Similarities and differences in the effects of sensitisation and challenge with Dermatophagoides farinae and Dermatophagoides pteronyssinus extracts in a murine asthma surrogate.

Patients with atopic asthma may become sensitised to the grain storage mite Dermatophagoides farinae (Der f), the house dust mite Dermatophagoides pteronyssinus (Der p) or both, but thus far little attention has been paid to date to possible variation in their pathophysiological effects. Here we present a side by side comparison of the effects of extracts of these two dust mites in a murine surrogate of atopic asthma. Compared with the Der p-challenged mice, however, the mice-challenged with Der f had favour changes in lung tissue elasticity and expression in matrix metalloproteinases in lung tissue, while the mice challenged with Der p showed more neutrophils infiltrating around the airway and stronger expression of steroid-resistant related cytokines in the lung tissue. Our data suggest that different dust mite crude extracts might lead different pathological characteristics, at least in murine models of asthma.

Imported fire ant immunotherapy prescribing patterns in a large health care system during an 11-year period.

BACKGROUND: The first large-scale evaluation of prescribing patterns for imported fire ant (IFA) in a large US health care system was published by Haymore et al in 2009. In this first evaluation of prescriptions from 1990 to 2007, the most often prescribed maintenance IFA prescription was 0.5 mL of 1:200 wt/vol. OBJECTIVE: To provide an updated description of IFA prescribing patterns over the ensuing 11 years from same large health care system. METHODS: We reviewed 1349 new IFA prescriptions written from 2007 to 2018, from a large nationwide health care system, with primary end points being maintenance prescription strength and prescribing patterns. RESULTS: In comparison to the data published by Haymore et al in 2009, which reported that 17% of the prescriptions were written for 0.5 mL of 1:100 wt/vol maintenance, we found that 69% (95% CI: 66.4%-71.4%) of IFA prescriptions written in the past 11 years were for the maintenance concentration of 0.5 mL of 1:100 wt/vol. We further studied the linear trend over time of percentage of prescriptions written for individual concentrations and observed that the percentage of 1:100 wt/vol prescriptions increased 3.5% yearly (R2 = 0.68, P < .001) from 2007 (40.0%, 95% CI: 24.6%-57.7%) to 2018 (84.4%, 95% CI: 77.4%-89.5%). CONCLUSION: Our study shows significant improvement in the accuracy and precision of IFA immunotherapy dosing for patients with IFA hypersensitivity, with ascendancy of 0.5 mL 1:100 wt/vol as the predominant treatment dose. A total of 87% of patients within our study were treated within the parameter recommendations, a stark improvement from findings in the 2009 Haymore study.

Subcutaneous Immunotherapy With High-Dose Cat and Dog Extracts: A Real-life Study.

BACKGROUND AND OBJECTIVE: Background: Data on the efficacy of immunotherapy administered to patients with cat or dog allergy are scarce. Objective: We aimed to evaluate the safety and efficacy of subcutaneous immunotherapy (SCIT) in patients with allergy to cat and dog dander. METHODS: Consecutive patients with rhinitis and/or asthma related to sensitization to cat or dog dander were included in a pragmatic, real-life, prospective, observational study. All patients had specific IgE to cat, dog, or both. SCIT was administered using an infusion pump over 3 sessions as part of a rush protocol, followed by monthly administration over 12 months. We recorded adverse events, clinical outcomes, pulmonary function, FeNO, symptoms of rhinitis and asthma, quality of life (QoL), Asthma Control Test (ACT) score, and visual analog scale (VAS) score at baseline, 6 months, and 12 months. RESULTS: The study population comprised 66 patients (38 females, 46 allergic to cat and 20 to dog), with ages ranging from 9 to 59 years. During the up-dosing phase, in which the infusion pump was used, 8.1% of doses elicited a systemic reaction and 5.4% caused a local reaction, while 9.3% of doses administered during the maintenance phase (ie, without an infusion pump) induced a systemic reaction. No local reactions were recorded. A significant improvement in FEV1, symptoms of rhinitis and asthma, QoL, use of medication, VAS score, and ACT score was observed at 6 months and continued at 12 months. Clinical improvement with cat extract was significantly better than with dog extract. CONCLUSIONS: High-dose SCIT has substantial clinical value in many cat- and dog-allergic patients.

