Systemic ketoconazole is an effective treatment of atopic dermatitis with IgE-mediated hypersensitivity to yeasts

BACKGROUND: IgE-mediated hypersensitivity to yeasts is often seen in atopic dermatitis (AD) patients, especially when dermatitis is located in the head, neck, and shoulder regions. Two studies have shown the efficacy of ketoconazole in the treatment of this type of AD, in contrast to results of topical treatment. The objective was to assess the clinical efficacy of antifungal treatment in AD in a randomized, double-blind, placebo-controlled study with oral ketoconazole and yeast-specific IgE levels and saprophytic yeast growth monitored simultaneously. METHODS: Eighty patients with AD and positive P. ovale and/or C. albicans RAST/skin prick test results were randomized to receive ketoconazole or placebo for 30 days. The yeast growth of skin and pharynx; P. ovale, C. albicans, andS. cerevisiae RAST; serum total IgE; and the severity of the eczema (SCORAD) were assessed at day 0 and thereafter at 1 and 3 months. RESULTS: A significant improvement was seen in the SCORAD scale in the ketoconazole group at the second visit in comparison to the first visit (P<0.0005; n=36), but not in the placebo group (n=39). Of the individual determinants of the SCORAD, itching (P<0.005), the extent of dermatitis (area percentage), excoriation, lichenification (P<0.01), erythema, papulation, and dryness (P<0.05) improved significantly in the ketoconazole group. In the placebo group, only the extent of dermatitis (area percentage) decreased significantly (P<0.05). In the ketoconazole group, the number of positive P. ovale cultures decreased from 60% to 31% (n=35) compared to the placebo group (64% to 56%; n=39). The clinical response was most significant in female patients with positive yeast cultures. CONCLUSION: Saprophytic yeasts may be a source of allergens in AD. Thus, patients with AD, yeast growth, and elevated IgE levels to yeasts should be offered antifungal treatment.     

Cross-reacting IgE and IgG antibodies to Pityrosporum ovale mannan and other yeasts in atopic dermatitis.

Atopic dermatitis (AD) patients often demonstrate positive skin prick test results and serum IgE antibodies to a range of different yeasts. This has been thought to be due to cross-reactivity. In this study, the cross-reactivity of IgE and IgG antibodies between mannan and crude antigens of Pityrosporum ovale, Candida albicans, and Saccharomyces cerevisiae and crude antigens of Cryptococcus albidus and Rhodotorula rubra was examined by RAST and ELISA inhibition with two serum pools of AD patients. We found cross-reacting IgE and IgG antibodies. In the IgE response, the main cross-reacting pattern was the mannan region, although inhibition could be achieved also with crude antigens of C. albicans, S. cerevisiae, and, to some extent, C. albidus. P. ovale was the most potent inhibitor of IgE-binding components, and against it the highest IgE antibody levels were detected in AD serum pools. In contrast, C. albicans was found to be the most important inducer of IgG antibodies, since the IgG level against P. ovale mannan in both AD serum pools was very low. Cross-reacting antibodies were also seen in ELISA inhibition with both crude and mannan antigens, but since the IgG antibody level of P. ovale mannan in AD serum pools was low, further studies are needed to confirm the IgG results.     

Candida albicans mannan- and protein-induced humoral, cellular and cytokine responses in atopic dermatitis patients

