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Friday, July 30, 2010

Nudging forward a new paradigm for food allergies

Characterization of food allergies has produced abundant data on their clinical presentation, but the biological construct driving the pathology is a ghost in the machine. In this month’s issue, Vassallo and Camargo make an intrepid, but logical, gesture at a syncretic hypothesis of food allergy evolution.

The authors start with the epidemiologic observation that societal decreases in exposure to sunshine have led to a rise in vitamin D deficiency (VDD) and that these two changes are coeval with the increase in food allergies. They offer a hypothesis linking evidence of VDD to atopic progression, i.e., eczema, respiratory compromise, and food allergy. Specifically, they propose that VDD results in increased infection susceptibility and altered microbiota in the gut, which in turn, cause higher levels of mucosal barrier damage, permitting excessive exposure to food allergens through the “leaky gut.”

Vassallo and Camargo mention known associations between VDD, childhood obesity, and food allergy. Physiologic support for their hypothesis comes from recent evidence that vitamin D is critical to induction of tolerance, as well as antimicrobial peptide (AMP) production in the epithelium, and suppression of inflammatory responses.

It is the AMP factor that the authors use to tie VDD to the effector mechanism. In addition to increased susceptibility to infection, VDD causes dysregulation of AMPs in the intestine, supporting an abnormal intestinal flora. They also point to recent data that suggest VDD has a primary role in mucosal barrier compromise.

Wrapping up, Vassallo and Camargo note several incongruities, including that VDD prevalence is higher than food allergy prevalence and genetic atopic predispositions may act independently to increase food allergy risk, from which they suggest that multiple “hits” are required to result in food allergy. They urge future, cross-disciplinary research designed to examine their hypothesis that correction of VDD during pregnancy and early childhood will lead to improved tolerance, mucosal immunity, and balanced intestinal flora.

We asked the authors whether their hypothesis could be applied to adults as well as children:
JACI: You make the distinction between failure to develop tolerance as seen in childhood and loss of tolerance associated with adult onset of food allergy. Is there reason to believe that vitamin D supplementation might mitigate loss of tolerance as well?

Carlos Camargo: We have chosen to focus on children because that’s when most food allergy begins -- and where we have found supportive epidemiologic data (eg, our recent publication on season of birth and food allergy -- citation below). In brief, we found increased risk of food allergy among children born in fall/winter, as compared to spring/summer. We found no association between season of birth and food allergy in older patients. (Vassallo MF, Banerji A, Rudders SA, Clark S, Mullins RJ, Camargo CA Jr. Season of birth and food allergy in children. Ann Allergy Asthma Immunol 2010; 104: 307-313.)

While it’s theoretically possible that vitamin D supplementation might mitigate loss of tolerance in adults, we will continue to focus our research efforts on childhood food allergy.

Do you have any questions for the authors, or comments about this study? We want to hear from you. Please feel free to post your own questions or comments. All questions and comments will be forwarded to the authors for a response.

Thursday, July 1, 2010

Guest post: A primer on human IgE antibody serology

Dear Readers:

In response to the concerns about an increase in the remote practice of allergy, which may possibly be influenced by direct allergy laboratory marketing campaigns, the AAAAI and ACAAI, in conjunction with Don Aaronson from the JCAAI formed a joint taskforce in 2006 on allergy diagnostic testing called the Specific IgE Test Task Force (SETTaF) . SETTaF’s purpose has been to develop educational materials that focus on the appropriate diagnostic evaluation of the allergic/suspected patient. In late 2008 SETTaF published a monograph directed at health care professionals, who care for allergic patients entitled the “Pearls and pitfalls of allergy diagnostic testing: report from the American College of Allergy, Asthma and Immunology/American Academy of Allergy, Asthma and Immunology Specific IgE Test Task Force.”(1) The task force is currently engaged in the development of a PowerPoint presentation and several monographs on diagnostic allergy testing that are directed at primary care professionals.

The key message throughout these educational materials is that the allergy evaluation must be initiated by and culminated with the patient's clinical history. It must be directed by a health care professional with sufficient understanding of diagnostic allergy testing to use the information obtained from his/her evaluation of the patient to determine: 1. What diagnostic allergy tests to order. 2. How to interpret the diagnostic allergy test results. 3. How to use the information obtained from the allergy evaluation to develop an appropriate therapeutic treatment plan.

