Meat allergy associated with galactosyl-α-(1,3)-galactose (α-Gal)-Closing diagnostic gaps by anti-α-Gal IgE immune profiling.

BACKGROUND: Glycoproteins and glycolipids of some mammalian species contain the disaccharide galactosyl-α-(1,3)-galactose (α-Gal). It is known that α-Gal is immunogenic in humans and causes glycan-specific IgG and also IgE responses with clinical relevance. α-Gal is part of the IgE-reactive monoclonal therapeutic antibody cetuximab (CTX) and is associated with delayed anaphylaxis to red meat. In this study, different α-Gal-containing analytes are examined in singleplex and multiplex assays to resolve individual sensitization patterns with IgE against α-Gal. METHODS: Three serum groups, α-Gal-associated meat allergy (MA) patients, idiopathic anaphylaxis (IA) patients with suspected MA, and non-meat-allergic healthy control individuals (HC), were analyzed via singleplex allergy diagnostics and a newly established immunoblot diagnostic system. The new dot blot detection system resolved individual IgE sensitization profiles for α-Gal-containing analytes CTX, bovine thyroglobulin (Bos d TG), and human serum albumin (HSA)-conjugated α-Gal. RESULTS: Singleplex allergy diagnostics using the α-Gal analytes CTX and Bos d TG confirms the history of MA patients in 91% and 88% of the cases, respectively. A novel dot blot-based assay system for the detection of IgE against α-Gal reveals individual IgE sensitization profiles for α-Gal-containing analytes. An α-Gal-associated IgE cross-reactivity profile (IgE against CTX, Bos d TG, and HSA-α-Gal) was identified, which is associated with MA. CONCLUSIONS: Detection of individual sensitization patterns with different α-Gal-containing analytes provides the basis for an individual allergy diagnosis for α-Gal-sensitized patients. Higher amounts of α-Gal in pork and beef innards compared to muscle meat as indicated by a higher staining intensity are a plausible explanation for the difference in allergic symptom severity.

Recent advances in the use of nanoparticles for allergen-specific immunotherapy.

The number of patients suffering from allergic asthma and rhinoconjunctivitis has increased dramatically within the last decades. Allergen-specific immunotherapy (AIT) is the only available cause-oriented therapy so far. AIT reduces symptoms, but has also a disease-modifying effect. Disadvantages are a long-lasting procedure, and in a few cases potential systemic adverse reactions. Encapsulation of allergens or DNA vaccines into nanostructures may provide advantages compared to the conventional AIT with noncapsulated allergen extracts: The protein/DNA molecule can be protected from degradation, higher local concentrations and targeted delivery to the site of action appear possible, and most importantly, recognition of encapsulated allergen by the immune system, especially by IgE antibodies, is prevented. AIT with nanoparticles (NPs) may offer a safer and potentially more efficient way of treatment for allergic diseases. In this review, we summarize the use of biodegradable NPs consisting of synthetic or natural polymers, liposomes, and virus-like particles as well as nonbiodegradable NPs like dendrimers, and carbon- or metal-based NPs for AIT. More or less successful applications of these NPs in prophylactic as well as therapeutic vaccination approaches in rodents or other animals as well as first human clinical trials are discussed in detail.

BASALIT trial: double-blind placebo-controlled allergen immunotherapy with rBet v 1-FV in birch-related soya allergy.

BACKGROUND: Conflicting results exist on the effect of allergen immunotherapy (AIT) on pollen-related food allergy. We aimed to investigate the efficacy of one-year AIT with the folding variant (FV) of recombinant (r) Bet v 1 on birch-related soya allergy. METHODS: Of 138 subjects with Bet v 1 sensitization, 82 were positive at double-blind placebo-controlled food challenge (DBPCFC) with soya. A total of 56 of 82 were randomized in the ratio of 2:1 (active: placebo). Per-protocol population (PPP) had received ≥150 µg of allergen or placebo preparation. OUTCOME MEASURES: lowest observed adverse effect levels (LOAEL), postinterventional occurrence of objective signs (objS) at any dose level, sIgE/IgG4 against Bet v 1 and Gly m 4. Between-group changes were investigated (ancova, Mann-Whitney U-test, Fisher exact test). RESULTS: Baseline characteristics including LOAELs were comparable in both groups with objS and subjS occurring in 82% and 95% of active (n = 38) vs 78% and 83% of placebo group (n = 18). After AIT, objS occurred in 24% and 47%, respectively. LOAEL group differences showed a beneficial tendency (P = 0.081) for LOAELobjective in PPP (30 active, 15 placebo). sIgG4 raised only in active group (Bet v 1: P = 0.054, Gly m 4: P = 0.037), and no relevant changes occurred for sIgE. Only 56% of the intended sample size was recruited. CONCLUSION: For the first time, we present data on the effect of rBet v 1-FV on birch-related soya allergy. rBet v 1-FV AIT induced significant immunogenic effects. Clinical assessment showed a tendency in favour of the active group but did not reach statistical significance.