BACKGROUND: The cytokine observed most often in atopic dermatitis (AD) is IL-4, but a role for IL-5 and IFN-gamma in the late and delayed phase reactions has been suggested. In AD with head, neck and shoulder distribution, hypersensitivity to saprophytic yeasts is an important pathogenetic factor. The yeast allergens include both the mannan polysaccharides and the proteins. Mannans are major cross-reacting allergens likely to be involved in the pathogenesis of AD. OBJECTIVE: To characterize the humoral, lymphoproliferative and cytokine (IL-2, 4, 5 and IFN-gamma) responses of peripheral blood mononuclear cells (PBMCs) induced by Candida albicans mannan and protein antigens in AD. METHODS: Fifteen AD patients and seven healthy controls were included. Ficoll-isolated PBMCs were stimulated by PHA and laboratory-generated mannan and protein extracts of C. albicans. Lymphocyte proliferation was measured and cytokine production was studied by ELISA. The antigen-specific IgG and IgE antibodies were analysed by ELISA and nitrocellulose RAST. RESULTS: In AD mannan (P < 0.005) and protein (P < 0.002), specific IgE levels were higher than in healthy controls. Both mannan and protein-specific lymphoproliferations (both: P < 0.02) were higher in AD than in healthy controls. Mannan, but not protein, induced long lasting IL-2 and IL-4 productions from 24 h lasting up to 66-96 h and IL-5 and IFN-gamma productions with elevated levels at 66 and 96 h. The mannan-induced IL-2 (P = 0.015) and IFN-gamma (P < 0.005) were increased in AD as compared with healthy controls. Significant correlations were seen between the protein-induced proliferation responses and both serum total IgE (r = 0.59, P < 0.01) and protein-specific IgE (r = 0.65, P < 0.005). The mannan-induced IL-2 responses correlated with the specific IgE (r = 0.62, P < 0.01) and proliferation (r = 0.51, P < 0.02) and S-IgE level (r = 0.71, P < 0. 002). Mannan-induced IL-4 and IFN-gamma productions also correlated (r = 0.43, P < 0.05). CONCLUSIONS: C. albicans mannan induced elevated IL-2 and IFN-gamma responses in AD patients. The correlations of the cytokine responses with mannan-induced IgE and proliferation responses suggest that C. albicans mannan induced TH1 type cytokine responses are involved in AD.     

Allergens of Pityrosporum ovale and Candida albicans

In atopic dermatitis (AD), a high prevalence has been reported of type I reactions and specific IgE to extracts of the commensal lipophilic skin yeast Pityrosporum ovale. In the present study, a highly significant correlation (r=0.77) was found between levels of anti- P , ovale IgE and of IgE reacting with extracts of Candida albicans , both measured by a sensitive ELISA method. In a series of 128 AD sera, 34 sera reacted positively with both yeast extracts, 38 reacted with P. ovale but not with C. albicans, and only one of the 56 anti-F. ovafe-negative sera showed a very weak reaction with C. albicans . The correlation was due to a marked cross-reactivity, as shown by inhibition ELISA. Eluid-phase preincubation of double-positive sera with either of the two yeast extracts resulted in a dose-dependent, and at high concentrations complete, inhibition of the IgE reactions with both coated P. ovale and C. albicans allergens. Mutual inhibition of IgE-binding could also be achieved with pools of glycoproteins and/or polysaccharides isolated from the crude extracts by Con A affinity chromatography. P. ovale allergens were, however, more potent fiuid-phase inhibitors than the corresponding C. albicans components. The apparently higher avidity for P. ovale allergens suggests that these antiyeast IgE antibodies in AD result from sensitization to P. ovale and cross-react with C albicans .     

IgE antibodies to protein and mannan antigens of pityrosporum ovale in atopic dermatitis patients

Background Pityrosporum ovale is a common saprophyte on the skin capable of inducing IgE antibody production in atopic dermatitis (AD) patients. Allergens ofP. ovale have been examined in several studies, but consensus on them is lacking. Objective This study was carried out to obtain more information about the IgE antibody response against P. ovale. including niunnun. Methods Sera from 64 AD patients and 10 healthy controls were analysed with immuno-blotting and the nitrocellulose radio allergosorbent test (RAST) method specifically developed to detect antimannan P. ovale IgE antibodies. Results In immunoblotting a total of 39 different IgE stained protein bands were seen. A high molecular weight staining was also seen especially in patients who displayed elevated mannan P. ovale RAST values. The most commonly stained protein bands in immunoblotting were 9 and 96 kD bands with antibodies in 73 and 65% of AD patients who had been positive in commercial P. orbiculare RAST with total serum IgE less than 4000 kU/I. Mannan RAST appeared positive in 77% of them. Positive immunoblotting to either of these bands was seen in 90% and, if added with staining with ihe 20 kD band, in 100% of these AD patients. A comhination of 9 kD IgE staining and mannan P. ovale RAST was positive in 92% of the patients and % kD and mannan P. ovale RAST in 85% of the patients. Conclusion It is evident that P. ovale has several allergens, the 9. 96 and 20 kD regions being the most important. According to our results mannan is also an important allegen of P. ovale     

Immediate hypersensitivity to Malassezia furfur and Candida albicans mannans in vivo and in vitro