This issue of the Journal of Allergy and Clinical Immunology presents a monograph developed by SETTaF on serological measurement of allergen-specific IgE testing specifically for the North American allergist. Per the protocol of a joint AAAAI/ACAAI task force, the SETTaF’s monographs are reviewed by the Board of Directors/Regents of both organizations. During this review, a question and answer dialogue evolved and the organizations' Boards suggested including the ‘Q&A’ with the document.

The members of SETTaF hope you find the monograph educational. The following questions and answers represent the views of the individuals and not SETTaF or the Board of Directors/Regents of the AAAAI/ACAAI.

Linda Cox, MD
SETTaF chair

Allergy Diagnostic Testing Primer: Questions and Answers

Below are some questions that were raised by ACAAI reviewers that will highlight some of the questions readers may have and an attempt by the authors to provide answers, where they are available.

Issue 1: Use of serum specific-IgE in clinical decision-making

Dana Wallace , MD (ACAAI president-elect): This report on the evolution of serum specific- IgE (s-IgE) testing is a timely and much needed document for the clinical allergist. While technically well written, it brings up perhaps more questions and confusion than it answers or clarifies. While the authors and many other academicians may find it clear and easy to understand, I am not sure that this will be the case for most clinicians. Particularly difficult will be the translation of this information into clinical decision-making support tools. It would be very helpful if an additional section could be added: “When and how to use specific IgE in vitro tests to improve patient care”. I would suggest that in this section, the authors try to answer the following questions (among others):

Linda Cox, MD ( SETTaF chair): I think one has to consider s-IgE similar to skin testing in terms of when and how it would be used in patient care and the decision on which type of test(s) would vary with the patient and other circumstances.

Brock Williams, PhD (Primer author and SETTaF member): The hard question to answer is when and how to use s-IgE testing to improve patient care. To me, this deals mostly with how to interpret the results and this is very patient dependent. Again the problem stems from the fact that allergic symptoms are variable both within and between individuals. This and the large number of variables (age, gender, exposure, infection, etc.) clearly need to be taken into account for the interpretation of s-IgE tests. We do know that s-IgE is closely linked to symptoms but levels and the allergens it is directed against differ among different individuals. Thus, in today’s world, s-IgE results will best be utilized by considering them as a risk factor rather than an absolute diagnostic fact.

Robert Hamilton, PhD: The ultimate decision of when to use a confirmatory IgE antibody test (skin test or serology) comes down to the physician deciding that there is sufficient clinical evidence to warrant a possible diagnosis of allergic disease. This concept is integral to the diagnostic algorithm built into the diagnostic practice parameters. As such, to try to list all the patient and physician decisions to determine when a diagnostic IgE antibody confirmatory test (skin test or serology) should be run is (in my opinion) out of the intended scope of this article. This article summarizes what we know and what we do not know about the assays used to detect allergen-specific IgE antibody. I agree with Brock that prudent use of a positive specific IgE antibody result is as a risk factor for allergic disease and not as a definitive indicator of the presence of allergic disease.

Issue 2: Which is the ‘best’ specific IgE assay?

Dana Wallace, MD: Which of the three methods of specific-IgE measurement described within the Primer is most beneficial for making clinical decisions or should they all be viewed as equal?

Linda Cox, MD: There is agreement that the results are not assay inter-changeable. One thought is the different assays may be measuring different antibodies.

Brock Williams, PhD: Comparability of different methods – most studies indicate that the Phadia ImmunoCAP is far above the other two tests in overall performance (accuracy, precision, and quantitative ability). The problem is that for certain allergens and levels of s-IgE the assays are somewhat in agreement. For others, they are very different. I find the explanation that these assays are sometimes measuring different populations of antibodies to be rather absurd. To begin with I have seen no proof of this concept and our studies with chimeric antibodies (where the total IgE = specific IgE) indicate that for those allergens investigated the Siemens assay (formerly DPC-Alastat/Immulite) has major problems with its calibration and the Hycor assay (Agilent) has problems with under-representation of allergen on some of their solid phases. The major user of the Hycor assay is Labcorp and we know in the past some have suggested that they have modified this test in house to make it more profitable. The ImmunoCAP results (done in triplicate with blinded samples) was ‘spot on’ in these cases.

