Anti-IgE autoantibody in patients with atopic dermatitis

The anti-IgE autoantibody in the IgG class was detected in 39/45 (86.7%) patients with atopic dermatitis by using a newly established solid-phase enzyme immunoassay. The epsilon-chain specificity of the anti-IgE autoantibody was confirmed by competitive inhibition by using human IgG, IgA, IgM, IgD, IgE, and heat-denatured IgE protein. Significant correlations were observed between the levels of the anti-IgE autoantibody and the serum IgE. Gel filtration studies indicated that the anti-IgE autoantibody in sera from atopic dermatitis was mainly present in the form of an immune complex with self IgE. The role of the IgE-anti-IgE immune complex and the role of the anti-IgE autoantibody in the modulation of the IgE-mediated immune system in atopic dermatitis are discussed.     

Two-dimensional electrophoretic display of restriction fragments from genomic DNA

We have developed a procedure for the resolution of restriction enzyme digests of mammalian genomic DNA in two dimensions. Fragments from a first digestion are separated on a column of purified agarose containing a second restriction enzyme in the absence of the divalent cation required for enzyme activity. After enzyme activation and digestion, the fragments are resolved on an agarose slab gel. We have digested rat genomic DNA and found in the ethidium-stained pattern a variety of features which have not been described previously.     

Two-dimensional electrophoretic proteome study of serum thermostable fraction from patients with various tumor conditions

One of the problems of plasma proteomics is a presence of large major components. In this work, we use the thermostable fraction as a way to deplete these major proteins. The thermostable fraction of serum samples from patients with ovarian, uterus, and breast cancers and benign ovarian tumor was analyzed using two-dimensional electrophoresis combined with MALDI-TOF(-TOF)-mass spectrometry. Of them, alpha-1-acid glycoprotein and clusterin are expressly down-regulated in breast cancer, whereas transthyretin is decreased specifically in ovarian cancer. Apolipoprotein A-I forms have decreased spot volumes, while haptoglobin alpha1, in contrast, is elevated in several tumors. These data are partly consistent with previous art studies on cancer proteomics, which involve mass-spectrometry-based serum profiling techniques. Serum thermostable fraction may be recommended as a good tool for medium and small protein proteome investigation, in particular, by 2D-electrophoresis.     

Two-dimensional spectroscopy of electrophoretic gels

Optical techniques are described which permit one to analyze two-dimensional electrophoretic gels in a fashion which is analogous to the one-dimensional spectroscopy of solutions. In the methods described, an electrophoretic gel is irradiated with monochromatic light and isozyme patterns are detected by the absorption of light or the fluorescent emission of light. The system described can both generate and detect monochromatic light in a range from 200 to 1100 nm. Without the use of histochemical stains, several isozymes have been visualized by purely optical means. Five methods for the visualization of lactate dehydrogenase and five methods for the demonstration of trypsin isozymes are described. In addition, general methods have been formulated for hydrolases and oxidases. Gel spectroscopy should permit the investigation of a wide range of new isozymes.This work was supported in part by NIH Grants CA 19017 and GM 21433.     

Allergic sensitivity in different groups of patients with atopic dermatitis

69 patients with atopic dermatitis, aged 16-42 years, were examined. The patients were divided into two groups: group 1 consisted of patients with atopic dermatitis and bronchial asthma and/or allergic rhinitis (25 patients), group 2 consisted of patients without the respiratory syndrome (44 patients). Scarification skin tests made it possible to find out the essential difference in the sensitivity of the examinees in these two groups. In group 1 the prevalence of sensitization to house-dust mites in 23 patients (92%) with monosensitization in 8 patients (32% of the group) was observed. In group 2 sensitivity of house-dust mites was lower: it was registered in 16 patients (36%) with monosensitization in 2 patients (4% of the group). The presence of cross-sensitivity between mite allergens and Candida albicans was established. In accordance with the results of scarification skin tests, treatment included the use of antihistamine preparations, antipruritic remedies and hormonal ointments as well as the elimination of sensitizing allergens. Improvement was registered in 21 patients.     