BACKGROUND: Elevated and correlative Malassezia furfur (M. furfur) and Candida albicans (C. albicans) mannan-specific IgE have been demonstrated in atopic eczema dermatitis syndrome (AEDS) of the head, neck and shoulder (HNS) region of the skin. The significance of these antibodies in vivo has not been demonstrated. METHODS: Sixty-five AEDS patients with HNS distribution were included. Serum total IgE (S-IgE) and yeast antigen-specific (Cetavlon-purified mannan and whole extract antigens of M. furfur and C. albicans) IgE were measured and skin prick tests (SPT) were performed with the yeast antigens. RESULTS: Mannan-specific IgE and SPT were positive in 51 and 48% of patients with M. furfur and in 42 and 22% with C. albicans, respectively. Whole extract-specific IgE and SPT were positive in 85 and 95% of patients with M. furfur and in 91 and 57% with C. albicans, respectively. The highest correlation between specific IgE and SPT was seen with M. furfur mannan (r = 0.60; P < 0.0001). Both M. furfur mannan-specific IgE (r = 0.76; P < 0.0001) and SPT (r = 0.44; P = 0.0005) correlated with S-IgE. CONCLUSIONS: Mannan-induced immediate hypersensitivity in vivo was demonstrated in SPT. The significant correlation between M. furfur mannan-specific IgE and SPT suggests that mannan is an important allergen in yeast hypersensitive AEDS in vivo.     

Study of the Th1/Th2 balance, including IL-10 production, in cultures of peripheral blood mononuclear cells from birch-pollen-allergic patients

BACKGROUND: Excessive production of interleukin (IL)-4, IL-5, IL-10, and IL-13 is thought to be important in the development of allergy and asthma. The objective of this investigation was to study Th1/Th2-like cytokine profiles in vitro in seven patients allergic to birch pollen and six nonallergic controls during the birch-pollen season. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated and cultured with birch-pollen extract (BPE) or tetanus toxoid (TT) for 7 days, harvested, and restimulated with the mitogens phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) for 24 h. Cytokine production was determined by ELISA, and logarithmic cytokine ratios were compared between the two groups and between the antigens. RESULTS: In the allergic group, the cultures prestimulated with BPE had a more Th2-like cytokine response than the TT-prestimulated cultures; i.e., lower IFN-gamma and higher IL-10 production (P<0.05), as well as higher IL-5/IFN-gamma and IL-13/ IFN-gamma ratios (P<0.05). There were also significantly higher IL-4/IFN-gamma (P<0.005) and IL-5/IFN-gamma (P<0.05) ratios in BPE-stimulated cultures in the allergic group than in the control group. The IL-4 and IL-13 production in vitro correlated with the specific serum IgE levels. CONCLUSIONS: BPE stimulation induces a Th2-like cytokine response by PBMC isolated during the pollen season from birch-pollen-allergic patients, indicating a Th2-type immune response to birch pollen in vivo.     

The relevance of skin prick tests for Pityrosporum ovale in patients with head and neck dermatitis

BACKGROUND: Patients with atopic dermatitis often develop immunoglobulin E antibodies against the yeast Pityrosporum ovale. This organism may produce positive skin prick reactions in a higher rate in patients with atopic dermatitis of the head, scalp, and neck region. METHODS: We investigated whether positive prick tests to P. ovale were associated with a specific localization in the head and neck region. A total of 589 consecutive patients were prick tested with a P. ovale extract from ALK Abelló. RESULTS: Thirty-seven patients (6.3%) showed a positive reaction to the P. ovale extract. We could not find significant differences in the localizations of the dermatitis and the pattern of reaction to the tested intracutaneous allergens between the 37 patients positive to P. ovale and the control group of 55 subjects with negative reactions. In a subgroup, we found an elevated level of P. ovale-specific IgE in 100.0% of the patients with head and neck dermatitis, compared with 13.6% in the atopic dermatitis patients with lesions in any other localization. CONCLUSIONS: Our results clearly show that P. ovale-specific IgE is strongly related to the head and neck localization of atopic dermatitis, but RAST seems more sensitive than a prick test with the extract we used.     