Adjuvant effects of aluminium hydroxide-adsorbed allergens and allergoids - differences in vivo and in vitro.

Allergen-specific immunotherapy (SIT) is a clinically effective therapy for immunoglobulin (Ig)E-mediated allergic diseases. To reduce the risk of IgE-mediated side effects, chemically modified allergoids have been introduced. Furthermore, adsorbance of allergens to aluminium hydroxide (alum) is widely used to enhance the immune response. The mechanisms behind the adjuvant effect of alum are still not completely understood. In the present study we analysed the effects of alum-adsorbed allergens and allergoids on their immunogenicity in vitro and in vivo and their ability to activate basophils of allergic donors. Human monocyte derived dendritic cells (DC) were incubated with native Phleum pratense or Betula verrucosa allergen extract or formaldehyde- or glutaraldehyde-modified allergoids, adsorbed or unadsorbed to alum. After maturation, DC were co-cultivated with autologous CD4(+) T cells. Allergenicity was tested by leukotriene and histamine release of human basophils. Finally, in-vivo immunogenicity was analysed by IgG production of immunized mice. T cell proliferation as well as interleukin (IL)-4, IL-13, IL-10 and interferon (IFN)-γ production were strongly decreased using glutaraldehyde-modified allergoids, but did not differ between alum-adsorbed allergens or allergoids and the corresponding unadsorbed preparations. Glutaraldehyde modification also led to a decreased leukotriene and histamine release compared to native allergens, being further decreased by adsorption to alum. In vivo, immunogenicity was reduced for allergoids which could be partly restored by adsorption to alum. Our results suggest that adsorption of native allergens or modified allergoids to alum had no consistent adjuvant effect but led to a reduced allergenicity in vitro, while we observed an adjuvant effect regarding IgG production in vivo.

Gram-positive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory T cell responses.

BACKGROUND: Recently, it has been established that pollen grains contain Th2-enhancing activities besides allergens. OBJECTIVE: The aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert. METHODS: Timothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed. RESULTS: A complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram-negative bacteria that are known to favour Th1-directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram-positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram-positive bacteria induced maturation of DC as measured by up-regulation of CD80, CD83 and CD86 and by enhanced production of IL-6, IL-12p40 and TNF-α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4(+) T cells with supernatants of homogenized Gram-positive bacteria plus grass pollen allergen-pulsed DC led to an enhanced proliferation and production of IL-4, IL-13, IL-10, IL-17, IL-22 and IFN-γ production compared with T cells that were stimulated with allergen-pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram-positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.

An alternative allergen:adjuvant formulation potentiates the immunogenicity and reduces allergenicity of a novel subcutaneous immunotherapy product for treatment of grass-pollen allergy.

