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1.  A Multi-Center Ring Trial of Allergen Analysis using Fluorescent Multiplex Array Technology 
Journal of immunological methods  2012;387(0):89-95.
Consistent performance of allergen assays is essential to ensure reproducibility of exposure assessments for investigations of asthma and occupational allergic disease. This study evaluated intra- and inter-laboratory reproducibility of a fluorescent multiplex array, which simultaneously measures eight indoor allergens in a single reaction well.
A multi-center study was performed in nine laboratories in the US and Europe to determine the inter-laboratory variability of an 8-plex array for dust mite, cat, dog, rat, mouse and cockroach allergens. Aliquots of 151 dust extract samples were sent to participating centers and analyzed by each laboratory on three separate occasions. Agreement within and between laboratories was calculated by the concordance correlation coefficient (CCC).
Results were obtained for over 32,000 individual allergen measurements. Levels covered a wide range for all allergens from below the lower limit of detection (LLOD=0.1 - 9.8ng/ml) to higher than 6800ng/ml for all allergens except Mus m 1, which was up to 1700ng/ml. Results were reproducible within as well as between laboratories. Within laboratories, 94% of CCC were ≥0.90, and 80% of intra-laboratory results fell within a 10% coefficient of variance (CV%). Results between laboratories also showed highly significant positive correlations for all allergens (∼0.95, p<0.001). Overall means of results were comparable, and inter-laboratory CV% for all allergens except Rat n 1 ranged between 17.6% and 26.6%.
The data indicate that performance criteria for fluorescent multiplex array technology are reproducible within and between laboratories. Multiplex technology provides standardized and consistent allergen measurements that will streamline environmental exposure assessments in allergic disease.
PMCID: PMC3955085  PMID: 23085532
Allergen measurement; asthma; indoor air quality; immunoassay; multiplex array; occupational health
2.  Biomarkers of Eosinophil Involvement in Allergic and Eosinophilic Diseases: review of phenotypic and serum markers including a novel assay to quantify levels of soluble Siglec-8 
Journal of immunological methods  2012;383(1-2):39-46.
There remains considerable controversy in the management of eosinophilic disorders, mainly due to a paucity of information regarding the clinical interpretation of total blood eosinophil counts versus surface activation markers versus eosinophil-derived or eosinophil-influencing mediator levels. Regrettably, few tests have been validated that define a unique clinical or prognostic phenotype that is more useful than simply monitoring total blood eosinophil counts. In this manuscript, phenotypic (cell surface) markers, along with serum and tissue-based markers that have been examined in the context of disease activity, are reviewed. We also report the development of a novel assay for detecting soluble Siglec-8 (sSiglec-8), a protein likely derived largely from eosinophils, as a potential serum biomarker. The assay consists of a competitive ELISA using a recombinant Siglec-8-Fc fusion protein. The goal of this preliminary study was to determine if sSiglec-8 is a useful biomarker that differentiates among patients with various eosinophil-associated diseases. In the final analysis, it is fair to say that further research is sorely needed to fully understand and validate the utility of various biomarkers, including sSiglec-8, before their use in clinical practice can be recommended with confidence.
PMCID: PMC3411856  PMID: 22683541
Eosinophil; granule proteins; ELISA; soluble Siglec-8
3.  A randomized, double-blind, placebo-controlled study of milk oral immunotherapy for cow’s milk allergy 
Orally administered, food-specific immunotherapy appears effective in desensitizing and potentially permanently tolerizing allergic individuals.
We sought to determine whether milk oral immunotherapy (OIT) is safe and efficacious in desensitizing children with cow’s milk allergy.
Twenty children were randomized to milk or placebo OIT (2:1 ratio). Dosing included 3 phases: the build-up day (initial dose, 0.4 mg of milk protein; final dose, 50 mg), daily doses with 8 weekly in-office dose increases to a maximum of 500 mg, and continued daily maintenance doses for 3 to 4 months. Double-blind, placebo-controlled food challenges; end-point titration skin prick tests; and milk protein serologic studies were performed before and after OIT.
Nineteen patients, 6 to 17 years of age, completed treatment: 12 in the active group and 7 in the placebo group. One dropped out because of persistent eczema during dose escalation. Baseline median milk IgE levels in the active (n = 13) versus placebo (n = 7) groups were 34.8 kUa/L (range, 4.86–314 kUa/L) versus 14.6 kUa/L (range, 0.93–133.4 kUa/L). The median milk threshold dose in both groups was 40 mg at the baseline challenge. After OIT, the median cumulative dose inducing a reaction in the active treatment group was 5140 mg (range 2540-8140 mg), whereas all patients in the placebo group reacted at 40 mg (P = .0003). Among 2437 active OIT doses versus 1193 placebo doses, there were 1107 (45.4%) versus 134 (11.2%) total reactions, with local symptoms being most common. Milk-specific IgE levels did not change significantly in either group. Milk IgG levels increased significantly in the active treatment group, with a predominant milk IgG4 level increase.
