Eiichiro Ueda

The ROR nuclear orphan receptor subfamily: Critical regulators of multiple biological processes

The nuclear receptor superfamily, a group of structurally related, ligand-dependent transcription factors, includes a large number of orphan receptors for which no ligand has yet been identified. These proteins function as key regulators of many physiological processes that occur during embryonic development and in the adult. The retinoid-related orphan receptors (RORs) alpha, beta, and gamma comprise one nuclear orphan receptor gene subfamily. RORs exhibit a modular structure that is characteristic for nuclear receptors; the DNA-binding domain is highly conserved and the ligand-binding domain is moderately conserved among RORs. By a combination of alternative promoter usage and exon splicing, each ROR gene generates several isoforms that differ only in their amino terminus. RORs bind as monomers to specific ROR response elements (ROREs) consisting of the consensus core motif AGGTCA preceded by a 5-bp A/T-rich sequence. RORE-dependent transcriptional activation by RORs is cell type-specific and mediated through interactions with nuclear cofactors. RORs have been shown to interact with certain corepressors as well as coactivators, suggesting that RORs are not constitutively active but that their activity is under some regulatory control. RORs likely can assume at least two different conformations: a repressive state, which allows interaction with corepressor complexes, and an active state, which promotes binding of coactivator complexes. Whether the transition between these two states is regulated by ligand binding and/or by phosphorylation remains to be determined. Ca2+/calmodulin-dependent kinase IV (CaMKIV) can dramatically enhance ROR-mediated transcriptional activation. This stimulation involves CaMKIV-mediated phosphorylation not of RORs, but likely of specific nuclear cofactors that interact with RORs. RORalpha is widely expressed. In the cerebellum, its expression is limited to the Purkinje cells. RORalpha-/- mice and the natural RORalpha-deficient staggerer mice exhibit severe cerebellar ataxia due to a defect in Purkinje cell development. In addition, these mice have thin long bones, suggesting a role for RORalpha in bone metabolism, and develop severe atherosclerosis when placed on a high-fat diet. Expression of RORbeta is very restricted. RORbeta is highly expressed in different parts of the neurophotoendocrine system, the pineal gland, the retina, and suprachiasmatic nuclei, suggesting a role in the control of circadian rhythm. This is supported by observations showing alterations in circadian behavior in RORbeta-/- mice. RORgamma, which is most highly expressed in the thymus, plays an important role in thymopoiesis. Thymocytes from RORgamma-/- mice undergo accelerated apoptosis. The induction of apoptosis is, at least in part, due to a down-regulation of the expression of the antiapoptotic gene Bcl-XL. In addition to the thynic phenotype, RORgamma-/- mice lack lymph nodes, indicating that RORgamma is essential for lymph node organogenesis. Overexpression of RORgamma has been shown to inhibit T cell receptor-mediated apoptosis in T cell hybridomas and to repress the induction of Fas-ligand and interleukin 2. These studies demonstrate that RORs play critical roles in the regulation of a variety of physiological processes. Further characterization of the mechanisms of action of RORs will not only lead to the identification of ROR target genes and provide additional insight into their normal physiological functions, but will also determine their roles in disease.

Successful Treatment of Stevens-Johnson Syndrome with Steroid Pulse Therapy at Disease Onset

PURPOSE To evaluate the visual prognosis of patients with Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), followed by general and topical high-dose corticosteroids administration from disease onset. DESIGN Prospective, observational case series. METHODS Between May 1, 2003 and June 30, 2005, we enrolled 5 patients with SJS or TEN with ocular complications at the acute stage. Intravenous pulse therapy with methylprednisolone (steroid pulse therapy; 500 or 1000 mg/day for 3 to 4 days) was initiated within 4 days from disease onset. Topically, 0.1% betamethasone was applied over 5 times daily for at least 2 weeks. Visual acuity (VA) and slit-lamp microscopic appearance 1 year from disease onset were evaluated. RESULTS At the first examination, corneal or conjunctival epithelial defects and pseudomembranous conjunctivitis were present in all cases. Skin eruptions dramatically improved after steroid pulse therapy. Although ocular inflammation increased for several days, pseudomembranes disappeared and corneal and conjunctival epithelium regenerated within 6 weeks. At the chronic stage, all eyes had clear corneas with the palisades of Vogt (POV), implying the presence of corneal epithelial stem cells. Best-corrected VA was 20/20 or better in all eyes. Five eyes showed superficial punctate keratopathy. No eye had cicatricial changes except for 1 with slight fornix shortening. No significant adverse effects of steroid occurred during all clinical courses. CONCLUSIONS Steroid pulse therapy at disease onset is of great therapeutic importance in preventing ocular complications. Topical betamethasone also shows great promise for preventing corneal epithelial stem cell loss in the limbal region and cicatricial changes.

The Role of Interleukin-33 in Chronic Allergic Conjunctivitis

PURPOSE The authors discovered a genetic association between the ST2L gene and atopy. The ST2L gene encodes a membrane-bound functional marker for Th2 cells. Recently, a novel Th2 cytokine, interleukin-33 (IL-33), was discovered to be a specific ligand for ST2L. The authors investigated the role of IL-33 in chronic allergic conjunctivitis. METHODS Immunohistochemical analysis was carried out using giant papillae samples obtained from patients with atopic keratoconjunctivitis. The authors used proinflammatory stimuli to clarify IL-33 mRNA/protein-inducing signals with cultured human conjunctival epithelial cells, fibroblasts, human umbilical vascular endothelial cells, and mast cells. These cells were also used to examine the expression of ST2L (IL-33R). Finally, cultured mast cells were stimulated with recombinant IL-33 (rIL-33) to examine the downstream signals. RESULTS The authors found IL-33 protein expression in human vascular endothelial cells in the giant papillae and in the control conjunctivae. IL-33 expression was also observed in conjunctival epithelium of the giant papillae but not in the control conjunctivae. IL-1 beta stimulation upregulated IL-33 mRNA expression in conjunctival fibroblasts. The authors also confirmed mature IL-33 protein expression in ocular resident cells by Western blot analysis. Preferential ST2L expression was observed in human mast cells, and phosphorylation of p38 MAPK and IL-13 mRNA induction was observed in human cultured mast cells after rIL-33 stimulation. Phosphorylation of p38 MAPK was inhibited by soluble ST2 protein. CONCLUSIONS The IL-33-ST2 signaling cascade plays some roles in the pathophysiology of chronic allergic conjunctivitis through the activation of mast cells.