The skin fungus-induced Th1- and Th2-related cytokine, chemokine and prostaglandin E2 production in peripheral blood mononuclear cells from patients with atopic dermatitis and psoriasis vulgaris

BACKGROUND: It is suggested that skin fungi may be involved in the development of atopic dermatitis (AD) and psoriasis vulgaris (PV). OBJECTIVE: We studied skin fungus-induced Th1- or Th2-related cytokine, chemokine and prostaglandin E2 (PGE2) secretion in peripheral blood mononuclear cells (PBMC) from patients with AD and PV and normal subjects. METHODS: PBMC were cultured with the extracts of Malassezia furfur (MF), Candida albicans (CA) and Trichophyton rubrum (TR). The cytokine, chemokine and PGE2 amounts in the supernatants were measured by enzyme-linked immunosorbent assays. RESULTS: MF induced IL-4 and macrophage-derived chemokine (MDC) secretion in AD patients, while induced IFN-gamma and interferon-inducible protein of 10 kDa (IP-10) secretion in PV patients, however, did not induce either secretion in normal subjects. CA induced IL-4, MDC, IFN-gamma and IP-10 secretion in AD and PV patients and normal subjects. In AD patients, the magnitude of IL-4 and MDC responses to CA was higher than that to MF. Compared with PV patients and normal subjects, the magnitude of IL-4 and MDC responses to CA was higher while that of IFN-gamma and IP-10 responses to CA was lower in AD patients. TR induced moderate IL-4 and MDC secretion only in AD patients. The three fungi induced higher levels of PGE2 secretion in AD patients than in PV patients and normal subjects. Cyclooxygenase-2 inhibitor NS-398 suppressed PGE2 responses to MF, CA and TR, and partially suppressed IL-4 and MDC responses to MF, CA and TR, while enhanced IFN-gamma and IP-10 responses to CA in AD patients, and these effects of NS-398 were reversed by cyclic AMP analogue. CONCLUSION: AD patients manifest Th2-skewed responses to MF, CA and TR, which may be partially attributable to the enhanced PGE2 responses to these fungi. PV patients manifest Th1-skewed responses to MF.     

PPARgamma expression profile and its cytokine driven regulation in atopic dermatitis

BACKGROUND: Recent studies point to the pathophysiological role of the peroxisome proliferators-activated receptor gamma (PPARgamma) in the inflammatory immune response. We have showed that activation of PPARgamma by specific ligands attenuates the allergic immune response via monocytes and lymphocytes. The objective of this study was to analyse the PPARgamma expression and its regulation via inflammatory cytokines. METHODS: We examined the PPARgamma expression in the lesional and nonlesional skin of atopic patients by immunohistochemistry. The expression patterns of PPARgamma mRNA and its isoforms were investigated in peripheral mononuclear blood cells of atopic and nonatopic donors and in cytokine-stimulated populations by quantitative real-time RT-PCR. RESULTS: Our data show an increased PPARgamma expression in lesional skin from atopic dermatitis patients. The analysis of PPARgamma mRNA reveals a significantly up-regulated expression of PPARgamma1 but not of PPARgamma2 in monocytes and CD4(+) T-cells from atopic dermatitis patients. Furthermore, we demonstrate that Th-cytokines, like IL-4, IL-13 and IFNgamma, which regulate the biphasic atopic immune response, directly regulate the expression of PPARgamma1. CONCLUSION: Taken together, these data demonstrate that PPARgamma isoforms are differently expressed and regulated by the local cytokine-milieu. Whether the increased expression of the PPARgamma1 receptor may be beneficial or not for a PPARgamma ligand-based treatment of atopic dermatitis, is currently under investigation.     

Use of specific IgE in assessing the relevance of fungal and dust mite allergens to atopic dermatitis: a comparison with asthmatic and nonasthmatic control subjects