Milk OIT appears to be efficacious in the treatment of cow’s milk allergy. The side-effect profile appears acceptable but requires further study.
PMCID: PMC3764488  PMID: 18951617
Cow’s milk; food allergy; IgE; prognosis; desensitization; tolerance; oral immunotherapy
4.  Allergic agonists in apheresis platelet products are associated with allergic transfusion reactions 
Transfusion  2011;52(3):575-581.
The mechanisms that underlie allergic transfusion reactions (ATRs) are not well characterized, but likely involve recipient, donor, and product factors. To assess product factors associated with ATRs, we investigated candidate mediators in apheresis platelet products associated with ATRs and controls.
Using bead-based and standard ELISA immunoassays, we tested supernatants from 20 consecutive apheresis platelet transfusions associated with ATRs and 30 control products for concentrations of mediators in 3 categories: acute inflammatory mediators, direct agonists of basophils and mast cells, and growth/priming factors of basophils and mast cells.
Median concentrations of the direct allergic agonists C5a, brain derived neurotrophic factor (BDNF), and CCL5 (RANTES) were 16.6%, 41.8%, and 13.9% higher, respectively, in the supernatant of apheresis platelet products that were most strongly associated with ATRs (P < 0.05 for each mediator). Other direct agonists (MIP-1α, MCP-1, eotaxin-1, IL-8) were similar between groups. Concentrations of acute inflammatory mediators and basophil growth/priming factors were also similar between groups (P > 0.2 for all associations).
The allergic agonists C5a, BDNF, and CCL5 may be mediators of ATRs in apheresis platelet products. Acute inflammatory proteins and basophil/mast cell growth and priming factors do not appear to be associated with apheresis platelet products that cause ATRs.
PMCID: PMC3711211  PMID: 21883267
allergy; transfusion reaction; IgE; platelet
5.  Atopic predisposition of recipients in allergic transfusion reactions to apheresis platelets 
Transfusion  2011;51(11):2337-2342.
The biologic mechanisms of allergic transfusion reactions (ATRs) are largely unknown. We sought to compare the atopic predisposition of platelet recipients who experienced an ATR to non-reactive control recipients.
We identified 37 consecutive apheresis platelet recipients who experienced an ATR and 26 matched controls. Total IgE and aero- and food-allergen-specific IgE were quantified in plasma by ImmunoCAP (Phadia, Phadiatop and Fx5). IgE testing of apheresis platelet supernatants was also performed.
Pruritus and urticaria were manifest in 91.9% and 83.8% of all ATRs, with more severe respiratory symptoms and angioedema occurring in <15% of cases. No subject had anaphylaxis. Sex, age, and primary diagnosis were balanced between the two groups. Total and aero-allergen specific IgE was higher among subjects experiencing an ATR in comparison to control subjects (median total IgE 55.5 kU/L vs. 8.3 kU/L, P=0.002; and median aero-allergen specific IgE 0.57 kUa/L vs. 0.36 kUa/L, P=0.046). IgE antibody levels in apheresis products associated with ATRs were similar to control products (P>0.1 for all IgE tests).
Recipient atopic predisposition, as defined by IgE sensitization, is a risk factor associated with ATRs.
PMCID: PMC3711221  PMID: 21569041
allergy; transfusion reaction; IgE; platelet
6.  Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin 
BioMed Research International  2013;2013:756789.
Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing.
PMCID: PMC3708386  PMID: 23878817
7.  Role of exhaled nitric oxide as a predictor of atopy 
Respiratory Research  2013;14(1):48.
The fractional exhaled nitric oxide (FeNO) is a quantitative, noninvasive and safe measure of airways inflammation that may complement the assessment of asthma. Elevations of FeNO have recently been found to correlate with allergic sensitization. Therefore, FeNO may be a useful predictor of atopy in the general population. We sought to determine the diagnostic accuracy of FeNO in predicting atopy in a population-based study.
We conducted a cross-sectional study in an age- and sex- stratified random sample of 13 to 15 year-olds in two communities in Peru. We asked participants about asthma symptoms, environmental exposures and sociodemographics, and underwent spirometry, assessment of FeNO and an allergy skin test. We used multivariable logistic regression to model the odds of atopy as a function of FeNO, and calculated area-under-the-curves (AUC) to determine the diagnostic accuracy of FeNO as a predictor of atopy.
