Road map for the clinical application of the basophil activation test in food allergy.

The diagnosis of IgE-mediated food allergy based solely on the clinical history and the documentation of specific IgE to whole allergen extract or single allergens is often ambiguous, requiring oral food challenges (OFCs), with the attendant risk and inconvenience to the patient, to confirm the diagnosis of food allergy. This is a considerable proportion of patients assessed in allergy clinics. The basophil activation test (BAT) has emerged as having superior specificity and comparable sensitivity to diagnose food allergy, when compared with skin prick test and specific IgE. BAT, therefore, may reduce the number of OFC required for accurate diagnosis, particularly positive OFC. BAT can also be used to monitor resolution of food allergy and the clinical response to immunomodulatory treatments. Given the practicalities involved in the performance of BAT, we propose that it can be applied for selected cases where the history, skin prick test and/or specific IgE are not definitive for the diagnosis of food allergy. In the cases that the BAT is positive, food allergy is sufficiently confirmed without OFC; in the cases that BAT is negative or the patient has non-responder basophils, OFC may still be indicated. However, broad clinical application of BAT demands further standardization of the laboratory procedure and of the flow cytometry data analyses, as well as clinical validation of BAT as a diagnostic test for multiple target allergens and confirmation of its feasibility and cost-effectiveness in multiple settings.

The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease.

The basophil activation test (BAT) has become a pervasive test for allergic response through the development of flow cytometry, discovery of activation markers such as CD63 and unique markers identifying basophil granulocytes. Basophil activation test measures basophil response to allergen cross-linking IgE on between 150 and 2000 basophil granulocytes in <0.1 ml fresh blood. Dichotomous activation is assessed as the fraction of reacting basophils. In addition to clinical history, skin prick test, and specific IgE determination, BAT can be a part of the diagnostic evaluation of patients with food-, insect venom-, and drug allergy and chronic urticaria. It may be helpful in determining the clinically relevant allergen. Basophil sensitivity may be used to monitor patients on allergen immunotherapy, anti-IgE treatment or in the natural resolution of allergy. Basophil activation test may use fewer resources and be more reproducible than challenge testing. As it is less stressful for the patient and avoids severe allergic reactions, BAT ought to precede challenge testing. An important next step is to standardize BAT and make it available in diagnostic laboratories. The nature of basophil activation as an ex vivo challenge makes it a multifaceted and promising tool for the allergist. In this EAACI task force position paper, we provide an overview of the practical and technical details as well as the clinical utility of BAT in diagnosis and management of allergic diseases.

Vitamins A and D have antagonistic effects on expression of effector cytokines and gut-homing integrin in human innate lymphoid cells.

BACKGROUND: Retinoic acid (RA), the main biologically active metabolite of vitamin A, is known to promote gut homing of lymphocytes, as well as various regulatory and effector immune responses. In contrast, the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), is predominantly immunosuppressive. Little is known about the direct effects of these vitamins on the recently identified innate lymphoid cells (ILCs). OBJECTIVE: We sought to characterize the effects of RA and 1,25D3 on human ILCs. METHODS: Peripheral blood mononuclear cells were isolated from 27 non-selected blood donor buffy coats, and ILCs were sorted by FACS. ILC1, ILC2, and ILC3 cells were cultured for 5 days with RA, 1,25D3, and various cytokines known to activate ILCs (IL-2, IL-7, IL-12, thymic stromal lymphopoietin (TSLP), IL-25, and IL-33). Cytokines produced by ILCs were measured in culture supernatants, and surface receptor expression was analysed by flow cytometry. RESULTS: Retinoic acid acted synergistically with IL-2 and other activating cytokines to induce expression of the gut-homing integrin α4ß7 in ILCs, as well as production of IL-5 and IL-13 in ILC2 cells, and IFN-γ in ILC1 and ILC3 cells. Expression of integrin α4ß7 and cytokine production in ILCs stimulated with RA + IL-2 was increased at least fourfold as compared to ILCs cultured with RA or IL-2 alone. In contrast, RA completely inhibited the IL-2-induced expression of cutaneous lymphocyte antigen (CLA) in ILCs. Moreover, addition of 1,25D3 to ILCs cultured with RA + IL-2 inhibited cytokine production and expression of integrin α4ß7 by at least 30%. CONCLUSIONS: Retinoic acid and 1,25D3 have antagonistic effects on the expression of effector cytokines and gut-homing integrin by human ILCs. The balance between these vitamins may be an important factor in the functioning of ILCs and the diseases in which ILCs are implicated, such as allergic inflammation.

Pathogen induced chemo-attractant hepoxilin A3 drives neutrophils, but not eosinophils across epithelial barriers.

Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator, hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces, eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids, however, it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study, neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient, whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly, while pathogen induced neutrophil trans-epithelial migration was substantial, pathogen induced eosinophil trans-epithelial migration was not observed. Further, gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers, whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection, HXA3 does not appear to exhibit chemotactic activity toward eosinophils.

Evidence of pathway-specific basophil anergy induced by peanut oral immunotherapy in peanut-allergic children.

BACKGROUND: In Westernized countries, over 1% of the population is allergic to peanuts or tree nuts, which carries a risk of severe allergic reactions. Several studies support the efficacy of peanut oral immunotherapy (OIT) for reducing the clinical sensitivity of affected individuals; however, the mechanisms of this effect are still being characterized. One mechanism that may contribute is the suppression of effector cells, such as basophils. Basophil anergy has been characterized in vitro as a pathway-specific hyporesponsiveness; however, this has not been demonstrated to occur in vivo. OBJECTIVE: To evaluate the hypothesis that basophil anergy occurs in vivo due to chronic allergen exposure in the setting of a clinical oral immunotherapy trial. METHODS: Samples of peripheral blood were obtained from subjects during a placebo-controlled clinical trial of peanut OIT. Basophil reactivity to in vitro stimulation with peanut allergen and controls was assessed by the upregulation of activation markers, CD63 and CD203c, measured by flow cytometry. RESULTS: The upregulation of CD63 following stimulation of the IgE receptor, either specifically with peanut allergen or non-specifically with anti-IgE antibody, was strongly suppressed by active OIT. However, OIT did not significantly suppress this response in basophils stimulated by the distinct fMLP receptor pathway. In the subset of subjects with egg sensitization, active peanut OIT also suppressed CD63 upregulation in response to stimulation with egg allergen. Allergen OIT also suppressed the upregulation of CD203c including in response to stimulation with IL-3 alone. CONCLUSION: Peanut OIT induces a hyporesponsive state in basophils that is consistent with pathway-specific anergy previously described in vitro. This suggests the hypothesis that effector cell anergy could contribute to clinical desensitization.

Immunology in the Clinic Review Series; focus on allergies: basophils as biomarkers for assessing immune modulation.

Allergen-specific immunotherapy is an effective clinical treatment for hypersensitivity to many allergens. Studies of basophils during immunotherapy have provided insight into underlying immune mechanisms and support the potential use of basophil activation as a biomarker of clinical outcomes. This review examines the evidence for different pathways of basophil modulation associated with various forms of immunotherapy. Better understanding the molecular mechanisms of basophil activation and desensitization and the relationship between suppression of these effector cells to clinical outcomes holds promise for further development and improvement in potential therapies for allergic diseases.

Genes controlling ion permeability in both motorneurons and muscle.

Genetic data suggest that unc-8 is a member of the epithelial sodium channel (ENaC) gene family (shreffler et al., 1995). Consistent with this idea, cosmid R13A1, containing an ENaC homolog, can restore normal locomotion to unc-8 mutants after germline transformation. To identify other genes encoding proteins that regulate ENaC function, extragenic unc-8 suppressor and enhancer mutations were sought. This report describes two new unc-8 suppressor mutations, sup-42(lb88) X and sup-43(lb141) II, and an enhancer mutation, enu-2(lb140) III. sup-43(lb141) and enu-2(lb140), cause vacuoles within body wall muscle, similar in appearance to those of unc-105(n490) II mutants, consistent with their proposed role in ENaC function. Single and double mutant phenotypes observed in this and previous work suggest that sodium channels in different tissues utilize an overlapping set of gene products: at least six in motorneurons, unc-8, mec-6, and sup-40-43, and at least five in muscle, sup-43, unc-8, enu-2, unc-105, and mec-6.

unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C. elegans locomotion.

Mechanically gated ion channels are important modulators of coordinated movement, yet little is known of their molecular properties. We report that C. elegans unc-8, originally identified by gain-of-function mutations that induce neuronal swelling and severe uncoordination, encodes a DEG/ENaC family member homologous to subunits of a candidate mechanically gated ion channel. unc-8 is expressed in several sensory neurons, interneurons, and motor neurons. unc-8 null mutants exhibit previously unrecognized but striking defects in the amplitude and wavelength of sinusoidal tracks inscribed as they move through an E. coli lawn. We hypothesize that UNC-8 channels could modulate coordinated movement in response to body stretch. del-1, a second DEG/ENaC family member coexpressed with unc-8 in a subset of motor neurons, might also participate in a channel that contributes to nematode proprioception.

The unc-8 and sup-40 genes regulate ion channel function in Caenorhabditis elegans motorneurons.