BACKGROUND: Subcutaneous specific immunotherapy (SCIT) has proven sustained clinical efficacy against allergy. The recommended regimen for SCIT is a gradual updosing over a period of weeks. Commonly, in commercial products for SCIT, the specific allergen is formulated with an adjuvant, most often in the form of aluminium hydroxide (AlOH). It has been shown that allergen-specific IgG antibodies are induced as a result of successful SIT. OBJECTIVE: To investigate the possibility of optimizing the formulation of AlOH-based grass-pollen allergy vaccines for SCIT in a way that allows for shorter updosing regimens while maintaining the immunogenicity of the vaccine. METHODS: Mice were immunized with various concentrations of Phleum pratense (Phl p) allergen extract and AlOH or a fixed dilution of the maintenance doses of one conventional and one alternatively formulated vaccine. The kinetics of Phl p-specific IgG antibody responses in serum and spleen T cell responses were determined. Allergenicity, measured as the ability of the formulations to activate human basophils, was also determined. In addition, human T cell responses and the expression of dendritic cell surface markers after vaccine challenge in vitro were analysed. RESULTS: Specific IgG antibody responses were shown to depend on the AlOH concentration, but not on the allergen concentrations. The immunogenicity of the conventional formulation and the alternative formulation was shown to be similar with regard to the in vivo-induced IgG and T cell responses. In contrast, the allergenicity of the alternative formulation was significantly reduced compared with the conventional formulation. CONCLUSION: The optimization of the formulation allows for administration of a lower dose of allergen while maintaining the immunogenicity of the product and at the same time reducing allergenicity. CLINICAL RELEVANCE: This study indicates that the optimization of the allergen and the adjuvant formulation could benefit the safety/efficacy profile and allow for shorter updosing.

Induction of hapten-specific tolerance by interleukin 10 in vivo.

Interleukin 10 (IL-10) is released during the induction phase of contact sensitivity and was shown in prior functional studies to convert epidermal Langerhans cells (LC) from potent inducers of primary immune responses to specifically tolerizing cells in vitro. To investigate whether IL-10 also subserves the function of a tolerizing agent in vivo ears of BALB/c or C3H mice were injected intradermally with 1-2 micrograms of recombinant mouse (rm)IL-10 8 h before epicutaneous application of 3% trinitrochlorobenzene (TNCB; a contact allergen). As a control, mice were injected with phosphate-buffered saline or IL-10 plus neutralizing amounts of anti-IL-10 mAb. 5 d later, mice were challenged with 1% TNCB on contralateral ears and ear swelling response was measured 24 h later. Whereas control-treated mice showed a normal ear swelling response to epicutaneous challenge (delta mm-2 = 25 +/- 5), ear swelling response of IL-10-treated animals was significantly inhibited (delta mm-2 = 3 +/- 2). Coinjection of IL-10-specific mAb together with rmIL-10 completely abrogated this effect. To differentiate between a state of nonresponsiveness and induction of tolerance by IL-10, mice initially treated with IL-10 and TNCB were resensitized with 3% TNCB in the absence of any treatment after 14 d of rest (group 1). Again mice were challenged 5 d later and ear swelling responses were tested. Whereas control mice treated with allergen alone (group 2) showed a good swelling response (delta mm-2 = 28 +/- 6), IL-10-treated mice (group 1) showed a minimal response towards application of allergen (delta mm-2 = 4 +/- 2). To show that anergy induction by IL-10 was antigen-specific, mice initially treated with IL-10 plus TNCB were exposed to 0.5% dinitrofluorobenzene (DNFB) 14 d later (group 1). After challenge with 0.1% DNFB, IL-10-treated mice showed an ear swelling response (delta mm-2 = 13 +/- 3; group 1) similar to that of control mice only sensitized with DNFB (delta mm-2 = 14 +/- 3; group 3). In an attempt to show the induction of antigen-specific tolerance in these mice in vitro, regional lymph nodes of mice initially treated with TNCB plus IL-10 (group 1) and control-treated mice (groups 2 and 3) were prepared and cultured in the presence of TNBS, dinitrobenzene sulfonate (DNBS), or medium to measure antigen-specific proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)

T cells expressing specific V beta elements regulate immunoglobulin E production and airways responsiveness in vivo.

The role of T cells expressing specific V beta elements was examined in the regulation of allergen-specific immunoglobulin (Ig)E production and airways responsiveness (AR). In BALB/c mice, inhalation of the allergen ovalbumin (OVA) induced an IgE anti-OVA response, immediate cutaneous reactivity, and increased AR. These results were associated with an expansion of V beta 8.1/8.2 T cells in local draining lymph nodes of the airways and the lung. Transfer of V beta 8.1/8.2 T cells from sensitized mice stimulated an IgE anti-OVA response, immediate cutaneous hypersensitivity, and increased AR in naive syngeneic recipients. In contrast, OVA-reactive V beta 2 T cells inhibited these effects. These data demonstrate for the first time that T cells with different V beta specificities play a critical role in the in vivo regulation of allergen-specific IgE production and AR.

Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 3.

BACKGROUND: Cockroach allergens play a very important role in allergic diseases, especially asthma. The major allergen of the American cockroach (Periplaneta americana), Per a 3, naturally occurs as isoforms of hexamers. OBJECTIVE: The aim of this study was to investigate whether the hexameric structures of Per a 3 influence their allergenicity and immunogenicity. METHODS: Therefore, we compared the different effects of native hexamers and dissociated monomers of cockroach haemolymph (HL), containing almost only Per a 3 proteins (HL-Per a 3), on proliferation and T-helper type 1 (Th1)/Th2 cytokine production of human CD4(+) T cells in co-culture with allergen-pulsed monocyte-derived autologous dendritic cells (DC) as well as the leukotriene release of basophils. RESULTS: In P. americana-sensitized and non-sensitized donors the HL-Per a 3 monomers were internalized faster by immature DC and induced higher proliferation and IFN-gamma production than the hexamers. While in non-sensitized donors IL-4 and IL-5 as well as IL-10 production were also increased after stimulation with monomeric HL-Per a 3-pulsed DC, Th2 cytokine and IL-10 production were only enhanced in P. americana-sensitized donors using hexameric HL-Per a 3-pulsed DC. Furthermore, in the leukotriene release assay the monomers were less effective than the hexamers. CONCLUSION: Our data indicate that the quaternary structure can influence both allergenicity and immunogenicity, also depending on the sensitization status. The monomeric variant of Per a 3 allergens could be a possible candidate for a specific immunotherapy because the IgE-mediated allergic reaction and the Th2-inducing capacity are diminished while the Th1-inducing capacity is retained.

Identification and induction of human keratinocyte-derived IL-12.

Interleukin 12 is a heterodimeric molecule that serves as a potent co-stimulator enhancing the development of Th1 cells. As one of the classical Th1 cell-mediated responses is contact sensitivity in skin, we wondered whether IL-12 might be produced by epidermal cells and serve as a mediator of this immune response. Using a sensitive, quantitative PCR technique we demonstrate that p35 chain mRNA of IL-12 is produced constitutively by human epidermal cells, whereas p40 chain mRNA can only be detected in epidermis treated with contact allergen, but not epidermis exposed to irritants or tolerogens. Time course studies showed a dramatic induction of IL-12 p40 mRNA 4 h after in vivo allergen treatment reaching peak strength after 6 h. In cell depletion assays we show that epidermal keratinocytes are the major source of this cytokine in the epidermis. This was further supported by analysis of mRNA derived from the human keratinocyte cell line HaCat expressing IL-12 p35 and p40 mRNA upon stimulation. The presence of bioactive IL-12 in supernatants derived from allergen-stimulated epidermal cells was demonstrated by IL-12-specific bioassay. Additional evidence for the functional importance of IL-12 in primary immune reactions in skin was obtained in allogeneic proliferation assays using human haptenated epidermal cells containing Langerhans cells as APC and allogeneic CD4+ T cells as responders. Anti-IL-12 mAb inhibited the proliferation of T cells by approximately 50%. In aggregate our data demonstrate that nonlymphoid keratinocytes are capable of producing functional IL-12 and provide evidence for the functional significance of IL-12 in primary immune responses in skin.

Development and transfer of immediate cutaneous hypersensitivity in mice exposed to aerosolized antigen.