Production of Inflammatory Cytokines and Nitric Oxide by Human Mast Cells Incubated with Toxoplasma gondii Lysate.

The roles of mast cells in allergic diseases and helminth infections are well known. However, the roles of mast cells in T. gondii infection is poorly understood. This study was focused on the production of pro-inflammatory cytokines (TNF-α, IL-4), chemokines (CXCL8, MCP-1) and nitric oxide (NO) by mast cells in response to soluble lysate of T. gondii tachyzoites. Production of CXCL8 (IL-8), MCP-1, TNF-α and IL-4 were measured by RT-PCR and ELISA. Western blot were used for detection of CXCR-1 and CXCR2. Our results showed that T. gondii lysates triggered mast cells to release CXCL8, MCP-1, TNF-α, IL-4 and to produce NO. This suggests that mast cells play an important role in inflammatory responses to T. gondii.

Accurate quantification of 5 German cockroach (GCr) allergens in complex extracts using multiple reaction monitoring mass spectrometry (MRM MS).

BACKGROUND: German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy. OBJECTIVE AND METHODS: The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC-MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts. RESULTS: We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high-resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD < 10%), linear over a wide range (r > 0.99, 0.01-1384 fmol/µL), and sensitive (LLOD and LLOQ <1 fmol/µL). Having established the parameters for LC-MRM MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy. CONCLUSIONS AND CLINICAL RELEVANCE: Our data demonstrate that the LC-MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies.

Fungal raw materials used to produce allergen extracts.

OBJECTIVE: To review the topic of fungal raw materials used for the production of allergen extracts and the associated challenges and highlight candidate areas for development before standardized fungal allergen extracts can be commercially produced. DATA SOURCES: A PubMed search was performed using focused keywords and combined with a review of regulatory documents and industry guidelines. Several books on mycology also were consulted. STUDY SELECTIONS: The information obtained through the literature, books, and industry was scrutinized and combined with personal experience and expertise to write this article. RESULTS: Fungi are complex ubiquitous organisms on Earth. They are beneficial and detrimental for humans. Fungi can cause hypersensitivity reactions, including types I, III, and IV. The procurement of fungal raw materials to prepare allergen extracts for diagnosis and possible allergen immunotherapy is complex owing to the intrinsic nature of fungi and their complex genome. Allergen manufacturers produce allergen extracts with variable qualitative and quantitative compositions, which can lead to unpredictable clinical outcomes. CONCLUSION: The clinician should be aware of the factors responsible for the qualitative and quantitative compositions of fungal allergen extracts and the reasons that currently preclude their standardization. Scientific advances and collaboration and cooperation between allergen manufacturing companies and regulatory agencies are necessary to improve the quality and consistency of fungal extracts. Moreover, clinicians should understand the limitations of currently available fungal extracts.

Evaluation of methods for the estimation of threshold concentrations by the skin prick test.

BACKGROUND: The allergen dose-response curve is flat; thus, small changes in wheal size reflect large differences in skin sensitivity. The sensitivity as measured by provocation tests is given by the threshold concentration that causes symptoms and/or objective signs. The threshold concentrations differ by several magnitudes between the most and the least sensitive individuals clinically allergic to the same allergen. Variation in technique can be minimized by relating allergen responses to that to histamine. The aim here is to present and validate simple methods for estimation of the skin sensitivity given as the concentration inducing a wheal of the same size as that with the positive reference, 10 mg/ml of histamine HCl, in the same patient. METHODS: Data from previously reported trials on the biological equilibration of allergen extracts were used to document a method to calculate the concentration of allergen required to induce a wheal of the same size as that with 10 mg/ml of histamine dihydrochloride in the same patient, and to validate the methods using the parallel line bioassay as the gold standard. RESULTS: The validated methods correlated well with the results obtained using the gold standard method and provide results of skin prick testing based on threshold concentrations of allergen. CONCLUSIONS: The validated methods reduce the error of differences in testing techniques and make it possible to report skin sensitivity at threshold concentrations. A simple method to be used in clinical practice and a method suitable to describe changes in skin reactivity over time or during treatment are proposed.

An Adjuvant-Free Mouse Model of Transdermal Sensitization and Oral Elicitation of Anaphylaxis to Shellfish.

