Daniela Breda

Allergen microarrays on high-sensitivity silicon slides

We have recently introduced a silicon substrate for high-sensitivity microarrays, coated with a functional polymer named copoly(DMA-NAS-MAPS). The silicon dioxide thickness has been optimized to produce a fluorescence intensification due to the optical constructive interference between the incident and reflected lights of the fluorescent radiation. The polymeric coating efficiently suppresses aspecific interaction, making the low background a distinctive feature of these slides. Here, we used the new silicon microarray substrate for allergy diagnosis, in the detection of specific IgE in serum samples of subjects with sensitizations to inhalant allergens. We compared the performance of silicon versus glass substrates. Reproducibility data were measured. Moreover, receiver-operating characteristic (ROC) curves were plotted to discriminate between the allergy and no allergy status in 30 well-characterized serum samples. We found that reproducibility of the microarray on glass supports was not different from available data on allergen arrays, whereas the reproducibility on the silicon substrate was consistently better than on glass. Moreover, silicon significantly enhanced the performance of the allergen microarray as compared to glass in accurately identifying allergic patients spanning a wide range of specific IgE titers to the considered allergens.

Effect of sublingual immunotherapy with grass monomeric allergoid on allergen-specific T-cell proliferation and interleukin 10 production

BACKGROUND Sublingual immunotherapy (SLIT) is safe and efficacious in the treatment of patients with allergic rhinitis. Although favorable clinical effects have been observed with controlled trials as early as a few months since the beginning of treatment, few biological changes induced by SLIT have been demonstrated. OBJECTIVE To investigate in grass-allergic patients the effect of a 2-month SLIT regimen, administered with a simplified protocol without up-dosing, on proliferation and production of cytokines characteristic of the regulatory T-cell phenotype (interleukin 10 [IL-10] and transforming growth factor beta [TGF-beta]) by allergen-specific T cells. METHODS Patients were recruited to the study in January 2006. SLIT was performed by self-administration and was continued for 60 days from February to April 2006. Eleven grass pollen-allergic patients with seasonal rhinitis were treated daily before the pollen season for 2 months with a modified allergen (monomeric allergoid) derived from a 3-grass pollen extract. Allergen-specific proliferation and production of IL-10 and TGF-beta were measured on peripheral blood mononuclear cells at baseline and treatment end. Tetanus toxoid served as the control antigen. RESULTS After SLIT, allergen-specific (P = .002) but not tetanus toxoid-specific proliferation decreased, whereas IL-10 transcription increased (P < .001). TGB-beta transcription was also increased after treatment, although not statistically significantly (P = .06). Changes in proliferation to allergen and in IL-10 transcription were correlated (r = -0.82, P = .003). CONCLUSIONS A short-term course of SLIT with modified allergen in grass-allergic patients is associated with the reduction of allergen-specific proliferation and with the up-regulation of the IL-10 regulatory cytokine.

Human monocytoid THP-1 cell line versus monocyte-derived human immature dendritic cells as in vitro models for predicting the sensitising potential of chemicals.

Immature dendritic cells (DCs) modulate differentiation markers following in vitro exposure to chemicals generating contact allergies. THP-1 is a monocytoid cell line maintaining some differentiating plasticity. In this study, human DCs and THP-1 cells were compared as in vitro models to predict contact sensitisation of chemicals with different sensitising potential. Expression of CD80 and CD86 was assessed by flow cytometry after exposure to subtoxic concentrations of potent (2,4-dinitrochlorobenzene, DNCB and p-phenylendiamine, PPD), strong (thimerosal, TMS), moderate (sodium tetrachloroplatinate, Na2PtCl4) sensitising compounds as well as of non-sensitising, irritating sodium dodecyl sulphate (SDS) as compared to a vehicle of sensitising substances (dimethyl sulphoxide, DMSO). Up-regulation of CD86 following in vitro incubation of DCs and THP-1 cells with DNCB, PPD, TMS and Na2PtCl4 but not with SDS, was observed. The CD80 membrane marker was up-regulated on DCs following in vitro incubation with DNCB and PPD, but not with TMS, Na2PtCl4 and SDS. On THP-1 cells, only DNCB up-regulated CD80 expression. In conclusion, both the cell line THP-1 and DCs are promising in vitro models for assays aiming at predicting the sensitisation potential of chemicals. THP-1 cell line is by far easier to handle and offers relevant advantages from the practical point of view.

T-Cell Receptor-Mediated Cross-Allergenicity

Background: Profilins are conserved and ubiquitous plant and animal proteins. We wanted to discover whether the T-cell response to conserved epitopes on birch and grass profilins could account for cross-allergenicity in subjects allergic to these two pollens. Methods: Thirty-one patients allergic to grass and birch were recruited for the study. Grass and birch reactive T lymphocytes were studied by measuring proliferation to birch and grass allergen, respectively, followed by Vβ T-cell receptor family-specific polymerase chain reaction and heteroduplex analysis. T-cell clones were derived from patients with cross-proliferating T cells. Results: In 25 of 31 subjects the T-cell response to grass was quite distinct from that to birch. In contrast, in 6 of 31 individuals grass T cells cross-proliferated to birch and this was reproduced in 4 patients by birch profilin. CD4 Th2 cell clones were derived which promiscuously recognized homologously conserved regions on birch and grass profilins. Conclusion: We conclude that a functionally relevant T-cell response to conserved regions of panallergens underlie cross-allergenicity in a subset of allergic patients. These results suggest that a reciprocal modulation of the response to one sensitizing allergen can occur following natural exposure to or immunotherapy with another allergen. These results have relevance in the management of patients with multiple allergies.