Robert Hamilton, PhD: I respectfully disagree with Brock on this issue. The most impartial examination of the inter-assay agreement issue indicates that all three methods display excellent precision, reproducibility and linearity. They do differ in the levels of IgE antibody that they detect and we currently do not know the reason for this. It is possibly a calibration issue, however, if there was a systematic bias caused by a calibration issue, we would see a consistent bias which we do not. My belief is that it has more to do with allergen heterogeneity between the methods. Thus to call the suggestion that the assays are measuring different populations of IgE antibody specificities "absurd" is too ‘heavy handed’. Brock, your data with the chimeric antibody are interesting and informative but not conclusive on a calibration issue.

We need to construct a better and more relevant experiment with human sera to try to dissect the answer to this question. Possibly this can be done with milk and peanut where we can identify the component specificities of IgE antibody using the ISAC and the components that are available on the ImmunoCAP. Then sera with defined distributions of IgE antibody can be analyzed by all 3 methods. So the binding of a serum containing 95% IgE anti-casein can be compared to a serum that contains 95% IgE anti-alpha-lactalbumin as a limited illustration of this concept. If we characterize many sera with a sufficient number of these component specificity distributions, we can examine this issue for a restricted number of allergen specificities.

In reality, we will probably not be able to definitively answer this question (at least to my satisfaction). So we are left with the conclusion that is stated in the primer article (which I believe is true) that these 3 assays measure different populations of IgE antibody. The causes are almost irrelevant as the 3 manufacturers are not in a position to change their assay formats. I discussed this issue personally with representative of each company during the last AAAAI meeting. At this point, we cannot say for sure which IgE antibody assay result is more "clinically relevant".

Issue 3: Is one assay too sensitive?

Dana Wallace, MD: Should one consider the Immulite too sensitive as it gives a higher reading of specific-IgE than the Hycor or ImmunoCap? Is there any general guide on comparing the results as the graph seems to show a trend?

Linda Cox, MD: One study did suggest Immulite overestimated s-IgE when the results of total IgE were compared with the hybridoma s-IgE results, which were all s-IgE. Brock and Bob are likely to have different answers

Brock Williams, PhD: The idea that if an assay gives a higher result it is better is fallacious. Again, the chimeric antibody results clearly demonstrate this. Accuracy is more important. Again, the calibration curve (total IgE tied to WHO standard) is very different for the Immulite system. This causes the amount of s-IgE measured to be overestimated particular at the high end with a number of different allergens. A requirement for quantification is that different dilutions (corrected for dilution) give you the same answer. In other words, they are linear and we have only seen this with the ImmunoCAP system.

The idea of sensitivity is also an area of great misunderstanding. This is because analytical sensitivity is a very different concept than clinical sensitivity and the two have often been confused. Analytical sensitivity deals with the low end of the assay, before you run into the background and is determined experimentally with statistical analysis in the laboratory. Clinical sensitivity deals with determining what level of s-IgE is capable of causing clinical symptoms upon exposure and in the near future, what particular allergenic substance it is directed against. Since the determination of clinical sensitivity depends upon the comparison to standards (patient history, puncture skin test, allergen organ challenges, etc.), which themselves have not been standardized and many of the cutoffs are arbitrary. In addition, the studies are dependent on the patient population studied so we really have very poor information in this area. Nevertheless, we mistakenly continue to use this concept to describe how well in a clinical sense these assays work. The same arguments can be used with regard to clinical specificity and, in my opinion, this has been very misleading in this field for quite some time.

