Background-There is little knowledge about clinical variables associated with vitamin D (vitD) insufficiency in asthmatic children.

Glucocorticoid (GC)-insensitive asthma is a challenging clinical problem that can be associated with life-threatening disease progression. The molecular basis of GC insensitivity is unknown. Alternative splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCRbeta, which does not bind GC but antagonizes the transactivating activity of the classic GCR. Thus increased expression of GCRbeta could account for glucocorticoid insensitivity. Bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC) were examined for GCRbeta immunoreactivity using a GCRbeta-specific antibody by immunohistochemical staining. Cell localization of GCRbeta expression was performed using a double immunostaining technique. Patients with GC-insensitive asthma expressed a significantly higher number of GCRbeta-immunoreactive cells in their BAL and peripheral blood than GC-sensitive asthmatics or normal control subjects. Furthermore, GCRbeta expression in GC-insensitive asthma was particularly high in airway T cells, which are thought to play a major role in the pathogenesis of asthma. We also examined the expression of GCRbeta in specimens from the airways of patients with chronic bronchitis. In chronic bronchitis, few cells were GCRbeta-positive and their numbers did not differ significantly from normal control subjects. We conclude that GC-insensitive asthma is associated with increased expression of GCRbeta in airway T cells.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Via next generation sequencing on a cohort of patients with severe atopic dermatitis, some with comorbid infections, we found 8 individuals from 4 families with novel heterozygous mutations in CARD11, a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant expression constructs into T cell lines demonstrated both loss of function and dominant interfering activity upon antigen receptor-induced NF-κB and mTORC1 activation. Patient T-cells had similar defects, as well as diminished IFN-γ cytokine production. The mTORC1 and IFN-γ production defects could be partially rescued by supplementing with glutamine, which requires CARD11 for import into T cells. Our findings indicate a single hypomorphic gene mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRα and GRβ, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRβ does not bind glucocorticoids and is an inhibitor of GRα activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform.The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRα was 475 ± 62 and 985 ± 107 for PBMCs and neutrophils, respectively. For GRβ, the MFI was 350 ± 60 and 1,389 ± 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRβ staining to 2,497 ± 140 (P < 0.05). No change in GRα expression was observed. This inversion of the GRα/GRβ ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRβ mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRα/GRβ heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRβ, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRβ by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

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Background The cause of corticosteroid resistant asthma is unknown. Objective To perform gene microarray analyses using BAL cells from well-characterized corticosteroid resistant (CR) and sensitive (CS) asthmatics to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance. Methods The patients were characterized as CR or CS based on FEV1% predicted improvement after one week course of oral prednisone. Expression of selected gene targets was verified by real time PCR and by ELISA. Results Microarray analyses demonstrated significantly higher levels (over three-fold increase, p<0.05) of transcripts for TNFα, IL-1α, IL-1β, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, CCL20 in BAL cells of CR asthmatics. These findings, confirmed by RT-PCR in additional BAL samples, were consistent with classical macrophage activation by bacterial products. In contrast, markers of alternatively-activated macrophages, Arginase I and CCL24, were decreased. Genes associated with activation of the LPS signaling pathway (EGR1, DUSP2, MAIL, TNFAIP3) were significantly elevated in CR BAL samples (p<0.05). These patients had significantly higher amounts (1444.0±457.3 pg per mg of total protein) of LPS in BAL fluid than CS asthmatics (270.5±216.0 pg; p<0.05) as detected by LAL assay and confirmed by gas chromatography mass spectrometry analysis. Pronged exposure to LPS induced functional steroid resistance to dexamethasone (DEX) in normal monocytes, demonstrated by persistently elevated IL-6 levels in the presence of DEX. Conclusions Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from CR asthmatics suggest that LPS exposure may contribute to CR asthma.

The environmental factors that contribute to the homing of T cells in skin disease is unknown. The skin lesions of atopic dermatitis (AD) are frequently colonized with superantigen (SAg), producing strains of Staphylococcus aureus. In vitro, these superantigens have the capacity to activate and expand T cells expressing specific T cell receptor BV gene segments, and also to increase their skin homing capacity via upregulation of the skin homing receptor, cutaneous lymphocyte-associated antigen (CLA). These activities have been proposed to enhance the chronic cutaneous inflammation of AD, but an in vivo role for SAg has not been conclusively demonstrated. In this study, we sought direct evidence for in vivo SAg activity by comparing the SAg profiles of S. aureus cultured from the skin of AD subjects to the T cell receptor Vbeta repertoire of their skin homing (CLA+) T cells in peripheral blood. SAg secreting S. aureus strains were identified in six of 12 AD patients, and all of these subjects manifested significant SAg-appropriate Vbeta skewing within the CLA+ subsets of both their CD4+ and their CD8+ T cells. T cell receptor Vbeta skewing was not detectable among the overall CD4+ or CD8+ T cell subsets of these subjects, and was not present within the CLA+ T cell subsets of five patients with plaque psoriasis and 10 normal controls. T cell receptor BV genes from the presumptively SAg-expanded populations of skin homing T cells were cloned and sequenced from three subjects and, consistent with a SAg-driven effect, were found to be polyclonal. We conclude that SAg can contribute to AD pathogenesis by increasing the frequency of memory T cells able to migrate to and be activated within AD lesions.

Although it is well established that immune mechanisms contribute to the pathogenesis of chronic inflammatory skin diseases such as atopic dermatitis (AD) and psoriasis, the actual events that trigger the immunological pathways resulting in these skin diseases are not well understood. Colonization and infection with Staphylococcus aureus and streptococci has been reported to exacerbate AD and psoriasis. Recent studies demonstrating that bacterial toxins can act as superantigens provide mechanism(s) by which S. aureus and streptococci could mediate an inflammatory skin lesion that consists predominantly of activated T-cells and monocytes. This review will explore the diverse mechanisms by which bacterial superantigens can induce skin inflammation following systemic or local infection. These observations provide a new direction for the development of novel approaches for the treatment of skin inflammation.