymes in relationship to PKCζ when each isozyme was considered as a ratio to PKCζ (ie, PKCα:ζ, PKCβI:ζ, and so forth). This was most evident for PKCα (P = .023), ε (P = .015), and θ (P = .046).

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  • Fig 5. 

    Neonatal PKC isozyme expression is shown in relation to fish oil supplementation, comparing expression in 31 children receiving fish oil (shaded bars) and 36 control group children (open bars) by using the Student t test. The results are presented as means and SEMs. P < .05 was considered statistically significant.

Relationship between fatty acid levels and PKC isozyme expression 

In general terms, PKCζ was consistently positively correlated with specific and total ω-3 LCPUFA measured in maternal (Table II) and fetal (Table III) red cell membranes. In contrast, there were inverse correlations between PKCζ levels and ω-6 fatty acids. The opposite relationship was seen for most other isozymes, which tended to be negatively correlated with the ω-3 LCPUFA and/or positively correlated with the n-6 LCPUFA. This was most evident for PKCβII, PKCε, and PKCθ (also shown in Table II, Table III).

Table II. Correlations between cord blood fatty acid levels and PKC expression: maternal red cell membrane LCPUFA
Table III. Correlations between cord blood fatty acid levels and PKC expression: fetal red cell membrane LCPUFA

Specificity and sensitivity of neonatal PKCζ in predicting allergic disease 

We generated ROC curves to determine the optimal cutoff levels for predicting allergic disease (at 1 year) and demonstrated that PKCζ expression below 62.3% predicted allergic disease with a sensitivity of 80% and a specificity of 63% (and accuracy of 71%; the number of children correctly classified as negative and positive) in this high-risk population.

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Discussion 

This is the first study to identify neonatal T-cell PKC isozyme expression (namely PKCζ) as a potential predictor of allergic disease. These relationships were seen with multiple independently assessed allergic outcomes, such as sensitization and different allergic disease states. Moreover, the reduced neonatal PKCζ expression in subsequently allergic children was detected in resting cells without the need for stimulation or culture. Although neonates at high allergy risk have been previously shown to have relative immaturity of TH1 IFN-γ cytokine production,1, 2, 3, 4, 5, 6 this has not been reliable as a predictive marker because these measures rely on inherently variable in vitro culturing systems with potent polyclonal stimulants that have the potential to distort patterns of response. Accordingly, we demonstrate here that the predictive effect of PKCζ is far greater than IFN-γ TH1 responses. Because there are no reliable early markers to predict allergic disease, our findings provide a strong basis for exploring PKCζ further in this context, particularly because this approach avoids in vitro manipulations and could be more readily standardized. It remains to be determined whether variable levels in isozyme expression are a function of genetic polymorphisms, because this could provide polymorphic markers in molecular screening for disease susceptibility. Further studies are also needed to determine whether PKC isozymes are differentially expressed in different MC and T-cell subsets.

In this high-risk (family history positive) population, PKCζ predicted allergic disease with a sensitivity of 80% and a specificity of 63% (and accuracy of 71%; the number of children correctly classified as negative and positive). This suggests that PKC is superior to any other biological marker assessed in this context, including cord blood IgE (which has a reported specificity of only 26% to 47%21, 22, 23, 24). Although other parameters have been examined previously (including eosinophil levels, histamine reactions, and leukocyte phosphodiesterase levels), none had an accuracy of more than 58%.7 Thus, family history is a reasonable but imperfect predictor of allergic disease,21, 22, 23 and here we have shown that PKC further predicted disease in this high-risk subgroup. This suggests that combining PKC and family history (and potentially other markers) could enhance the capacity to predict allergic disease. We now need to test this in a general population with mixed allergy risk to develop predictive algorithms.

The study provides novel data that neonatal PKC isozymes are differentially expressed, with PKCζ varying inversely with most other isozymes (particularly PKCα). Although PKCζ expression had a protective relationship in allergic disease, PKCα was associated with an increased risk of developing subsequent TH2 (IL-5) responses to allergens and other stimuli. Our findings support intrinsic T-cell differences in neonates at high atopic risk, which are independent of any secondary differences as a result of antigen presenting cell function.25 Indeed we have observed that functional competence of neonatal T cells is preceded by maturation of neonatal T-cell PKC isozymes during culture, and this maturation is dependent on PKC activation (Hii and Ferrante, unpublished data, January 2005). Together these novel data suggest that variable inadequacy in PKC kinase signaling may be responsible, at least in part, for the variations in neonatal T-cell phenotype and subsequent disease risk.

Another key finding is that PKC expression can be modified through intervention. In our previous studies, we noted that ω-3 LCPUFA fish oil supplementation in pregnancy could modify neonatal T-cell function (as detected by differences in cytokine responses).9 Consistent with this, we now demonstrate that fish oil supplementation is also associated with differential expression of neonatal PKC isozymes. Although other isozymes tended to be consistently downregulated by fish oil, PKCζ was significantly increased. Because PKCζ was shown to be an independent predictor of reduced subsequent allergic disease, this could present an important pathway through which ω-3 LCPUFA could have clinical effects in reducing allergic disease. Although some relationships were noted between PKC expression and neonatal cytokine responses, these were not strong or consistent. This may be a reflection of (1) low-level cytokine responses at this age, (2) the unclear significance of antigen-specific cytokine responses at birth, and/or (3) inherent variability in the cell culture assays used to detect cytokine responses.

These findings provide further support for the capacity of these early dietary exposures to influence immune development favorably. We propose that interventions during an early critical window of immune development can alter the risk of subsequent sensitization and disease. At this stage, the timing of this critical window is not clear, although perinatal differences in immunity of individuals with allergy indicate that this is likely to be early in development.5, 26, 27 It is therefore logical that to be most effective, preventive interventions also need to be targeted early. This could be an explanation for the lack of any clinical benefit in the only large-scale intervention study that was designed to assess the preventive effects of fish oil supplementation in children at high risk of allergic disease.28 Although the current study was too small to detect any clinical benefits of fish oil in allergy prevention, there was a trend for lower allergic sensitization in children whose mothers had fish oil in pregnancy.9 There are currently a number of other larger studies (still ongoing) that will address this more definitively.

In conclusion, we have shown for the first time that neonatal T-cell PKCζ expression is lower in children with allergy, and that this isozyme is also significantly enhanced by increased membrane ω-3 LCPUFA content. This not only provides further evidence that environmental interventions can significantly modify neonatal T-cell function but also suggests that PKCζ should be explore further as a marker of disease risk. As disease prevention strategies become more refined, risk markers will become of increasing importance to target interventions and to assess their effects. The findings open new horizons in developmental immunology in which levels of intracellular signaling molecules may be amenable for use as markers of T-cell development and variations in these characteristics may be relevant to immune phenotype and disease development.

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We acknowledge the staff and volunteers who assisted in this study. We thank Elaine Pascoe for her assistance and statistical advice and Jasmine Hale, Paul Noakes, Liza Breckler, Heidi Lehmann, and Angie Taylor for assistance in the follow-up clinics.

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 Supported by the National Health and Medical Research Council of Australia. J.A.D. is supported by the Child Health Research Foundation of Western Australia.Disclosure of potential conflict of interest: The authors have received grant support from the National Health and Medical Research Council of Australia.

PII: S0091-6749(07)00644-6

doi:10.1016/j.jaci.2007.03.045

The Journal of Allergy and Clinical Immunology
Volume 120, Issue 1 , Pages 200-206, July 2007

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