Robert Hamilton, PhD: I agree with Brock that higher results produced by any one of the 3 assays are not more clinically relevant. The predictive power of the quantitative level of IgE antibody is discussed in the Primer article, with a caution that these published values need to be used judiciously. . On this issue I agree with what Brock has said about analytical versus clinical sensitivity. Analytically, all three assays can technically detect IgE antibody levels down to 0.1 kUa/L. Historically, the 0.35 kUa/L positive cutpoint was set based on the fact that levels above 0.35 had some clinical significance. We do not know and may never know the clinical significance of levels between 0.1 and 0.35 kUa/L. Interpretation also differs by allergen specificity as some clinicians judge low level Hymenoptera or peanut specific IgE levels differently than a ragweed specific IgE of the same level, because of their clinical relevance in relation to systemic reactions. Thus, a specific IgE level between 0.1 and 0.35 kUa/L needs to be cautiously and judiciously interpreted within the context of the patient’s clinical history.

Issue 4: Can the clinician compare a patient’s s-IgE if they were performed utilizing different assays?

Dana Wallace, MD: If one needs to compare levels of s-IgE and different methods have been used, should the clinician insist that they be repeated so that at least the methods match?

Recognizing that the ImmunoCap has the most published literature in food allergy and predictive levels for a negative food challenge, the clinician has often looked to this as preferred method. Is this still correct?

Linda Cox, MD: Yes. When the laboratory findings are inconsistent with the clinical history, it is recommended to repeat the analysis at the same or a different laboratory. If the results are different, how does the clinician interpret this data? Also recommend repeating with a different method-skin test if sIgE or vice versa. Clinical history is generally the deciding factor. This was a key message in the first SETTaF paper.

I believe the predictive value established in the earlier studies were somewhat specific to a particular population and cannot be universally applied across other age groups/disease.

Robert Hamilton, PhD: We know from the College of American Pathologists Proficiency Survey data that all 3 assay methods from 90% of the laboratories agree well in producing dichotomous measures of IgE antibody (presence/positive vs absence/negative). Therefore, if you are using IgE antibody measurements as a “risk factor” to consider with all the other variables in making the diagnosis, then the Immulite can detect the presence of IgE anti-peanut in a serum as well as an ImmunoCAP. So, if you are asking about the presence of IgE antibody as a risk factor, then “No”, re-analysis by a second assay method is not necessary.

However, there are the occasional spurious results generated by the occasional laboratory or spurious skin test results generated by the occasional clinic. These do occur. So, if the IgE antibody result (skin test or serology) is inconsistent with the clinical history, then it should be repeated, possibly with a different method. We learned this from Dr. Shapiro’s legal case involving Hymenoptera venom sensitivity.

If you are attempting to use the quantitative level to make a prediction about the presence of a clinically evident food allergy or wheeze in an asthmatic child, then published data will dictate what assay needs to be used. In this case the published quantitative predictive data to date are all from the ImmunoCAP. One cannot use the Immulite or Hycor data to make a decision based on published criteria generated with the ImmunoCAP. This will change if new published data are generated with the other two IgE antibody methods.

Issue 5: How important are patient characteristics (age, disease state, etc.) in interpreting s-IgE results?

Dana Wallace, MD: However, the article seems to imply that without knowing the age of the patient, the disease state, the allergen exposure mode and extent, and ratio of Specific IgE/Total IgE that was used in the published research studies and in the individual patient, these published guidelines may be inaccurate and perhaps useless. If this is the correct interpretation, it should be more clearly stated.

Just how important are the lack of affinity and clonality measurements in the overall clinical usefulness of interpreting the results of specific-IgE measurements?

Should we be dividing patient into groups below and above the 4% specific- IgE/Total IgE ratios? Would this help to predict severity of allergic reaction and/or response to treatment?

Linda Cox, MD: I not sure how important sIgE/total IgE. Again Bob and Brock can comment further. I think the importance of affinity and clonality is still be elucidated. This is an area that needs further research but an area of particular interest for Bob.

Brock Williams, PhD: I agree with Linda, that affinity and clonality are parameters that still need to be evaluated. I believe that the realization that certain allergens are much more relevant to symptom production (component resolved diagnosis) is likely to rectify this.