Two Caenorhabditis elegans genes, unc-8 and sup-40, have been newly identified, by genetic criteria, as regulating ion channel function in motorneurons. Two dominant unc-8 alleles cause motorneuron swelling similar to that of other neuronal types in dominant mutants of the deg-1 gene family, which is homologous to a mammalian gene family encoding amiloride-sensitive sodium channel subunits. As for previously identified deg-1 family members, unc-8 dominant mutations are recessively suppressed by mutations in the mec-6 gene, which probably encodes a second type of channel component. An unusual dominant mutation, sup-41 (lb125), also co-suppresses unc-8 and deg-1, suggesting the existence of yet another common component of ion channels containing unc-8 or deg-1 subunits. Dominant, transacting, intragenic suppressor mutations have been isolated for both unc-8 and deg-1, consistent with the idea that, like their mammalian homologues, the two gene products function as multimers. The sup-40 (lb130) mutation dominantly suppresses unc-8 motorneuron swelling and produces a novel swelling phenotype in hypodermal nuclei. sup-40 may encode an ion channel component or regulator that can correct the osmotic defect caused by abnormal unc-8 channels.

Serodiagnosis of Chagas' disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection.

Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes.

During feeding by infected mosquitoes, malaria sporozoites are injected into the host's bloodstream and enter hepatocytes within minutes. The remarkable target cell specificity of this parasite may be explained by the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. We have now identified these receptors as heparan sulfate proteoglycans (HSPG). The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. We have purified and partially characterized the CS-binding HSPGs from HepG2 cells. They have a molecular weight of 400,000-700,000, are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans.

Antibody responses of visceral leishmaniasis patients to gp63, a major surface glycoprotein of Leishmania species.

A major surface glycoprotein of Leishmania parasites, gp63, is highly conserved among species and is expressed in both infective and intracellular stages. Although much is known about its role in entry and survival in host macrophages, patient antibody responses to this glycoprotein have not been well characterized. The prevalence of anti-gp63 antibody in sera of leishmaniasis patients was evaluated by ELISA. Sera from most acute visceral leishmaniasis patients (84%) from Brazil and Sudan had notably high levels of antibody to recombinant (r) gp63. Sera from other forms of leishmaniasis and from other diseases did not contain significantly elevated levels of anti-gp63 antibody. These results indicate that rgp63 might be a useful constituent of a defined serologic test for visceral leishmaniasis.

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.

Identification and synthesis of a major conserved antigenic epitope of Trypanosoma cruzi.

A gene sequence encoding an immunodominant protein with a repetitive epitope from the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, was cloned and expressed. The identified 10-amino acid repeat is present within a high-molecular-weight trypomastigote antigen that appears specific to and conserved among T. cruzi isolates. More importantly, greater than 95% of T. cruzi infection sera, including both chronic and acute Chagas disease, contained elevated levels of antibody to a 15-amino acid synthetic peptide bearing the repetitive B-cell epitope. Considering the wide diversity of T. cruzi parasites, as well as the broad spectrum of clinical manifestations of Chagas disease, such a prevalent immune response among patients is significant and applicable to the control of Chagas disease through the diagnosis of T. cruzi infection.

A rapid and simple diagnostic test for active visceral leishmaniasis.

We have developed an immunodot assay for the serodiagnosis of active visceral leishmaniasis (AVL) which utilizes protein A colloidal gold as the visualizing agent. The test is simple, requires few reagents, and can be completed in two hours. It is sensitive and specific for active visceral leishmaniasis, and generally correlates with the ELISA. Either whole blood or sera in minute quantities may be used as test antibody. In addition, the use of the protein A gold immunodot is shown to detect anti-leishmania antibodies in infected dogs.

An improved serodiagnostic procedure for visceral leishmaniasis.

The enzyme-linked immunosorbent assay (ELISA) is a sensitive and specific serodiagnostic method for leishmaniasis. In this report, we describe how this versatile assay can be improved by the use of protein A or protein G conjugates for the specific detection of Leishmania antibody in the sera of patients with visceral leishmaniasis. In direct comparisons with anti-immunoglobulin conjugate, enzyme-linked protein A gave significantly higher absorbance values for positive sera without a corresponding increase in absorbance values for sera from normal individuals or from patients with other diseases known to cross-react with leishmaniasis. The effect was to increase the distance between positive and negative values, which aided in the interpretation of the results. This also permitted visual distinction between positive sera and negative or weakly reactive sera. The assay was effective using either blood or serum as the source of primary antibody. A further advantage of protein A over anti-Ig conjugate was its ability to detect specific antibody in dog as well as human sera. Finally, we demonstrated the usefulness of the protein A ELISA with a recombinant leishmania antigen, gp63.