BACKGROUND: Although allergens have been implicated as aggravating factors in atopic dermatitis (AD), there is little epidemiologic data on the significance of specific IgE. OBJECTIVE: We sought to compare sensitization to dust mite and fungi between patients with AD and asthmatic and nonasthmatic control subjects. METHODS: Total IgE and specific IgE to Dermatophagoides pteronyssinus, Alternaria alternata, Aspergillus fumigatus, Candida albicans, Malassezia furfur, and Trichophyton rubrum were measured in 73 patients with moderate to severe AD. Total IgE and IgE specific for D pteronyssinus, A alternata, and M furfur were also measured in sera from 156 asthmatic and 212 nonasthmatic control subjects. RESULTS: Positive correlations were found between total IgE and IgE antibodies specific for each of the antigens. IgE specific for M furfur was observed more frequently in adults compared with children with AD (P <.01). AD sera had higher levels of total IgE and a higher prevalence of positive sera to D pteronyssinus (95% vs 42% and 17% for subjects with AD, asthmatic subjects, and nonasthmatic subjects, respectively), M furfur (53% vs 1% and 0.5%), and A alternata (49% vs 29% and 18%). Among the sera from subjects allergic to mites, the contribution of IgE specific for D pteronyssinus to the total IgE levels was similar regardless of the clinical status. CONCLUSIONS: Our results demonstrate that moderate-to-severe AD is strongly associated with sensitization to dust mite andM furfur (odds ratios, 45.6 and 132 vs pooled control sera). These results suggest that both environmental allergens and colonizing fungi contribute to the severity of disease, which is consistent with the view that mite avoidance and antifungal treatment can be beneficial in the treatment of these patients.     

Reduced IFNγ production in response to IL-12 stimulation and/or reduced IL-12 production in atopic patients

BACKGROUND: Several studies have shown that interleukin (IL)-4 and interferon-gamma (IFNgamma) are important for the regulation of IgE production and that IL-12 induces IFNgamma. OBJECTIVE: IFNgamma production in response to IL-12 stimulation and IL-12 production were investigated in peripheral blood mononuclear cells (PBMCs) of atopic patients with various levels of serum IgE. METHODS: Cytokine production from PBMCs was measured following stimulation with a nonspecific stimulator (phytohemagglutinin: PHA), a specific stimulator (Der f 1) and IL-12 in 17 healthy controls and 23 atopic patients with various serum IgE levels. RESULTS: The IFNgamma production by IL-12-stimulated PBMCs in the atopic group was lower than that in the control group. Furthermore, the serum IgE level was negatively correlated with IFNgamma production by PBMCs stimulated with IL-12 (P < 0.001), and with IL-12 production by PBMCs stimulated with Der f 1 (P < 0.001). Although the IFNgamma concentrations by PHA-stimulated PBMCs were correlated with those by IL-12-stimulated PBMCs, there were differences in several patients. CONCLUSIONS: Our results indicated that atopic patients may have some abnormality in the IL-12-IFNgamma loop. It was shown that the elevation in IgE levels in atopic patients may be due to reduced IFNgamma production in response to IL-12 stimulation and/or due to reduced IL-12 production.     

Transforming growth factor‐bβ1 suppresses atopic dermatitis‐like skin lesions in NC/Nga mice

BACKGROUND: Atopic dermatitis is a chronic, relapsing inflammatory disorder characterized by pruritic and eczematous skin lesions. Transforming growth factor (TGF)-beta1 has been implicated in the suppression of inflammatory responses. OBJECTIVE: The purpose of this study is to determine whether TGF-beta1 suppresses skin lesions in a mouse model of atopic dermatitis. METHODS: We used the NC/Nga strain of mice as an in vivo model of atopic dermatitis. The effects of exogenous TGF-beta1 on atopic dermatitis-like skin lesions in NC/Nga mice were evaluated clinically, histologically and immunologically. RESULTS: Subcutaneous injection of recombinant TGF-beta1 macroscopically suppressed eczematous skin lesions in NC/Nga mice associated with reduced serum immunoglobulin E (IgE) levels. Histological analysis showed that TGF-beta1 significantly inhibited the infiltration of inflammatory cells such as mast cells and eosinophils into the skin of NC/Nga mice. Spontaneous interferon (IFN)-gamma production from splenocytes of NC/Nga mice was down-regulated by the treatment with TGF-beta1 and neutralizing antibody against IFN-gamma inhibited skin lesions in NC/Nga mice. The inhibitory effect of TGF-beta1 on the skin lesions lasted at least 1 week after cessation of the treatment. CONCLUSION: These findings indicate that TGF-beta1 suppressed atopic dermatitis-like skin lesions in NC/Nga mice at least in part through down-regulation of IFN-gamma. These results suggest that TGF-beta1 may have a therapeutic potential for atopic dermatitis.     