Of 1441 recruited participants, 1119 (83%) completed all evaluations. Mean FeNO was 17.6 ppb (SD=0.6) in atopics and 11.6 ppb (SD=0.8) in non-atopics (p<0.001). In multivariable analyses, a FeNO>20 ppb was associated with an increase in the odds of atopy in non-asthmatics (OR=5.3, 95% CI 3.3 to 8.5) and asthmatics (OR=16.2, 95% CI 3.4 to 77.5). A FeNO>20 ppb was the best predictor for atopy with an AUC of 68% (95% CI 64% to 69%). Stratified by asthma, the AUC was 65% (95% CI 61% to 69%) in non-asthmatics and 82% (95% CI 71% to 91%) in asthmatics.
FeNO had limited accuracy to identify atopy among the general population; however, it may be a useful indicator of atopic phenotype among asthmatics.
PMCID: PMC3654880  PMID: 23639047
Allergic sensitization; Asthma; Exhaled nitric; Allergic rhinitis
8.  Bovine and Porcine Gelatin Sensitivity in Milk and Meat-Sensitized Children 
Capsule Summary
Cross-reactive bovine and porcine gelatin-specific IgE antibody is more common in cow’s milk and beef or pork meat-sensitized individuals than previously known and a potential risk factor for allergic reactions to gelatin-containing products (foods, vaccines).
PMCID: PMC2784137  PMID: 19665767
gelatin; porcine; bovine; meat; cow’s milk; IgE antibody; prevalence; cross-reactivity
9.  Pollen-specific immunoglobulin E positivity is associated with worsening of depression scores in bipolar disorder patients during high pollen season 
Bipolar Disorders  2012;14(1):90-98.
An association between allergic disease and depression has been consistently reported, but whether the key mediating ingredients are predominantly biological, psychological, or mere artifacts remains unknown. In the current study, we examine the hypothesized relationship between allergen-specific immunoglobulin E (IgE) status and changes in allergy symptoms with worsening in depression scores in depressed sensitized individuals.
In patients with recurrent mood disorders, we individually coupled sensitization to specific seasonal aeroallergens (as assessed by allergen-specific IgE) with temporal windows of exposure to aeroallergens (low versus high tree or ragweed pollen counts measured according to the National Allergy Bureau guidelines). We compared Structured Interview Guide for the Hamilton Depression Rating Scale–Seasonal Affective Disorder Version (SIGH-SAD) depression score changes in 41 patients with mood disorders [25 with major depression and 16 with bipolar I disorder, diagnosed by Structured Clinical Interview for DSM (SCID)] seropositive for tree or ragweed pollen-specific IgE antibody versus 53 patients with mood disorders (30 with major depression and 23 with bipolar I disorder) seronegative for aeroallergen-specific IgE.
Worsening in total depressive scores from low to high pollen exposure was greater in allergen-specific IgE positive patients as compared to allergen-specific IgE antibody negative patients (p = 0.01). When stratified by polarity, the association was significant only in patients with bipolar I disorder (p = 0.004). This relationship was resilient to adjustment for changes in allergy symptom scores.
To our knowledge, this is the first report of coupling a molecular marker of vulnerability (allergen-specific IgE) with a specific an environmental trigger (airborne allergens) leading to exacerbation of depression in patients with bipolar I disorder.
PMCID: PMC3293700  PMID: 22329476
allergen; allergen-specific IgE antibody; allergy; bipolar disorder; depression; ragweed pollen; tree pollen
10.  The safety and efficacy of sublingual and oral immunotherapy for milk allergy 
Oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) are potential therapies for food allergy, but the optimal method of administration, mechanism of action, and duration of response remain unknown.
We sought to explore the safety and efficacy of OIT and SLIT for the treatment of cow’s milk (CM) allergy.
We randomized children with CM allergy to SLIT alone or SLIT followed by OIT. After screening double-blind, placebo-controlled food challenges and initial SLIT escalation, subjects either continued SLIT escalation to 7 mg daily or began OIT to either 1000 mg (the OITB group) or 2000 mg (the OITA group) of milk protein. They were challenged with 8 g of milk protein after 12 and 60 weeks of maintenance. If they passed the 60-week challenge, therapy was withdrawn, with challenges repeated 1 and 6 weeks later. Mechanistic correlates included end point titration skin prick testing and measurement of CM-specific IgE and IgG4 levels, basophil histamine release, constitutive CD63 expression, CD203c expression, and intracellular spleen tyrosine kinase levels.