We previously showed that BALB/c mice sensitized to ovalbumin (OVA) by brief daily inhalations of antigen over 10 consecutive days exhibit elevated antigen-specific serum IgE antibody levels and increased airways responsiveness. For the first time, we now show that animals sensitized in this fashion to either OVA or ragweed (RGW) develop immediate hypersensitivity skin test reactions when challenged 2 d after completion of the sensitization protocol. Skin testing, performed by direct assessment of wheal formation after intradermal injection of allergen, was sensitive and specific, since animals exposed to RGW by inhalation only responded to RGW, and OVA-sensitized animals responded only to OVA. Positive reactions were associated with mast cell degranulation, whereas control injections were not. Since only sensitized IgE high responder BALB/c mice but neither nonsensitized BALB/c mice nor OVA-sensitized IgE low responder SJL/J mice exhibited wheal responses, induction of OVA-specific IgE appeared to be essential for the mediation of OVA-specific immediate hypersensitivity reactions of the skin in this model. Passive cutaneous anaphylaxis (PCA) testing confirmed the presence of antigen-specific IgE in the serum. Mice that developed IgG (predominantly IgG2b) anti-OVA antibodies did not respond to OVA injection, indicating that OVA-specific IgG was not involved in this system. Further support for the role of IgE in the immediate hypersensitivity response included the wheal response to intradermal injection of anti-IgE antibody that occurred in OVA- and RGW-sensitized mice at 10-fold lower concentrations than in nonsensitized BALB/c mice and not in sensitized SJL/J mice. After transfer of mononuclear cells from peribronchial lymph nodes of OVA- or RGW-sensitized BALB/c mice, naive, syngeneic recipients developed antigen-specific IgE and specific immediate hypersensitivity responses, indicating that the local lymphoid tissue at the site of sensitization can transfer responsiveness to these allergens. These results demonstrate for the first time the ability to elicit and study IgE-mediated immediate skin hypersensitivity responses in the mouse and illustrate the association of increased antigen-specific and total serum IgE levels, airways hyperresponsiveness, and antigen-specific immediate cutaneous reactivity after sensitization to allergen via the airways.

Standardization of double blind placebo controlled food challenge with soy within a multicentre trial.

BACKGROUND: Multicentre trials investigating food allergies by double blind placebo controlled food challenges (DBPCFC) need standardized procedures, challenge meals and evaluation criteria. We aimed at developing a standardized approach for identifying patients with birch related soy allergy by means of DBPCFC to soy, including determination of threshold levels, in a multicentre setting. METHODS: Microbiologically stable soy challenge meals were composed of protein isolate with consistent Gly m 4 levels. Patients sensitized to main birch allergen Bet v 1 and concomitant sensitization to its soy homologue Gly m 4 underwent DBPCFC. Outcome was defined according to presence and/or absence of ten objective signs and intensity of eight subjective symptoms as measured by visual analogue scale (VAS). RESULTS: 138 adult subjects (63.8% female, mean age 38 years) underwent DBPCFC. Challenge meals and defined evaluation criteria showed good applicability in all centres involved. 45.7% presented with objective signs and 65.2% with subjective symptoms at soy challenge. Placebo challenge meals elicited non-cardiovascular objective signs in 11.6%. In 82 (59.4%) subjects DBPCFC was judged as positive. 70.7% of DPBCFC+ showed objective signs and 85.4% subjective symptoms at soy challenge. Subjective symptoms to soy challenge meal in DBPCFC+ subjects started at significantly lower dose levels than objective signs (p < 0.001). Median cumulative eliciting doses for first objective signs in DBPCFC+ subjects were 4.7 g [0.7-24.7] and 0.7 g [0.2-4.7] total soy protein for first subjective symptoms (p = 0.01). CONCLUSIONS: We present the hitherto largest group of adults with Bet v 1 and Gly m 4 sensitization being investigated by DBPCFC. In this type of food allergy evaluation of DBPCFC outcome should not only include monitoring of objective signs but also scoring of subjective symptoms. Our data may contribute to standardize DBPCFC in pollen-related food allergy in multicentre settings. TRIAL REGISTRATION: EudraCT: 2009-011737-27.

Epidermal cells enhance interleukin 4 and immunoglobulin E production after stimulation with protein allergen.