BACKGROUND: Shellfish (SF) allergy is a leading cause of systemic anaphylaxis in humans. An adjuvant-free mouse model to evaluate allergenicity and oral anaphylaxis to SF is currently unavailable. Here, we tested the hypothesis that transdermal exposure (TDE) to SF protein extract (SFPE) not only elicits a systemic allergic immune response but also will clinically sensitize mice for oral anaphylaxis. METHODS: Adult BALB/c female mice (6-8 weeks of age) were exposed to saline or SFPE once a week for 4 weeks using a transdermal sensitization method. Systemic SF-specific IgE, IgG1 and IgG2a and total (t)IgE responses were measured using ELISA. Systemic anaphylaxis upon oral SFPE administration was assessed according to clinical symptoms and the hypothermia shock response (HSR). Using individual mouse data, the correlation between the readouts of allergenicity was determined using Pearson's analysis. Spleen-cell IL-4 and IFN-x03B3; responses were determined using primary cell culture and ELISA. RESULTS: TDE to SFPE resulted in marked systemic specific (s)IgE, tIgE, IgG1 and IgG2a responses. Oral challenge with SFPE in sensitized mice (but not controls) elicited systemic anaphylactic clinical reactions and HSR. A strong correlation was observed between sIgE, tIgE and HSR. Spleen cells isolated from allergic mice (but not controls) exhibited memory IL-4 and IFN-x03B3; cytokine responses. CONCLUSION: We report a novel adjuvant-free mouse model of SF allergy with robust quantifiable and correlated readouts of allergenicity that may be used in basic biomedical, preclinical and applied food/nutrition research on SF allergy.

Objectifying the conjunctival provocation test: photography-based rating and digital analysis.

BACKGROUND: Patients with allergic rhinoconjunctivitis are susceptible to both nasal and ocular symptoms. The conjunctival provocation test (CPT) is an established diagnostic procedure used in allergic rhinoconjunctivitis, particularly to document a patient's current reactivity to allergens. To date, there are no international guidelines defining the CPT. No approved evaluation method exists for interpreting CPT results. This paper aims to establish the digital analysis of macroimages as an objective, validated and standardized method for interpreting CPT results. METHODS: In a clinical immunotherapy trial with 155 patients, treatment progress was documented based on the CPT. Local investigators used a symptom score to grade tearing, reddening and the patients' subjective perception of symptoms (mucosal irritation). A central observer rated conjunctival hyperemia via digital photography. Digital image analysis software was utilized to determine conjunctival hyperemia. RESULTS: Spearman's correlation between the local investigators' and the central observer's ratings was r = 0.729 (p < 0.001); the percentage of total agreement was 48% (based on 739 photos). Digital image analysis (based on 48 photos) had a high percentage of total agreement with the central observer's ratings (69%) but a low percentage of total agreement with the investigators' ratings (38%). The corresponding correlations were r = 0.264 and 0.064, respectively. CONCLUSION: Photography-based rating by a central observer may represent a valuable supplement to the local investigator's assessment for making an objective evaluation of CPT results. Digital image analysis possesses the potential of being an objective evaluation method compared to the wide-spread subjective evaluation by the investigators.

Non-digestible oligosaccharides modulate intestinal immune activation and suppress cow's milk allergic symptoms.

BACKGROUND: Cow's milk allergy is a common food allergy in childhood and no effective preventive or curative treatment is available. This study aimed at comparing single short-chain galacto- (scGOS), long-chain fructo- (lcFOS) or pectin-derived acidic oligosaccharides (pAOS) and/or mixtures of scGOS/lcFOS (GF) or scGOS/lcFOS/pAOS (GFA) to prevent or treat food allergy. METHODS: In the preventive protocol, C3H/HeOuJ mice were fed diets containing single oligosaccharides or mixtures GF or GFA throughout the study protocol. In the treatment protocol, GF or GFA was provided for 4 wk starting after the last sensitization. The allergic skin response and anaphylaxis scores were determined, after oral challenge whey-specific immunoglobulins were measured, and qPCR for T-cell markers and Foxp3 counts using immunohistochemistry were performed on the small intestine and colon. RESULTS: Only in the preventive setting, the GF or GFA mixture, but not the single oligosaccharides, reduced the allergic skin response and whey-IgG(1) levels in whey-sensitized mice, compared to the control diet. Both GF and GFA increased the number of Foxp3+ cells in the proximal small intestine of whey - compared to sham-sensitized mice. Expression of Th2 and Th17 mRNA markers increased in the middle part of the small intestine of whey-sensitized mice, which was prevented by GF. By contrast, GFA enhanced Tbet (Th1), IL-10 and TGF-ß mRNA expression compared to GF which was maintained in the distal small intestine and/or colon. CONCLUSIONS: Dietary supplementation with scGOS/lcFOS or scGOS/lcFOS/pAOS during sensitization, both effectively reduce allergic symptoms but differentially affect mucosal immune activation in whey-sensitized mice.