Thirty subjects with CM allergy aged 6 to 17 years were enrolled. After therapy, 1 of 10 subjects in the SLIT group, 6 of 10 subjects in the SLIT/OITB group, and 8 of 10 subjects in the OITA group passed the 8-g challenge (P = .002, SLIT vs OIT). After avoidance, 6 of 15 subjects (3 of 6 subjects in the OITB group and 3 of 8 subjects in the OITA group) regained reactivity, 2 after only 1 week. Although the overall reaction rate was similar, systemic reactions were more common during OIT than during SLIT. By the end of therapy, titrated CM skin prick test results and CD63 and CD203c expression decreased and CM-specific IgG4 levels increased in all groups, whereas CM-specific IgE and spontaneous histamine release values decreased in only the OIT group.
OIT was more efficacious for desensitization to CM than SLIT alone but was accompanied by more systemic side effects. Clinical desensitization was lost in some cases within 1 week off therapy.
PMCID: PMC3437605  PMID: 22130425
Food allergy; immunotherapy; milk allergy; basophil; spontaneous histamine release
11.  Asthma outcomes: Biomarkers 
Measurement of biomarkers has been incorporated within clinical research studies of asthma to characterize the population and associate the disease with environmental and therapeutic effects.
National Institutes of Health institutes and federal agencies convened an expert group to propose which biomarkers should be assessed as standardized asthma outcomes in future clinical research studies.
We conducted a comprehensive search of the literature to identify studies that developed and/or tested asthma biomarkers. We identified biomarkers relevant to the underlying disease process progression and response to treatment. We classified the biomarkers as either core (required in future studies), supplemental (used according to study aims and standardized), or emerging (requiring validation and standardization). This work was discussed at an National Institutes of Health–organized workshop convened in March 2010 and finalized in September 2011.
Ten measures were identified; only 1, multiallergen screening to define atopy, is recommended as a core asthma outcome. Complete blood counts to measure total eosinophils, fractional exhaled nitric oxide (Feno), sputum eosinophils, urinary leukotrienes, and total and allergen-specific IgE are recommended as supplemental measures. Measurement of sputum polymorphonuclear leukocytes and other analytes, cortisol measures, airway imaging, breath markers, and system-wide studies (eg, genomics, proteomics) are considered as emerging outcome measures.
The working group participants propose the use of multiallergen screening in all asthma clinical trials to characterize study populations with respect to atopic status. Blood, sputum, and urine specimens should be stored in biobanks, and standard procedures should be developed to harmonize sample collection for clinical trial biorepositories.
PMCID: PMC3390196  PMID: 22386512
Multiallergen screen; fractional exhaled nitric oxide; sputum eosinophils; total eosinophils; IgE; urinary leukotriene E4
12.  Elevation of IgE in children with sickle cell disease is associated with doctor diagnosis of asthma and increased morbidity 
A doctor diagnosis of asthma is associated with increased morbidity (pain and acute chest syndrome, ACS) among children with sickle cell disease (SCD). An association between IgE levels and asthma and morbidity has not been investigated in children with SCD.
We tested the hypothesis that elevated total and allergen-specific IgE levels are associated with asthma and SCD morbidity in children with SCD.
A cross-sectional study of children with SCD who participated in the Silent Cerebral Infarct Trial was conducted. Logistic regression and negative binomial regression were used to investigate potential associations of total and allergen-specific IgE levels with asthma diagnosis and SCD morbidity, both confirmed by medical record review. Elevation of total IgE was defined as age- and sex-adjusted IgE exceeding 90th percentile compared to a non-atopic reference population. IgE antibody positivity to Altermaria alternata (mold), Blatella germanica (cockroach), and Dermatophagoides pteronyssinus (dust mite) was assessed by ImmunoCAP analysis.
Children with SCD (140 asthmatics, 381 non-asthmatics) were evaluated. Elevations in total IgE (p = 0.04) and IgE antibody specific for Altermaria alternata (p = 0.0003), Blatella germanica (p = 0.008), and Dermatophagoides pteronyssinus (p = 0.01) were associated with asthma. ACS (p = 0.048) but not pain (p = 0.20) was associated with total IgE, but neither were associated with specific IgE levels.
Significantly increased levels of total and allergen-specific IgE levels are associated with asthma in SCD. High IgE levels are a risk factor for ACS and not pain rates.
PMCID: PMC3105194  PMID: 21388662
Total IgE; allergen-specific IgE; asthma risk indicator; acute chest syndrome; pain; sickle cell disease; hemoglobinopathies
13.  Phase 1 results of safety and tolerability in a rush oral immunotherapy protocol to multiple foods using Omalizumab 
Up to 30% of patients with food allergies have clinical reactivity to more than one food allergen. Although there is currently no cure, oral immunotherapy (OIT) is under investigation. Pilot data have shown that omalizumab may hasten the ability to tolerate over 4 g of food allergen protein.