Robert Hamilton, PhD: Four variables contribute to the effectiveness of the IgE antibody response in inducing effector cell function: IgE antibody concentration, affinity, epitope clonality and specific to total IgE ratio or IgE specific activity. This has been clearly shown by the Christensen et al article in the JACI.2 All of these variables are important and constantly changing as the humoral immune response matures with continuing allergen exposure. And one of these alone cannot be shown at present to be an exclusive risk factor for interpreting IgE antibody results in the diagnostic decision process. I agree with Brock that future modifications to our serological assays (e.g., use of components, microarrays) may help us understand this better at the time of interpreting the diagnostic data. The patient demographics will always be critical to the interpretation of the IgE antibody serology as the clinical history is the final arbiter of the definitive diagnosis.

Issue 6: Is the significance of the s-IgE level different when the total IgE is low vs. high?

Dana Wallace, MD: Is it fair to say that when the total IgE level is very high, a high specific-IgE has limited meaning while a high specific-IgE with a low total IgE level is very significant?

Linda Cox, MD: Probably.

Brock Williams, PhD: Specific activity (s-IgE/T-IgE). While this concept makes some intuitive sense, it hasn’t yielded any concrete benefits. This is likely to be important when low levels of t-IgE are seen but really falls apart when multiple allergens and higher levels of s-IgE are seen. While this could be important in immunotherapy, presently it is an area for investigation and not ready for prime time in my opinion. This is particularly true when you investigate monosensitized patients, which is a rare situation for U.S. allergists. Again, I believe we have to further evaluate this concept in lieu of s-IgE to the allergens that actually are relevant (component resolved diagnosis).

Robert Hamilton, PhD: I agree with you all. The IgE specific activity has its most relevance when the total IgE is low and the % specific to total IgE is high. Use of the 4% IgE specific activity as a general criterion can be useful for evaluating patients on Xolair as Johannson et al have shown. However, we need more data to confirm its utility in applying it across the board in the general interpretation of IgE antibody data.

Issue 7: Can the clinician use the s-IgE/t-IgE ratio to predict response to allergen immunotherapy?

Dana Wallace, MD: Is there enough data for the clinician to calculate the specific IgE to total IgE ratio and predict the response to specific immunotherapy? If so what is the magic ratio? If not is there ongoing research that will answer this question? One paper looked at this and 16% was ≥16% was the magic number.3

Robert Hamilton, PhD: No. we need more data to show the utility of this measure in assessing immunotherapy efficacy. It should however be computed to evaluate its utility in future immunotherapy studies.
Brock Williams, Ph.D: I agree with Bob that it is premature to use s-IgE results to guide immunotherapy. This area certainly needs more study and again will probably be better understood when patients and their responses are understood in terms of the specific allergen components that are responsible for symptoms.

Issue 8: Can s-IgE be measured in a patient on Xolair® (omalizumab)?

Dana Wallace, MD: Should the clinician be using ImmunoCAP s-IgE when assessing the patient onXolair? What would be the utility of doing so?Is one looking for a defined endpoint?

Linda Cox, MD: I believe one can get reliable results with ImmunoCAP on s-IgE testing in patients on Xolair, which might be useful in determining if you are adequately dosing (i.e., under) but again Bob is the better person to answer this question

Brock Williams, PhD: Bob has shown quite clearly that the ImmunoCAP can be used with confidence in patients on Xolair.

Robert Hamilton, PhD: The article Brock is referring to clearly shows that IgE (total and allergen specific) can be accurately measured in patients on Xolair with the ImmunoCAP and not with the other two assay methods.4 The important questions are (1) what utility does the measurement of total IgE and specific IgE provide for a patient on Xolair? and (2) where do “free IgE” measurements (IgE not bound with Xolair) fit into the evaluation of the Xolair patient? Examine the Primer article as it addresses these questions.

Issue 9: Will ImmuoCAP rapid®, a non-quantitative allergy test designed for the office setting useful tool for primary care professionals?

Dana Wallace, MD: Will the ImmunoCAP rapid be a good tool for primary care physicians? Should we embrace it?

Linda Cox, MD: The rapid screen office allergy test, which is similar to a pregnancy test is intended for the primary care office. It will likely bring more attention to allergy and to the allergist. Thus, indirectly, I think it is good for the specialty, but there needs to be some education about appropriate use and referral.