Dramatic declines in seropositivity as determined with crude extracts of Plasmodium falciparum schizonts between 2000 and 2010 in Dielmo and Ndiop, Senegal.

BACKGROUND: Programmes of pre-elimination of malaria have been implemented in Senegal since 2010, and the burden of malaria has decreased substantially. These changes in the epidemiology should be monitored with effective tools that allow changes in patterns of transmission to be estimated. In Dielmo and Ndiop, two villages of Senegal with different malaria endemicity, infections have been followed longitudinally for 20 years, during which time there have been several control interventions leading to substantial decreases of transmission. This study aimed to compare malaria antibody responses of the inhabitants of these two villages, between 2000 and 2010, using schizont crude extracts of a local strain of P. falciparum (Pf Sch07/03). METHODS: Sera collected from inhabitants of the two villages (141 from Dielmo and 79 from Ndiop in 2000; 143 from Dielmo and 79 from Ndiop in 2010) were used to assess the prevalence of antibodies against crude schizont extracts of Pf Sch07/03. Three ages groups were defined: [5-9] yrs, [10-14] yrs and [15-19] yrs. Statistical comparisons were performed. Seroprevalence and the magnitude of antibody responses were compared between age groups, villages and periods. RESULTS: Overall seroprevalence to P.fSch07/03 decreased between 2000 and 2010 in both villages: from 94.4% to 44.4% in Dielmo and from 74.4% to 34.6% in Ndiop. The difference between Dielmo and Ndiop was highly significant in 2000 (p<0.001) but not in 2010 (p >0.20). The decrease in seroprevalence was larger in younger (more than 40%) than older (less than 19%) inhabitants. Longitudinal monitoring of the younger group showed that seroprevalence decreased between 2000 and 2010 in Dielmo from 98.7 to 79.3, but not in Ndiop from 67.6 to 66.7. The magnitude of antibody responses in seropositive individuals was significantly higher in 2000 than 2010 for both villages. CONCLUSIONS: Crude extracts of P. falciparum are appropriate tools for evaluating malaria prevalence at different periods, and in both low and high endemic area. Using crude extracts from local strains to assess transmission may allow efficient evaluation of the consequences of control programs on malaria transmission.

Blomia tropicalis Blo t 5 and Blo t 21 recombinant allergens might confer higher specificity to serodiagnostic assays than whole mite extract.

BACKGROUND: Blomia tropicalis is a dust mite and an important source of allergens in tropical regions. Up to now, the assays to diagnose atopy to this mite use whole body extract as antigens. However, anti-B. tropicalis IgE antibodies cross-react with Ascaris lumbricoides antigens, hindering the diagnosis of allergy to this mite. In this study, B. tropicalis recombinant allergens were evaluated with the purpose of developing an immunodiagnostic assay for allergy to this mite with greater specificity than those commercially available. METHODS: Two B. tropicalis allergens (Blo t 5 and Blo t 21) were cloned into a plasmidial expression vector, expressed in Escherichia coli and purified by affinity chromatography. Sixty-three sera containing anti-B. tropicalis extract (BtE) IgE antibodies were used to investigate IgE reactivity to the recombinant Blot 5 and 21 allergens. Inhibition assays with 20 sera pre-adsorbed with A. lumbricoides extract were performed using rBlo t 5, rBlo t 21, and BtE as antigens. All the assays were carried using indirect ELISA. RESULTS: Eighty-two point nine percent and 80.0% of the sera with anti-BtE antibodies from 35 children reacted with rBlo t 5 and rBlo t 21, respectively, whereas 92.8% and 89.3% of the 28 sera with anti-BtE antibodies from adult asthma patients reacted with the same allergens, and 96.4% of these sera reacted with a mixture of rBlo t 5 and rBlo t 21. In an inhibition ELISA, the absorption of sera by A. lumbricoides extract affected less the reaction with rBlo t 5 and rBlo t 21 than with BtE. CONCLUSIONS: The rBlo t 5 and rBlo t 21 allergens contain important epitopes recognized by IgE antibodies of individuals allergic to B. tropicalis antigens. Moreover, the assays using the recombinant allergens had lower IgE cross-reactivity with A. lumbricoides antigens, a fact which would confers higher specificity to serodiagnostic assays than the crude mite extract. However, additional recombinant allergens should be evaluated in order to reach the same sensitivity of the commercially available assays based on mite extract.