To evaluate the safety and dose tolerability of a Phase 1 Single Site OIT protocol using omalizumab to allow for a faster and safe desensitization to multiple foods simultaneously.
Participants with multiple food allergies received OIT for up to 5 allergens simultaneously with omalizumab (rush mOIT). Omalizumab was administered for 8 weeks prior to and 8 weeks following the initiation of a rush mOIT schedule. Home reactions were recorded with diaries.
Twenty-five (25) participants were enrolled in the protocol (median age 7 years). For each included food, participants had failed an initial double-blind placebo-controlled food challenge at a protein dose of 100 mg or less. After pre-treatment with omalizumab, 19 participants tolerated all 6 steps of the initial escalation day (up to 1250 mg of combined food proteins), requiring minimal or no rescue therapy. The remaining 6 were started on their highest tolerated dose as their initial daily home doses. Participants reported 401 reactions per 7,530 home doses (5.3%) with a median of 3.2 reactions per 100 doses. Ninety-four percent (94%) of reactions were mild. There was one severe reaction. Participants reached their maintenance dose of 4,000 mg protein per allergen at a median of 18 weeks.
These phase 1 data demonstrate that rush OIT to multiple foods with 16 weeks of treatment with omalizumab could allow for a fast desensitization in subjects with multiple food allergies. Phase 2 randomized controlled trials are needed to better define safety and efficacy parameters of multi OIT experimental treatments with and without omalizumab.
PMCID: PMC3936817  PMID: 24576338
Food allergy; Oral immunotherapy (OIT); Specific Oral Tolerance Induction (SOTI); Multiple food allergy; Safety; Efficacy; Omalizumab; Desensitization
14.  Management of Patients With Cystic Fibrosis and Allergic Bronchopulmonary Aspergillosis Using Anti-Immunoglobulin E Therapy (Omalizumab) 
Omalizumab is a recombinant DNA-derived humanized immunoglobulin G (IgG) anti-IgE monoclonal antibody approved for use in patients with allergic asthma. However, it is not approved for allergic bronchopulmonary aspergillosis (ABPA). Conflicting reports exist about the effects of omalizumab on ABPA in patients with cystic fibrosis (CF). We report 2 patients with CF treated with omalizumab, in whom frequency of ABPA exacerbations was markedly reduced with treatment. Additionally, hospitalizations were reduced from 5 per year to once in 18 months in the first patient and from twice to once per year in the second patient. Free IgE decreased by 87.9% after 6 months of therapy in the first patient and by 95.6% after 7 months of therapy in the second patient. Neither of the two patients had evidence of asthma. Omalizumab may be useful in treating ABPA in patients with CF, and including free IgE in monitoring the response to therapy will be helpful.
PMCID: PMC3428193  PMID: 23118662
Allergic bronchopulmonary aspergillosis; cystic fibrosis; free IgE; omalizumab
15.  Frailty is associated with impairment of vaccine-induced antibody response and increase in post-vaccination influenza infection in community-dwelling older adults 
Vaccine  2011;29(31):5015-5021.
Annual immunization with a trivalent inactivated vaccine (TIV) is considered efficacious for prevention of seasonal influenza in older adults. However, significant controversy exists in the current literature regarding the clinical effectiveness of TIV immunization in this highly heterogeneous population. Frailty is an important geriatric syndrome characterized by decreased physiologic reserve and increased vulnerability to stressors. Using a validated set of frailty criteria, we conducted a prospective observational study to evaluate TIV-induced strain-specific hemagglutination inhibition (HI) antibody titers and post-vaccination rates of influenza-like illness (ILI) and infection in frail and nonfrail older adults. The results indicate that frailty was associated with significant impairment in TIV-induced strain-specific HI titers and increased rates of ILI and laboratory-confirmed influenza infection. These findings suggest that assessing frailty status in the elderly may identify those who are less likely to respond to TIV immunization and be at higher risk for seasonal influenza and its complications.
PMCID: PMC3129421  PMID: 21565245
Frailty; Influenza immunization; Antibody response; Influenza infection; Influenza-like illness; Older adults
16.  Safety and feasibility of oral immunotherapy to multiple allergens for food allergy 
Thirty percent of children with food allergy are allergic to more than one food. Previous studies on oral immunotherapy (OIT) for food allergy have focused on the administration of a single allergen at the time. This study aimed at evaluating the safety of a modified OIT protocol using multiple foods at one time.