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

Heat shock protein 60 (hsp60) is a highly conserved stress protein and target of self-reactive T cells in various inflammatory diseases. Not much is known about a possible role in atopic disease. As atopic diseases are considered to be the result of a disturbance in the balance between T helper cells type 2 and regulatory T cells, it is of interest to know whether hsp60 acts as a bystander antigen in atopic disease. Our aim was to investigate whether hsp60 is involved in the chronicity of inflammation of atopic dermatitis (AD). We studied the expression of hsp60 in skin tissue of adults with AD by immunohistochemistry. Peripheral blood mononuclear cells (PBMC) of children with AD were cultured with hsp60 and proliferative responses, cytokine secretion, surface markers, and functional assays were compared to responses of PBMC of healthy controls (HC). Hsp60 was detected more in lesional skin of AD patients compared to nonlesional skin. Furthermore, PBMC of children with AD proliferated more strongly in response to hsp60 compared to HC. hsp60-reactive T cells of atopic children produced high levels of IFNγ and low levels of IL-10. In vitro activation with hsp60 leads to the induction of CD4⁺CD25^bright T cells expressing FOXP3 in both HC as well as in atopic children. However, despite their regulatory phenotype, hsp60-induced CD4⁺CD25^brightCD127⁻FOXP3⁺ T cells of AD patients were incapable of suppressing effector T cells in vitro. hsp60 is recognized by proinflammatory (IFNγ high, IL-10 low) T cells in atopic patients and is more present in lesional AD skin. This suggests that hsp60-specific T cell responses contribute to local inflammation in AD.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-012-0361-3) contains supplementary material, which is available to authorized users.     

Increased acetylcholine levels in skin biopsies of patients with atopic dermatitis

Recent experimental evidence indicates that non-neuronal acetylcholine is involved in the regulation of basic cell functions. Here we investigated the cholinergic system in the skin of healthy volunteers and patients with atopic dermatitis (AD). The synthesizing enzyme, choline-acetyltransferase (ChAT), was studied by anti-ChAT immunohistochemistry and enzyme assay. Skin biopsies taken from healthy volunteers and from AD patients were separated into the 2 mm superfical (epidermis and upper dermis) and 3 mm underlying portion (deeper dermis and subcutis). ChAT enzyme activity was detected in homogenized skin and subcutaneous fat (about 13 nmol/mg protein/h). ChAT immunoreactivity was expressed in keratinocytes, hair papilla, sebaceous and eccrine sweat glands, endothelial cells and mast cells. In healthy volunteers the superficial and underlying portion of skin biopsies contained 130 +/- 30 and 550 +/- 170 pmol/g acetylcholine (n = 12), respectively. In AD patients (n = 7) acetylcholine was increased 14-fold in the superficial and 3-fold in the underlying biopsy portion. The present study demonstrates the widespread expression of ChAT protein in the vast majority of human skin cells. Tissue levels of acetylcholine are greatly (14-fold) enhanced in the superficial 2 mm skin of AD patients.     

Cell-cycle withdrawal in cultured keratinocytes

Cell-cycle withdrawal is the irreversible arrest of replication that occurs in keratinocytes early in terminal differentiation. According to the epidermal proliferation unit (EPU) model of renewal, withdrawal takes place in a subset of cells that have completed a final cycle of amplification replication. Using a recently developed double-labelling assay, we followed cell-cycle withdrawal in growing cultures of epidermal keratinocytes and correlated these results with population-growth kinetics. The levels of withdrawal measured were much too high to be consistent with the population-growth kinetics. These unexpectedly high levels could be explained by postulating that withdrawal takes place in a specific subset of cells, as described by the EPU model. Other possible explanations were entertained but, for various reasons, were considered unlikely. To learn whether withdrawal occurred in basal or suprabasal cells, confluent cultures were pulse labeled with 3H-thymidine, and the position of the labeled cells was monitored by autoradiography during the period of cold chase. The results indicated that, in cultured keratinocytes, withdrawal takes place while the cells are still in the basal compartment.     

In vitro interleukin 4 and interferon-gamma production by mononuclear cells from atopic dermatitis patients

In order to elucidate further the possible role of specific cytokines in the pathogenesis of atopic dermatitis (AD) the in vitro production of interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) in patients with severe atopic dermatitis (n = 4) was compared with that in a group of non-atopic healthy controls. Overall IL-4 production by PHA- and PWM-driven PBMNCs was increased in controls during the first 48 h in culture. Addition of interleukin 2 (IL-2) into parallel cultures generated an insignificant (p > 0.05) increase in IL-4 production in AD patients compared with that from controls. IFN-gamma production by PWM-stimulated PBMNCs was markedly decreased in AD patients compared with controls (p < 0.01). Addition of IL-2 (250 U/ml) to parallel cultures failed to restore IFN-gamma production in AD patients. Finally, no IL-4 or IFN-gamma activity could be detected in any of the sera. In conclusion, the data suggest a possible dysregulation of cytokine production in at least a subgroup of AD patients, with an impaired capacity to secrete IFN-gamma, but a partially intact IL-4 generating capacity.     