Interleukin-17 production and T helper 17 cells in peripheral blood mononuclear cells in response to ocular lysate in patients with birdshot chorioretinopathy.

PURPOSE: To determine the cytokine response to ocular lysates of peripheral blood mononuclear cells (PBMCs) from patients with birdshot chorioretinopathy (BSCR). METHODS: In the PBMCs of 19 patients with BSCR, T cell cytokine production in response to human retina and choroid lysates was analyzed with flow cytometry and compared to the responses against skin lysates. Five patients had active disease and had not yet been treated (naïve to systemic therapy); 14 patients had either immunomodulatory therapy (IMT) or inactive disease (referred as inactive/IMT). The PBMCs of 11 HLA-A29-positive healthy individuals were used as controls. RESULTS: The levels of interleukin-17 (IL-17) in supernatant of cultures stimulated with retina lysate were higher in patients with active BSCR compared to the HLA-A29 positive controls. The levels of other T cell cytokines (IL-10 and interferon-γ [IFN-γ]) in PBMC cultures did not change significantly after stimulation with ocular lysate. The frequency of CD4(+) IL-17(+) (T helper 17 [Th17]) T cells but not of CD4(+) IFN-γ (Th1) T cells was elevated in the PBMCs of patients with active BSCR stimulated by retina lysates compared to skin lysates. CONCLUSIONS: Our data demonstrate that PBMCs exhibit an IL-17-mediated immune response to retina lysate in patients with active disease naïve to systemic therapy. This is accompanied by the enrichment of IL-17-producing CD4(+) T cells. These findings support the current concept of chronic Th17-cell mediated inflammation and provide evidence that links the Th17 signatures to ocular-specific immune responses in BSCR.

ß-mannosyl linkages inhibit CAWS arteritis by negatively regulating dectin-2-dependent signaling in spleen and dendritic cells.

AIMS: CAWS, Candida albicans water-soluble fraction, is an extracellular mannoprotein produced by C. albicans NBRC1385. It is a ligand of dectin-2, the C-type lectin receptor for innate immunity, and has strong potency for induction of vasculitis in DBA/2 mice. The structure of this mannoprotein is known to be modulated by the culture conditions. To clarify the structure required for vasculitis, CAWSs were prepared in the two culture conditions with or without pH control, and biological properties were compared. METHODS: CAWSs prepared by the standard protocol and pH controlled at 7.0 were designated as CAWS and CAWS727, respectively. The antigenicity was detected by the anti-Candida mannan IgG. These chemical structures were assessed by nuclear magnetic resonance analysis and the lectin array system. The in vitro activity of CAWSs was tested by tumor necrosis factor-α (TNF-α) induction using bone marrow-derived dendritic cells and spleen cell cultures. RESULTS: The antigenicity of CAWS727 was similar to CAWS but the nuclear magnetic resonance analysis showed a higher ratio of ß-mannosyl linkages were detected in CAWS727. The lectin array showed relative affinities of CAWS727 to α-mannosyl specific lectins were weaker than those of CAWS. CAWS induced severe vasculitis in DBA/2 mice while CAWS727 did not. CAWS significantly induced TNF-α but CAWS727 did slightly. In addition, CAWS-induced TNF-α production was inhibited by mixing with CAWS727 in a concentration dependent manner. CONCLUSION: The α-mannosyl linkages of Candida mannan is a key molecule for the immunotoxicity. CAWS727, which conatins ß-mannosyl linkages, competitively bound to lectin receptors, and resulted in reductions in the inflammatory response.

The US Food and Drug Administration review of the safety and effectiveness of nonstandardized allergen extracts.