Participants underwent double-blind placebo-controlled food challenges (DBPCFC) up to a cumulative dose of 182 mg of food protein to peanut followed by other nuts, sesame, dairy or egg. Those meeting inclusion criteria for peanut only were started on single-allergen OIT while those with additional allergies had up to 5 foods included in their OIT mix. Reactions during dose escalations and home dosing were recorded in a symptom diary.
Forty participants met inclusion criteria on peanut DBPCFC. Of these, 15 were mono-allergic to peanut and 25 had additional food allergies. Rates of reaction per dose did not differ significantly between the two groups (median of 3.3% and 3.7% in multi and single OIT group, respectively; p = .31). In both groups, most reactions were mild but two severe reactions requiring epinephrine occurred in each group. Dose escalations progressed similarly in both groups although, per protocol design, those on multiple food took longer to reach equivalent doses per food (median +4 mo.; p < .0001).
Preliminary data show oral immunotherapy using multiple food allergens simultaneously to be feasible and relatively safe when performed in a hospital setting with trained personnel. Additional, larger, randomized studies are required to continue to test safety and efficacy of multi-OIT.
Trial registration NCT01490177
PMCID: PMC3913318  PMID: 24428859
Food allergy; Oral immunotherapy (OIT); Specific oral tolerance induction (SOTI); Multiple; Safety; Efficacy
17.  Subcutaneous Allergen Immunotherapy Restores Human Dendritic Cell Innate Immune Function 
We recently reported that human blood dendritic cells from allergic subjects have impaired IFN-α production following TLR9-dependent innate immune stimulation. It is not known how subcutaneous allergen immunotherapy (SCIT) affects dendritic cell immune responses.
The aim of this study is to determine how SCIT affects human dendritic cell function.
PBMC and plasmacytoid dendritic cells (pDCs) were isolated from the blood of 7 dust mite allergic subjects at baseline and upon reaching a standard SCIT maintenance dose that included dust mite and other aeroallergens. Cells were stimulated with various adaptive and innate immune receptor stimuli, or media alone for 20hrs with secreted cytokine levels determined by ELISA. A portion of the cells were used to measure intracellular signaling proteins by flow cytometry. Humoral immune responses were measured from plasma.
SCIT resulted in a 3-fold increase in PBMC production of IFN-α in response to CpG at 100 nM (P = 0.015) and at 500 nM (P = 0.015), n = 7. The predominant cell type known to produce IFN-α in response to CpG (CpG ODN-2216) and other TLR9 agonists is the pDC. As expected, a robust innate immune response from isolated pDCs was re-established among allergic subjects undergoing SCIT resulting in a 5-fold increase in IFN-α production in response to CpG at 500 nM (P = 0.046), n = 7. In contrast, IL-6 production was unaffected by SCIT (P = 0.468). Consistent with published reports, IgG4 blocking antibody increased 10-fold with SCIT (P = 0.031), n = 7. There was no significant increase in the frequency of pDCs or the expression of TLR9 that would account for the rise in IFN-α production.
Allergen immunotherapy increases dendritic cell TLR9 mediated innate immune function, which has previously been shown to be impaired at baseline in allergic subjects.
PMCID: PMC2837539  PMID: 20205698
allergy; immunotherapy mechanisms; SCIT; dendritic cell; pDC; IFN-α; TLR9; cytokines
18.  Effect of in vitro aspirin stimulation on basophils in patients with aspirin-exacerbated respiratory disease 
Basophil activation has been implicated in the pathogenesis of aspirin exacerbated respiratory disease. However, a comprehensive analysis of basophil responses to aspirin in terms of mediator release, cytokine secretion and increased expression of surface activation markers has not been performed.
To study the in vitro effects of aspirin on the concurrent release of histamine, leukotriene C4 and IL-4 from human basophils and to also evaluate changes in surface activation markers (CD63, CD69 and CD203c) expressed by these cells.
Basophil-enriched cell suspensions from 10 patients with aspirin exacerbated respiratory disease and 10 healthy volunteers were incubated with lysine-aspirin for up to 3 hours. Cells were analysed for expression of CD63, CD69 and CD203c using flow cytometry. Cell-free supernatants were evaluated for histamine, and leukotriene C4 release and for IL-4 secretion.
Aspirin-induced expression of CD63, CD69 and CD203c yielded 30%, 80% and 70% sensitivity, respectively, but with poor specificity. There was no significant difference in leukotriene C4 synthesis between groups. None of the patients with aspirin exacerbated respiratory disease (or controls) released IL-4 in response to aspirin. A higher dose of 5 mg/ml aspirin mediated non-specific effects on basophils.