Chromium (VI)-induced immunotoxicity and intracellular accumulation in human primary dendritic cells.

Chromium compounds, besides being occupational carcinogens, can also induce allergic contact dermatitis (ACD) and other immunomodulatory effects. In this study we investigate cell viability, uptake and intracellular distribution of chromium in human primary dendritic cells (DCs), either immature (iDCs) or driven to differentiate by a specific maturation stimulus (LPS) (mature DCs, mDCs), when exposed for 48 h to concentrations of soluble radiolabelled Na251CrO4 ranging from 5 to 0.5 μM. The modulation of the expression of membrane markers (CD80, CD86, MHC class II) correlated with the immunological functions of DCs was also measured. After 48 h of exposure the mean IC50 values in 4 donors were 36 and 31 μM in iDCs and mDC respectively, as detected by propidium iodide incorporation. Cellular uptake of chromium was nearly linear with increasing doses. At 48 h post-exposure chromium was accumulated preferentially in the nuclear and cytosolic fractions (44.1 to 66% and 13.1 to 31% of total cellular chromium, respectively). Although a high inter-individual variability was observed, an increase in the expression of CD86 and, to a lower extent, CD80 and MHC class II membrane markers was found in mDCs of single donors. These results highlight the relevance of searching for the biodistribution of trace metals in primary cells of the immune system. Moreover, they suggest that DCs differentiation markers can help in measuring the immunotoxicity of metal

In Search of a Vaccine for Mouse Allergy: Significant Reduction of Mus m 1 Allergenicity by Structure-Guided Single-Point Mutations

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

Platinum Group Elements Enhance the Allergic Immune Response by Acting on Dendritic Cells

Background: Atmospheric pollution may play a role in the immune response to allergens either directly or by entering the food chain. While particulate platinum group elements (PLGE) emitted by catalytic converters can be considered biologically inert, approximately 10% of these species accumulate in the environment as bioavailable soluble forms. Methods: We challenged in vitro human immature and mature monocyte-derived dendritic cells with subtoxic concentrations of soluble species of PLGE. Dendritic cells were studied both at baseline and following treatment with Na2PtCl6, Na2PdCl6 or Na3RhCl6. (NH4)6Mo7O24 was included as control. The following end-points were considered: expression of differentiation markers, effectiveness of allergen presentation and Th2 cytokine production by cocultured T lymphocytes, expression of IgE-type I receptor and efficiency of IgE-dependent endocytosis. Results: We found that treatment with PLGE (but not with the control metal) increased costimulatory molecule expression and antigen presentation, amplified IL-5 production by cocultured T lymphocytes, upregulated IgE-type I receptor membrane expression, and augmented IgE-type I receptor-mediated endocytosis. Conclusions: We conclude that PLGE have an adjuvant-like effect on dendritic cells that can favor and amplify the immune response to allergens.

Clinical and immunological correlates of pre-co-seasonal sublingual immunotherapy with birch monomeric allergoid in patients with allergic rhinoconjunctivitis.

Sublingual immunotherapy is safe and efficacious in the treatment of patients with allergic rhinitis. The clinical and biological efficacy of modified allergens (allergoids) has not been fully clarified. We investigated in birch allergic patients the effect of a pre-co-seasonal sublingual immunotherapy regimen with a modified allergen extract on clinical parameters and on T cell proliferation and regulatory cytokine production (IL-10, TGF-beta). We found that during the birch pollen season symptoms and drug usage scores were 30 and 40% improved, respectively, in treated versus control subjects (p<0.0001 for both comparisons) whereas well days were 23.5 (33%) versus 16.9 (23%) (p=0.0024), respectively. Bet v 1 allergen specific proliferation decreased (p = 0.0010), whereas IL-10 transcription increased (p = 0.0010) in treated, but not in control patients. Moreover, TGF-beta transcription was increased, although not significantly (p=0.066), following immunotherapy. Thus, sublingual immunotherapy with modified allergen in birch-allergic subjects was safe, clinically efficacious and associated with the reduction of allergen-specific proliferation and with the increased production of the IL-10 regulatory cytokine.

Unreliable Measurement of Basophil Maximum Leukotriene Release with the Bühlmann CAST 2000 Enzyme-Linked Immunosorbent Assay Kit

ABSTRACT The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

Broad-Spectrum Inhibition of HIV-1 by a Monoclonal Antibody Directed against a gp120-Induced Epitope of CD4

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.