Characterization of chemokine receptor expression and cytokine production in circulating CD4+ T cells from patients with atopic dermatitis: up-regulation of C-C chemokine receptor 4 in atopic dermatitis

BACKGROUND: Th2 and Th1 cells have been suggested to express CCR3/CCR4 and CCR5/CXCR3, respectively. OBJECTIVE: We examined CCR3, CCR4, CCR5 and CXCR3 expression and cytokine production in peripheral blood CD4+ T cells from patients with atopic dermatitis (AD), which has been postulated to be a Th2-type cell-mediated disease, and then analysed the possible correlation between these values and the levels of several clinical parameters. METHODS: Intracellular cytokine production and chemokine receptor expression in peripheral blood CD4+ T cells from 40 AD patients and 20 sex- and age-matched healthy control subjects were studied by flow cytometry. RESULTS: The frequencies of IL-4- and IL-13-producing CD4+ T cells from patients with AD were significantly higher than those from healthy control subjects (IL-4:3.9 +/- 2.1% vs. 1.6 +/- 0.7%, P = 0.0005, IL-13:4.0 +/- 2.1% vs. 1.8 +/- 0.8%, P = 0.0023), whereas the frequencies of IL-2- and IFN-gamma-producing CD4+ T cells were significantly decreased in AD patients (IL-2:38.1 +/- 10.3% vs. 51.3 +/- 6.3%, P = 0.0003, IFN-gamma: 9.9 +/- 3.5% vs. 26.4 +/- 4.6%, P < 0.0001). The percentage of CCR4+ cells in CD4+ CD45RO+ T cells in AD patients was significantly higher than that in healthy control subjects (24.4 +/- 8.0% vs. 10.9 +/- 2.3%, P < 0.0001) and was correlated positively with the total serum IgE, serum lactic dehydrogenase (LDH) level, eosinophil number, eruption score, and IL-4 and IL-13 secretion in CD4+ T cells, and inversely with IL-2 and IFN-gamma secretion in CD4+ T cells. In contrast, CCR3 was not detected on circulating CD4+ T cells even in AD patients. On the other hand, the percentage of CCR5+ or CXCR3+ cells in CD4+ CD45RO+ T cells in AD patients was significantly decreased (CCR5:23.2 +/- 7.0% vs. 28.4 +/- 5.4%, P = 0.023, CXCR3:29.9 +/- 11.4% vs. 38.5 +/- 6.7%, P = 0.028) and was positively correlated with eruption score (P < 0.05). Multiple regression analyses showed that the percentage of CCR4 expression highly correlated with serum IgE, LDH, eosinophil number and eruption in AD patients. CONCLUSION: CCR4+ cells might be involved in the aetiopathogenesis of AD.     

IgE responses to exogenous and endogenous allergens in atopic dermatitis patients under long‐term systemic cyclosporine A treatment

Atopic dermatitis (AD) patients mount IgE antibody responses to a variety of environmental allergens and also to autoantigens. We analyzed serum samples from four AD patients who had received oral cyclosporine A (CyA) treatment for up to 17 months regarding IgE autoreactivity to nitrocellulose‐blotted human epithelial cell extracts and IgE levels to environmental allergens by quantitative ImmunoCap measurements. Skin inflammation was assessed by SCORAD. During full‐dose treatment, a strong reduction in T‐cell‐mediated skin symptoms was observed which reappeared when CyA treatment was reduced or stopped. The intensity of IgE autoreactivity seemed to follow skin inflammation as it was reduced during full‐dose treatment and increased upon inflammation. Interestingly, IgE levels to exogenous allergens were boosted by allergen exposure, declined thereafter, and seemed to be unaffected by CyA. Our data thus indicate that allergen‐specific IgE production is boosted by allergen contact and cannot be reduced by CyA‐mediated T‐cell suppression.     

American cockroach Cr-PI allergen induces lymphocyte proliferation and cytokine production in atopic patients