Basophil responses to in vitro aspirin challenge are poor indicators of clinical sensitivity. Aspirin activates some basophils by means of mechanisms which differ from the classical IgE mediated pathway. Our study also shows that the use of 27 mM of aspirin (5 mg/ml) used by previous investigators causes nonspecific basophil activation, thereby eliminating its usefulness in a cell based diagnostic test for AERD. Evaluation of in vitro basophil activation has low clinical value in identifying aspirin- induced respiratory reactions
PMCID: PMC2788679  PMID: 19486029
Aspirin; asthma; aspirin exacerbated respiratory disease; basophil; CD63; CD69; CD203c; histamine; leukotriene C4; flow cytometry
19.  Open Label Maintenance Following Milk Oral Immunotherapy (MOIT) for IgE Mediated Cow's Milk Allergy 
Capsule Summary
MOIT can be effective in desensitizing children with severe IgE-mediated CMA, with most tolerating markedly increased amounts of cow’s milk protein over time and demonstrating changes in serologic markers.
PMCID: PMC2739248  PMID: 19665770
Cow’s milk; food allergy; IgE; prognosis; desensitization; tolerance; oral immunotherapy
20.  IVIg contains a broad repertoire of anti-carbohydrate antibodies that is not restricted to the IgG2 subclass 
Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects.
To examine commercial intravenous immunoglobulin (IVIg) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed.
We employed a glycan microarray to evaluate IVIg preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans courtesy of the Consortium for Functional Glycomics Core H ( Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIg using immunoaffinity chromatography and depletion confirmed using nephelometry and surface plasmon resonance.
Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (e.g., Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and non-pathogenic bacteria, including lipopolysaccharide and lipoteichoic acid, capsular polysaccharides and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anti-carbohydrate recognition patterns compared to the starting IVIg preparation. Little to no binding activity was detected to human endogenous glycans including tumor-associated antigens.
This novel, comprehensive analysis provides evidence that IVIg contains a much wider range than previously appreciated of anti-carbohydrate IgG antibodies, including those recognizing both pathogenic and non-pathogen-associated prokaryotic glycans.
PMCID: PMC2777748  PMID: 19443021
IgG subclasses; gamma globulin; anti-carbohydrate antibodies; glycans; IgG2; glycan microarray
21.  Anti-IgE Treatment of Eosinophil Associated Gastrointestinal Disorders 
Eosinophil Associated Gastrointestinal Disorders (EGIDs) are commonly associated with atopy and are being recognized with increasing frequency. Current therapy for EGIDs is inadequate.
We sought to determine the efficacy of anti-IgE therapy in EGIDs and investigate the role of IgE in disease pathogenesis.
Nine subjects with EGIDs received omalizumab every 2 weeks for 16 weeks while other therapy was held constant. Blood absolute eosinophil counts, tissue eosinophil counts, symptom scores, and free IgE were serially measured. Allergen skin testing, and flow cytometry for basophil activation and FcεRI were determined at baseline and at week 16.
Omalizumab was associated with a decrease in absolute eosinophil count at both the 16 week (34%, p=0.004) and combined weeks 12–16 (42%, p=0.012) time points. Tissue eosinophils decreased in the duodenum (59%) and gastric antrum (69%), but did not reach statistical significance (p=0.074 and 0.098, respectively). Esophageal eosinophil counts remained unchanged. Basophil and dendritic cell FcεRI expression, and free IgE were all significantly decreased (p<0.005). Omalizumab increased the concentration of allergen required to trigger half-maximal basophil activation by 170-fold. Allergen skin test wheal and erythema responses decreased by 78% and 82%, respectively. Symptom scores were decreased at both the midstudy (63%) and end of study (70%) time points (p<0.005 for both).
These results demonstrate that IgE-mediated processes contribute to the generation of eosinophilic inflammation in EGIDs, and suggest that anti-IgE therapy may be effective in these disorders.
Clinical implications
Anti-IgE may be a potential therapy for EGIDs.
PMCID: PMC2768344  PMID: 17765756
Eosinophil; eosinophilic gastroenteritis; eosinophilic esophagitis; omalizumab; IgE; food allergy; basophil
22.  Diagnostic evaluation of food-related allergic diseases 
Food allergy is a serious and potentially life-threatening problem for an estimated 6% of children and 3.7% of adults. This review examines the diagnostic process that begins with a patient's history and physical examination. If the suspicion of IgE-mediated food allergy is compelling based on the history, skin and serology tests are routinely performed to provide confirmation for the presence of food-specific IgE antibody. In selected cases, a provocation challenge may be required as a definitive or gold standard reference test for confirmation of IgE mediated reactions to food. Variables that influence the accuracy of each of the diagnostic algorithm phases are discussed. The clinical significance of food allergen-specific IgE antibody cross-reactivity and IgE antibody epitope mapping of food allergens is overviewed. The advantages and limitations of the various diagnostic procedures are examined with an emphasis on future trends in technology and reagents.