BACKGROUND: Nonstandardized allergen extracts have been used for a century. Until 1972, these products were regulated by the National Institutes of Health, and products were not required to have an individualized showing of effectiveness. Jurisdiction was then transferred to the US Food and Drug Administration (FDA), which established external review panels to make recommendations regarding safety and effectiveness. Two external panels deliberated, the first from 1974-1979 and the second from 1982-1983. OBJECTIVE: We sought to review external panels' recommendations and assess the safety and effectiveness of nonstandardized allergen extracts, FDA-reviewed available literature, and databases since 1972. METHODS: Currently licensed nonstandardized allergen extracts were reviewed according to extract type. Available data were collected from medical and nonscientific search engines. Nomenclature was ascertained by consulting www.itis.gov or www.atcc.org. The FDA's Adverse Event Reporting System was probed for events associated with extract use. Provisional threshold levels of safety and effectiveness were established, and extracts were sorted according to whether they met the thresholds. RESULTS: In the Adverse Event Reporting System, there were 178 adverse event reports, including 13 deaths, associated with allergen extract use over 23 years. No single group of extracts predominated. Among 1269 allergen extracts reviewed, there were 480 for which use in the diagnosis and treatment of allergic disease were addressed in the literature, 207 for which only diagnostic use was addressed, 565 for which minimal or no supportive literature was identified, and 17 for which potential safety concerns were found. CONCLUSIONS: When used according to professional guidelines, almost all nonstandardized allergen extracts for diagnosis and therapy appear to be safe. Provisional thresholds of effectiveness were met by 54% of extracts reviewed.

Mycobacterium tuberculosis Sonicate-Induced IFNγ, CXCL10 and IL10 can Differentiate Severity in Tuberculosis.

Improved tools are required to study immunopathogenesis of tuberculosis (TB). Mycobacterium tuberculosis antigen-stimulated T cell-based assays can detect TB but are less effective when responses are compromised such as in severe disease. We investigated immune responses to M. tuberculosis whole sonicate (MTBs), recombinant antigens ESAT6 and CFP10 in whole blood cells of healthy endemic controls (EC, n = 42) and patients with pulmonary (PTB, n = 36) or extrapulmonary (ETB, n = 41) disease. Biomarkers of T cell activation (IFNγ) or modulation (IL10) and chemokines, CXCL9, CXCL10 and CCL2, secretion were measured. MTBs, ESAT6 and CFP10 all induced IFNγ responses in TB. ESAT6-induced IFNγ was elevated in TB as compared with EC. MTBs stimulated the highest IFNγ levels but did not differentiate between TB and EC. However, MTBs-induced CXCL10 (P = 0.004) was reduced, while IL10 (P < 0.001) was raised in TB as compared with EC. Between sites, MTBs-induced CCL2 (P = 0.001) and IL10 secretion was higher in PTB than ETB (P < 0.001). In comparison of disease severity, MTBs-induced IFNγ (P = 0.014) and CXCL10 (P = 0.022) levels were raised in moderate as compared with far advanced PTB. In ETB, MTBs-induced IL10 levels were greater in less-severe (L-ETB) than in severe disseminated (D-ETB) cases, P = 0.035. Within the L-ETB group, MTBs-induced IFNγ was greater in patients with tuberculous lymphadenitis than those with pleural TB (P = 0.002). As immune responses to MTBs were differentially activated in TB of different sites and severity, we propose the utility of MTBs-induced IFNγ, CXCL10 and IL10 as biomarkers in TB.

Diet influences growth rates and allergen and endotoxin contents of cultured Dermatophagoides farinae and Dermatophagoides pteronyssinus house dust mites.

BACKGROUND: The house dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus are cultured to obtain material for the production of allergen extracts for research, diagnostic and immunotherapeutic purposes. METHODS: We cultured mites on two different diets that supported thriving populations and determined the population growth rates, dynamics of allergen accumulation, and endotoxin concentrations in extracts made from mites harvested from the cultures. RESULTS: D. farinae populations grew faster on a diet of rodent chow/yeast than on an egg/yeast diet but a larger peak population size was achieved on the egg/yeast diet. Diet influenced the dynamics of the production of groups 1 and 2 allergens and the group 1/2 ratios for both species. To population peak, Der f 1 was produced at a faster rate on the chow/yeast diet but greater amounts of Der f 1 were produced by mites grown on the egg/yeast diet. D. pteronyssinus populations grew faster and achieved greater density on the egg/yeast diet compared to the chow/yeast diet. D. pteronyssinus produced more Der p 1 than Der p 2 when grown on chow/yeast while more Der p 2 than Der p 1 was produced on egg/yeast. Endotoxin concentrations in extracts made from whole cultures for both species at maximum population density were very different in the two diets. Washing the mites resulted in the loss of up to 88% of the allergen. CONCLUSION: Mite-culturing diet directly effects population growth, the dynamics of allergen accumulation, the group 1/2 allergen ratio and the endotoxin contents in extracts of cultured house dust mites.