Exposure to certain allergens via epithelial tissues is the primary route for the induction of immunoglobulin E-dependent allergies of the immediate type associated with atopic diseases. In order to address the question whether and how epithelial cells might contribute to the induction or increase of TH2-dependent IgE production, we performed co-culture experiments of syngeneic epidermal cells and cells from the associated lymphoid tissue or spleen (responder cells) of BALB/c mice primed with ovalbumin in vivo. In the presence of ovalbumin in vitro, immunoglobulin E but not immunoglobulin G2a production was significantly enhanced by the addition of epidermal cells, and separation of epidermal cells from responder cells by a membrane that prevented cellular contacts or addition of antibodies against intercellular adhesion molecule-1 reduced the enhancement of immunoglobulin E production induced by epidermal cells. Depletion of major histocompatibility complex class II+ antigen presenting Langerhans cells from the epidermal cells prior to co-culture also reduced the enhancement of immunoglobulin E production induced by epidermal cells. The enhanced immunoglobulin E production was dependent on the induction of TH2 cell-derived interleukin-4 detected in co-cultures because it was completely inhibited after addition of anti-interleukin-4 antibodies that also lead to increased immunoglobulin G2a production. Whereas interleukin-4 was not produced by epidermal cells, interleukin-10 seemed to be one important mediator contributed by epidermal cells. Interleukin-10 skewed the response toward a TH2-mediated IgE response because antibodies against interleukin-10 inhibited interleukin-4 and immunoglobulin E production, whereas they enhanced interferon-gamma and immunoglobulin G2a production.

Cutaneous exposure to the superantigen staphylococcal enterotoxin B elicits a T-cell-dependent inflammatory response.

We analyzed the impact of superantigens secreted by skin-colonizing Staphylococci on the skin and the associated lymphoid tissue following epicutaneous application and intracutaneous injection of small amounts of staphylococcal enterotoxin B (SEB). A single intracutaneous injection of 50 ng of SEB elicited a strong inflammatory response in the skin of BALB/c mice. Three to 6 h later, we observed langerhans cell activation, mast cell degranulation, vasodilation, upregulation of ICAM-1, and induction of VCAM-1 on dermal blood vessels, with vascular adhesion of granulocytes. by 12 to 24 h, cell infiltration of the dermis increased, reaching the epidermis. Among the infiltrating leukocytes, a substantial number of eosinophils was found. After 48 h, the infiltrate was dominated by mononuclear cells. The response to SEB was dose-dependent, and signs of inflammation slowly disappeared over 5 to 7 days. Although the induction of VCAM-1 on dermal blood vessels suggested a role for interleukin-1/tumor necrosis factor-alpha in this reaction, the activation of monocytes/macrophages was not able to substitute for lymphocytes, as severe combined immunodeficiency (SCID) mice (which are lymphocyte-deficient) did not mount an inflammatory skin response to intradermal injection of SEB. The fact that nude mice (T-cell-deficient) also did not mount an inflammatory response to SEB indicated the T-cell dependency of the response. The V beta specificity of the SEB effect was demonstrated by the fact that SJL/J mice, which lack V beta 8+ T cells (the major SEB-reactive T cell population in mice), exhibited much weaker responses. Deletion or tolerization of SEB-reactive V beta T cells was not observed after a single intradermal injection of such minute amounts of SEB.

Modulation of contact sensitivity responses by bacterial superantigen.

Superantigens are potent modulators of the immune system, especially T cells. Therefore, we determined the influence of superantigens on the T-cell-mediated immune response, contact sensitivity. We chose the combination of staphylococcal enterotoxin B (SEB) as superantigen and 2,4-dinitrofluorbenzene (DNFB) as the contact sensitizer, because in BALB/c mice SEB reacts almost exclusively with V beta 8+ T cells, and these cells are capable of transferring contact sensitivity to DNFB from sensitized donors to naive syngeneic recipients. Pretreatment with a single intradermal injection of 50 ng SEB 24 h before DNFB exposure at the same site on the lower abdomen enhanced the induction of contact sensitivity: its intradermal injection permitted sensitization with non-sensitizing concentrations of DNFB as assessed by ear swelling responses after challenge with DNFB. In contrast, pretreatment with repeated intradermal injections of 50 ng SEB every other day over at least 1 week inhibited the induction of contact sensitivity following sensitization. The enhancing effect of SEB may be explained by the creation of a proinflammatory milieu in the skin after a single intradermal injection of the bacterial toxin, whereas the inhibitory effect may be due to tolerization of V beta 8+ T cells. The data indicate that products of skin-colonizing bacteria that can serve as superantigens are able to augment or inhibit the development of contact sensitivity.

Flow-cytometric screening for the modulation of receptor-mediated endocytosis in human dendritic cells: implications for the development of an in vitro technique for predictive testing of contact sensitizers.