PMCID: PMC2776233  PMID: 19946406
23.  Human blood dendritic cells from allergic subjects have impaired capacity to produce interferon-α via TLR9 
High affinity IgE receptor (FcεRI) expression on blood dendritic cells reportedly correlates with serum IgE levels. Our studies demonstrate that plasmacytoid dendritic cells (pDCs) secrete pro-inflammatory cytokines (IL-6, TNF-α) following FcεRI stimulation – a mode of activation that simultaneously reduces expression of Toll-like receptor 9 (TLR9). Whether or not TLR9 and/or FcεRI levels and their function on dendritic cells relate to allergic status is unknown.
The aim of this study is to compare the innate (TLR9-mediated) immune response of human plasmacytoid dendritic cells to TLR9 and FcεRIα receptor expression in allergic and nonallergic subjects.
Basophil depleted mononuclear cell fractions containing pDCs were prepared from peripheral blood of allergic and non-allergic subjects. Intracellular TLR9 and surface FcεRIα expression in BDCA-2 positive cells were determined by flow cytometry. Activating anti-IgE antibody, anti-FcεRIα antibody, and TLR9 agonist were used to stimulate cell suspensions, with cytokine levels determined by ELISA.
No difference in the frequency of pDCs was detected among allergic (n = 9) versus nonallergic (n = 11) subjects (P = 0.261). While there was also no difference in baseline expression of TLR9, pDCs from allergic subjects produced 6-fold less IFN-α when stimulated with CpG (P = 0.002). Conversely, there was higher FcεRIα expression (P = 0.01) on the pDCs of allergic subjects.
Impaired TLR9-dependent immune responses in human plasmacytoid dendritic cells are associated with allergic status and inversely correlate with FcεRIα expression. This impaired innate immune response among dendritic cells of allergic subjects may lead to more targeted therapeutic approaches and could provide a better understanding of the mechanisms underlying conventional and CpG-based immunotherapy.
PMCID: PMC2707903  PMID: 18318750
allergy; dendritic cells; IFN-α; plasmacytoid; TLR9; CpG-DNA; cytokines
24.  Allergen Specific IgE, Number and Timing of Past Suicide Attempts, and Instability in Patients with Recurrent Mood Disorders 
Suicide and decompensation of mental illness peak in spring and, to a lesser extent, in fall. Several recent studies reported that suicide and decompensation peaks coincided with spring and fall aeroallergen peaks. Allergic symptoms occur as the result of a complex biochemical cascade initiated by IgE antibodies (sensitization) and allergens (triggers). Animal models have shown molecular/neurochemical changes in the brain, as well as relevant behavioral changes associated with this IgE-mediated biochemical cascade. These factors suggest that seasonal allergy could precipitate suicidality and mood instability. In the current study, we compared the prior suicide attempt and history of decompensation of mood disorders in allergen sensitive vs nonsensitive patients. Further, we compared the ratio of events (attempts and decompensations) during the allergy season to events occurring during the rest of the year. Patients with Major Depressive Disorder or Bipolar I or II Disorder (n=80) were studied. There were no statistical differences in any measurement performed between the allergen sensitive and nonsensitive patients. These negative results are not consistent with recent epidemiological studies supporting a predictive association between allergy and categorical measures of suicidality (ideation, attempts, and completion). Clinical samples are likely not adequate to study less than strong predictive associations with suicide and suicide risk factors.
PMCID: PMC2586113  PMID: 19043595
Environment; allergy; mood disorders; suicide; psychiatry
25.  Human Metapneumovirus Infection Plays an Etiologic Role in Acute Asthma Exacerbations Requiring Hospitalization in Adults 
The Journal of infectious diseases  2005;192(7):1149-1153.
We determined the prevalence of human metapneumovirus (hMPV) infection in adults with asthma who were prospectively enrolled after hospitalization for an acute asthma exacerbation. Nasal wash specimens collected at admission and 3 months after discharge were tested for hMPV by real-time reverse-transcription polymerase chain reaction assays. hMPV was detected in 7 (6.9%) of 101 subjects at hospitalization and in 1 (1.3%) of 75 subjects at follow-up (odds ratio, 7 [95% confidence interval, 0.9–312]; P =.03). None of the patients with hMPV infection at hospitalization tested positive at follow-up, strongly suggesting that hMPV plays a direct etiologic role in acute asthma exacerbations.
PMCID: PMC1476781  PMID: 16136455

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