Robert Hamilton, PhD: The ImmunoCAP rapid is FDA cleared and we will see how it fits into the overall diagnostic process in the USA. It is too early to tell if it will be useful or abused. The Primer article simply introduces the ImmunoCAP rapid . Allergists need to know that the ImmuoCAP rapid®, data may be introduced by the patient at an allergist’s office visit.
Brock Williams, Ph.D. The ImmunoCap rapid is much like a laboratory test for cholesterol in that it can be interpreted as a measure of risk. The caveats mentioned above in interpreting the results in lieu of the patient’s history most certainly will have to be evaluated on a case by case basis.

1. Cox L, Williams B, Sicherer S, et al. Pearls and pitfalls of allergy diagnostic testing: report from the American College of Allergy, Asthma and Immunology/American Academy of Allergy, Asthma and Immunology Specific IgE Test Task Force. Ann Allergy Asthma Immunol 2008;101:580-92.
2. Christensen LH, Holm J, Lund G, Riise E, Lund K. Several distinct properties of the IgE repertoire determine effector cell degranulation in response to allergen challenge. J Allergy Clin Immunol 2008;122:298-304.
3. Di Lorenzo G, Mansueto P, Pacor ML, et al. Evaluation of serum s-IgE/total IgE ratio in predicting clinical response to allergen-specific immunotherapy. J Allergy Clin Immunol 2009;123:1103-10, 10 e1-4.
4. Hamilton RG. Accuracy of US Food and Drug Administration-cleared IgE antibody assays in the presence of anti-IgE (omalizumab). J Allergy Clin Immunol 2006;117:759-66.

A new genotype associated with abnormal NFκB function in immunodeficiency without ectodermal dyplasia

A scaffolding protein, NEMO, associated with a complex required in the NFκB translocation pathway has been previously associated with ectodermal dysplasia-associated immune deficiency (EDID) syndrome. The syndrome is X-linked, and presents clinically as a history of recurrent infections, hypogammaglobulinemia, and poor memory B cell function. Affected individuals have abnormalities of ectoderm-derived tissues, such as hair, skin, nails, and teeth. NEMO, in complex with inhibitor of NFκB kinase (IKK) proteins, is necessary for NFκB translocation and gene activation.

This month, Mooster et al. describe a male patient and brother with immunodeficiency, but without ectodermal dysplasia, that was traced to a novel gene mutation in the 5’ untranslated region in the NFκB essential modifier gene (NEMO) gene. They report that TLR-activated production of TNFα and IFNα in peripheral blood is significantly decreased in the index patient, consistent with impaired innate immune response.

The authors find that a G→T mutation in the +1 position of the intron 1B causes destruction of the exon 1B to exon 2 splice in the NEMO gene. Two abnormally sized NEMO mRNAs with intact coding regions are produced, causing inefficient translation that results in inadequate levels of functional NEMO proteins. NEMO mRNA is 4-fold lower and NEMO protein expression is 8-fold lower in the index patient as compared to normal controls. The brother of the index patient is also affected by the same mutation. Their report is the first description of an immunodeficiency that results from decreased levels of functional NEMO protein, rather than functionally impaired protein.

The authors comment that the absence of ectodermal involvement suggests that either normal ectodermal development has a less stringent requirement for NEMO than does immune function, or other NEMO isoforms are able to compensate for the reduction in the 1B isoform.

We asked the authors about the implications of their report:
JACI: In your conclusion, you suggest that clinical work-up of patients presenting with immunodeficiency consistent with EDID, but without ectodermal involvement, should be evaluated for NEMO mRNA and proteins levels, followed by coding analysis. What implications does the characterization of these patients have for their treatment?

Jana Mooster and Douglas McDonald: In patients with immunodeficiency consistent with EDID, but without ectodermal dysplasia or even with a normal NEMO coding sequence, one should consider evaluating NEMO protein levels and NFκB function. Patients with NEMO deficiency but without ectodermal dysplasia can appear clinically similar to patients with common variable immune deficiency. NEMO deficiency, however, is known to cause susceptibility to atypical mycobacterial infections. Thus, identification of a NEMO mutation identifies patients that require monitoring for potential atypical mycobacterial infections.

We want to hear from you. Please feel free to post your own questions or comments. All questions and comments will be forwarded to the authors for a response.