The aim of this study was to explore the usefulness of human blood dendritic cells (DC) in the development of an in vitro model for predictive testing of contact sensitizers. A method was established to monitor the influence of chemicals on the intracellular targeting of antibody-crosslinked MHC class II molecules after their uptake by human DC. Using a three-colour flow-cytometric technique, freshly prepared DC were distinguished from other MHC class II-bearing cell types such as B-cells and monocytes in unseparated mononuclear cell suspensions of healthy volunteers. The assay is based on the pH-sensitivity of internalized fluorescein-coupled MHC class II specific antibodies. Quenching of fluorescence intensity due to internalization into acidic intracellular compartments was observed with untreated DC whereas internalization into less acidic structures following stimulation with strong contact sensitizers ensured that the fluorescence intensity was conserved. The usefulness of this approach for predictive testing of the preservatives MI/MCI, imidazolidinyl urea, methyl-4-hydroxy-benzoate and 2-phenoxyethanol in comparison to the strong allergen DNFB and the irritants sodium lauryl sulphate and dithranol was explored. Whereas low concentrations of MI/MCI resembled the strong allergen DNFB, high concentrations of imidazolidinyl urea were required for a moderate response. Methyl-4-hydroxy-benzoate and 2-phenoxyethanol as well as the irritants SLS and dithranol failed to induce a significant effect in this assay. The non-responsiveness to the latter compounds reflected their minor or absent capacity to induce contact hypersensitivity in humans, whereas DNFB, MI/MCI and imidazolidinyl urea are well established contact sensitizers. These data suggest that the capacity of a chemical to modulate endocytotic mechanisms in dendritic cells in vitro seems to reflect the probability of that substance acting as a hapten in vivo.

Ultrastructural cytochemical visualization of chromium in the skin of sensitized guinea pigs.

Using a modified sulfide silver method for electron microscopy, the intraepidermal and intracellular localization of epicutaneously applied potassium dichromate was investigated at varying times in sensitized and nonsensitized guinea pigs. The hapten penetrated rapidly into the epidermis. There was a homogeneous extra- and intracellular staining of the keratinocytes in the upper epidermis. The basal and suprabasal cells, by contrast, exhibited a predominant extracellular and plasma membrane localization of the silver grains. This membrane staining pattern was also observed in the Langerhans cells showing cellular and endocytotic activation in the sensitized animals. No specific cellular uptake of the hapten by the Langerhans cells was found. These results demonstrate that the epicutaneous application of chromate resulted in a characteristic intraepidermal distribution which may be related to the epidermal conversion of the hexavalent chromate to the immunogenic trivalent form. Moreover, the absent intracellular localization of the hapten in the activated Langerhans cells supports the notion that contact allergens can be presented to T cells without prior intracellular processing.

Detection of increased tyrosine phosphorylation in murine Langerhans cells after stimulation with contact sensitizers.

The signalling pathways in epidermal Langerhans cells (LC) during activation by contact sensitizers are poorly understood. Recently, we have described an increased phosphorylation of tyrosine residues in human MHC class II-positive cells in vitro following stimulation with contact sensitizers. In the study reported here the formation of phosphotyrosine (p-tyr) in murine epidermal LC upon stimulation with contact sensitizers was examined. By the use of a flow cytometric technique a significant increase in p-tyr was demonstrated in LC stimulated in vitro with the strong contact sensitizers TNCB (2,4,6-trinitro-chlorobenzene) and MCI/MI (5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone) but not after treatment with the irritants sodium lauryl sulphate or benzoic acid. The protein tyrosine kinase inhibitors genistein and tyrphostin A9, but not tyrphostin AG 1288, were able to block this process significantly. Similar results were obtained using the LC-like dendritic cell line XS52. In addition, Western blot analysis on XS52 cells revealed a selective phosphorylation of two protein bands with a molecular weight between 50 and 60 kDa following stimulation with TNCB. These results demonstrate that contact sensitizers induce an increased phosphorylation of tyrosine residues in murine LC and can be used as the basis for in vivo studies using inhibitors for signal transduction pathways for prevention of primary sensitization to contact sensitizers.

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.