Background Previous studies have shown cockroach-induced antigen-specific IgF-mediated asthma. In cockroach-Infested areas, more then 50% of asthmatic subjects may have positive skin reactions to this allergen. Partial purified Cr-PI allergen from American cockroaches contains allergens with molecular weights of 72 and 78k Da; however, little is known about its eifect on the lymphoeyte proliferation and cytokine production. Objective IgE synthesis is known to be regulated by interleukin-4 (IL-4) and interferon gamma (IFN-γ). Therefore, we studied Cr-PI allergen-induced cytokine production in atopic patients and healthy normal controls to understand each factors’ role in the disease. Methods Peripheral blood mononuclear cells (PBMC) from cockroach skin-sensitive patients and controls were stimulated with mitogen and Cr-PI for proliferative response and cytokine production. Cr-PI antigen-specific T-cell cultures of atopic patients and healthy normal controls were used to test C r-PI-lnduced proliferation and cytokine mKNA expression. Results PMBC ot atopic subjects showed a significantly (P < 0.01) higher stimulation index for Cr-PI induced proliferation (SI = l l.8.3.7) when compared with that of non-atopic subjects (SI =4.1 ± 0.8) and cord bloods (SI = 2.1 ± 0.4). Cr-Pl-induced IL-4 was observed only in the PBMC of atopic patients, whereas Cr-PI-induced IFNγ was detected in both atopic patients and normal controls. Likewise, Cr-PI-induced IL-4 mRNA expression in T-cell cultures was detected in all atopies but only one of nine controls. Conclusion IL-4 mKNA expression and IL-4 production in PBMC and T-cell cultures of atopic patients showed good correlation with clinical symptoms, skin-reactivity, specific IgE and proliferative response to Cr-PL These results suggests that cockroach allergen may be a hidden cause of asthma and other atopie diseases.     

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8⁻ T cells, IL-2 and interferon-gamma (IFN-γ) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8⁺ T cells IL-2 was optimally induced after 10 h and IFN-γ after 6 h. The levels of IL-2 and IFN-γ in CD8⁺ and CD8⁻ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2⁺ (range 9.7–41.3) and CD3/IFN-γ⁺ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3⁺/CD8⁺ peripheral blood T cells expressing IFN-γ and an increased percentage of CD3⁺/CD8⁻ T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.     

IgE-, IgA- and IgG-antibody responses to carbohydrate and protein antigens of Candida albicans in asthmatic children

Analysis of IgE, IgA and IgG antibodies directed against Candida albicans antigens in 28 asthmatic children was performed with immunoblotting after SDS-PAGE. Analysis with the purified cytoplasmic protein fraction revealed a major protein allergen with an MW of 46 kD. In addition to the major allergen, 15 other antigenic bands with molecular weights between 16 and 135 kD bound IgE. Ten of 13 anti-C. albicans IgE-positive children had IgE towards the 46 kD major allergen. None of the subjects in the study group or in the non-atopic controls had IgA or IgG antibodies towards this protein. Analysis of the crude surface extract showed that mannan, a carbohydrate, was an intermediate allergen contrary to being the major antigen in IgA and IgG antibody responses.     

Impaired TLR-2 expression and TLR-2-mediated cytokine secretion in macrophages from patients with atopic dermatitis

Background:  In many patients with atopic dermatitis (AD), the disease is complicated by their enhanced susceptibility to bacterial skin infections, especially with Staphylococcus aureus . The pattern recognition receptor toll-like receptor (TLR)-2 recognizes components of S. aureus , for example, lipoteichoic acid (LTA) and peptidoglycan (PGN) and, therefore, might be crucial in the pathogenesis and flare-ups of AD.
Objective:  To investigate TLR-2 expression and cytokine secretion in macrophages from patients with AD compared to healthy controls upon TLR-2 stimulation with PGN, LTA and Pam3Cys.
Methods:  Macrophages were cultivated from highly purified peripheral blood monocytes of AD patients and nonatopic healthy controls and stimulated with PGN, LTA and Pam3Cys in a time and dose–dependent manner. Afterwards, TLR-2 expression and cytokine secretion were measured on protein and mRNA level. TLR-1 and TLR-6 expression were investigated on the mRNA level. Immunohistochemical stainings from punch biopsies were performed to investigate TLR-2 expression in skin macrophages .
Results:  We could clearly show that macrophages from patients with AD expressed significantly less TLR-2, whereas the expression pattern of TLR-1 and TLR-6 were not altered. Macrophages had a reduced capacity to produce pro-inflammatory cytokines such as IL-6, IL-8 and IL-1β after stimulation with TLR-2 ligands.
Conclusion:  Our findings clearly show an impaired TLR-2 expression and functional differences of TLR-2-mediated effects on macrophages of AD patients compared to healthy controls which might contribute to the enhanced susceptibility to skin infections with S. aureus in AD.