Varying allergen composition and content affects the in vivo allergenic activity of commercial Dermatophagoides pteronyssinus extracts.

BACKGROUND: Diagnosis and immunotherapy of house-dust mite (HDM) allergy is still based on natural allergen extracts. The aim of this study was to analyze commercially available Dermatophagoides pteronyssinus extracts from different manufacturers regarding allergen composition and content and whether variations may affect their allergenic activity. METHODS: Antibodies specific for several D. pteronyssinus allergens (Der p 1, 2, 5, 7, 10 and 21) were used to analyze extracts from 10 different manufacturers by immunoblotting. Sandwich ELISAs were used to quantify Der p 1 and Der p 2 in the extracts. Mite-allergic patients (n = 45) were skin-tested with the extracts and tested for immunoglobulin E (IgE) reactivity to a panel of 10 mite allergens (Der p 1, 2, 4, 5, 7, 8, 10, 14, 20 and 21) by dot blot. RESULTS: Only Der p 1 and Der p 2 were detected in all extracts but their concentrations and ratios showed high variability (Der p 1: 6.0-40.8 µg ml(-1); Der p 2: 1.7-45.0 µg ml(-1)). At least 1 out of 4 allergens (i.e. Der p 5, 7, 10 and 21) was not detected in 8 of the studied extracts. Mite-allergic subjects showed different IgE reactivity profiles to the individual mite allergens, the extracts showed different allergenic activity in skin-prick tests and false-negative results. CONCLUSIONS: Commercially available D. pteronyssinus extracts lack important allergens, show great variability regarding allergen composition and content and some gave false-negative diagnostic test results in certain patients.

House dust mite-specific immunotherapy alters the basal expression of T regulatory and FcεRI pathway genes.

BACKGROUND: Regulatory T (Treg) cells and IgE-mediated signaling pathways could play important roles in the induction of allergen tolerance during house dust mite-specific subcutaneous immunotherapy (HDM-SCIT). Our aim was to compare the basal expression levels of Treg, T helper 1 (Th1) and Th2 transcription factors and components involved in IgE-mediated signaling in healthy subjects with those in HDM-allergic patients both untreated and successfully treated with HDM-SCIT. METHODS: Thirty-nine HDM-allergic patients who completed a 3- to 5-year course of mite extract SCIT, 20 mite-allergic controls and 25 healthy controls participated in this study. The efficacy of SCIT was monitored using skin-prick tests (SPTs), total immunoglobulin E (tIgE), specific IgE (sIgE), sIgG(4), nasal challenge and visual analog scale (VAS) scores at several time points. The mRNA levels of forkhead box protein 3 (FOXP3), T-BET, GATA-3, FcεRI, spleen tyrosine kinase (Syk), phosphatidylinositol 3 kinase (PI3K) and SH2 domain-containing inositol phosphatase (SHIP) were quantified by real-time RT-PCR using nonstimulated whole blood samples. RESULTS: Decreased wheal sizes and VAS scores, negative challenges and increased sIgG(4) levels indicated that SCIT was effective in the treated patients. Basal expression levels of FOXP3 and GATA-3 decreased and T-BET levels increased in both treated patients and in healthy controls compared to untreated patients. The IgE-mediated pathway kinases Syk and PI3K exhibited reduced expression, whereas SHIP phosphatase levels were elevated in both treated patients and healthy controls relative to untreated patients. The expression levels of FcεRI were not significantly altered. CONCLUSIONS: Immunotherapy using HDM extracts results in a modification of the basal expression levels of several IgE-related signaling factors and induces a highly significant upregulation of Th1-response and downregulation of Th2-response transcription factors. Interestingly, this therapy also appears to reduce the basal expression of FOXP3.