Comparative study of Staphylococcus aureus isolated from lesional and non-lesional skin of atopic dermatitis patients

The skin of patients with atopic dermatitis (AD) is often colonized by Staphylococcus aureus, and superantigenic exotoxins produced by the organism are thought to be an important precipitating factor of AD. However, there are few reports comparing the characteristics of S. aureus isolated from the lesional and non-lesional skin of identical AD patients. In this study, therefore, we examined whether the presence of superantigen-producing S. aureus correlates with the formation of eczematous lesion of AD patients. The detection rate of S. aureus on the lesional skin of AD patients was higher than on the non-lesional skin of AD patients. Furthermore, the bacterial cell count of S. aureus on the lesional skin of AD patients was also significantly higher than that of the non-lesional skin of AD patients. However, there was no significant difference between the detection rate of superantigenic exotoxin-producing S. aureus on the lesional and nonlesional skin of AD patients. These results suggest that the number of S. aureus present is more important in the formation of eczematous lesion of AD patients than the presence of superantigenic exotoxin-producing S. aureus strains per se.     

Biofeedback treatment of atopic dermatitis

To investigate the feasibility of a behaviorally oriented intervention program with atopic dermatitis, 12 patients were exposed to a fixed sequence of treatment phases including a no-treatment baseline phase, a phase incorporating nonspecific treatment factors, and a phase involving frontal electromyographic (EMG) feedback and relaxation instructions. Photographic analyses of involved skin areas revealed significant remission of dermatological problems across the entire program, although significant changes could not be attributable to any specific phase. Ratings of itching level decreased within but not across treatment sessions, and variable correlations across subjects were found between frontal EMG and itching level. MMPI results from the dermatitis subjects were within normal limits. Overall, the results provided mixed support for the hypothesis that atopic dermatitis may be amenable to intervention through behaviorally oriented treatment procedures.     

基于微生物视角的“皮-肠”轴与特应性皮炎

特应性皮炎(atopic dermatitis,AD)是一种难治易复发皮肤病,由于病因复杂且患病率逐年增加,该病已经成为公共卫生领域关注的问题。随着高通量测序、元基因组学和代谢组学等技术的应用,发现AD的发生与发展与微生物群落息息相关,“微生物-皮-肠”轴及它们之间的串扰机制也逐渐被验证。“微生物-皮-肠”轴在过敏性皮肤炎症中扮演了重要角色。本文综述了“微生物-皮-肠”轴与AD的关系,及其可能交流的信号分子和潜在途径,重点关注了涉及益生菌、菌群移植和抗菌肽等微生物缓解AD的潜在机制,为靶向微生物群治疗过敏性皮肤炎症提供了一个新的视角。     

Sphingosylphosphorylcholine is upregulated in the stratum corneum of patients with atopic dermatitis

To clarify the functional relevance of sphingomyelin (SM) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [(14)C]acetic anhydride to yield [(14)C-C(2)]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase. These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD.     

Comparative proteomic analysis of peripheral blood mononuclear cells from atopic dermatitis patients and healthy donors

Atopic dermatitis (AD) is a chronic inflammatory skin disease that induces changes in various inflammatory skin cells. The prevalence of AD is as high as 18% in some regions of the world, and is steadily rising. However, the pathophysiology of AD is poorly understood. To identify the proteins involved in AD pathogenesis, a comparative proteomic analysis of protein expression in peripheral blood mononuclear cells isolated from AD patients and healthy donors was conducted. Significant changes were observed in the expressions of fourteen proteins, including the vinculin, PITPNB, and Filamin A proteins. Among the proteins, alpha-SNAP and FLNA decreased significantly, and PITPNB increased significantly in AD patients compared with control subjects; these findings were further confirmed by real-time PCR and Western blot analysis. The comparative proteome data may provide a valuable clue to further understand AD pathogenesis, and several differentially regulated proteins may be used as biomarkers for diagnosis and as target proteins for the development of novel drugs.