Elevated Platelet Activation in Patients with Atopic Dermatitis and Psoriasis: Increased Plasma Levels of β-Thromboglobulin and Platelet Factor 4

BACKGROUND Beyond their role in hemostasis and thrombosis, platelets are important for modulating inflammatory reactions. Activated platelets play a role in the pathomechanism of inflammatory diseases such as asthma, but little is known about platelet activation in chronic skin inflammation, including atopic dermatitis (AD) and psoriasis. Furthermore, the relationship between platelet activation and disease severity is not understood. This work was performed to investigate plasma levels of β-thromboglobulin (β-TG) and platelet factor 4 (PF4) as platelet activation markers in patients with AD or psoriasis, and to determine the relationships between these markers and disease severity. METHODS Plasma levels of β-TG and PF4 were measured by enzyme-linked immunoassay in 22 healthy controls, 44 patients with AD, and 16 patients with psoriasis. The relationships between these markers and the scoring AD (SCORAD) index, blood eosinophilia, serum IgE and serum lactate dehydrogenase were investigated in AD patients, and relationships with the psoriasis area and severity index (PASI) score were examined in psoriatic patients. RESULTS Plasma β-TG and PF4 levels were significantly higher in patients with AD or psoriasis compared with healthy controls. β-TG and PF4 levels correlated with the SCORAD index, and PF4 levels correlated with PASI scores. Elevated β-TG and PF4 levels were significantly reduced after treatments. CONCLUSIONS Our results show that blood platelets are activated in patients with AD or psoriasis, suggesting that activated platelets play a role in the pathomechanism of chronic skin inflammation. Furthermore, plasma β-TG and PF4 may be markers for the severity of AD and psoriasis.

Platelet-derived microparticles and soluble P-selectin as platelet activation markers in patients with atopic dermatitis☆

Plasma levels of platelet-derived microparticles (PDMPs) and soluble P-selectin (sP-selectin) were investigated as markers of platelet activation in 46 atopic dermatitis (AD) patients, 20 non-atopic urticaria patients and 22 healthy controls. The relationships between these markers and the scoring AD (SCORAD) index, blood eosinophil number, serum IgE, and serum lactate dehydrogenase were also investigated in AD patients. Plasma PDMPs and sP-selectin levels were significantly higher in AD patients compared with the other two groups. In multiple regression analysis, the SCORAD index was the most significant factor associated with the platelet activation markers. Among the SCORAD index components, excoriations were most closely related to the markers. The elevated levels of PDMPs and sP-selectin were significantly reduced following skin lesion improvement by drug treatment. Our results show that blood platelets are activated in AD patients upon aggravation of eruption, suggesting that activated platelets may be involved in the pathomechanism of AD.

Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate-pulsed mature, monocyte-derived dendritic cells

We performed a clinical trial to assess the feasibility and efficacy of immunotherapy with peptides, tumor lysate or both peptides and tumor lysate‐pulsed mature, monocyte‐derived dendritic cells (DC) for advanced malignant melanoma patients that are resistant to conventional therapies. Sixteen patients were enrolled in this trial. All patients received DC vaccines i.d. in the proximal thigh, close to the inguinal lymph nodes, one treatment per week or 2 weeks. Several factors such as clinical findings, computed tomography (CT) images, delayed type hypersensitivity (DTH) response, enzyme‐linked immunosorbent spot (ELISPOT) assay, and immunohistochemistry in primary, metastatic lesions and the DTH site were evaluated. Clinical results through DC vaccination were as follows: in 11 evaluable cases, three stable disease, six progression of disease and two disease‐free from the time of study entry to the completion of one vaccination course. One patient showed reduction of the tumors in the metastases on chest CT during the first and second course of DC vaccination. Ten out of 14 evaluable cases showed positive DTH responses to more than one treatment with melanoma peptides or tumor lysate. Eight out of 13 evaluable cases showed positive immunological responses to more than one treatment with melanoma peptides or tumor lysate in an ELISPOT assay. As for the experiences with toxicity and adverse reactions, autosensitization dermatitis‐like eruptions appeared in five cases during DC vaccination. No severe adverse effects were seen in any of the patients. In our study, the clinical efficacy in prolongation of the patients’ survival was confirmed. At the same time, cancer immunoediting of the tumor was also found. It will be necessary to improve the tumor‐specificity of this therapeutic approach and to analyze the mechanism(s) of tumor escape from immunosurveillance in melanoma.

Narrow‐band ultraviolet B phototherapy in patients with recalcitrant nodular prurigo

Management of nodular prurigo has been less than satisfactory. Conventional therapies such as systemic antihistamines and topical steroids have not been particularly successful. The effects of narrow‐band ultraviolet B (NB‐UVB) phototherapy in the treatment of various inflammatory dermatoses have been proven, however, no data exist on the efficacy and the duration of remission in NB‐UVB monotherapy for nodular prurigo. The aim of this study was to evaluate the effect of NB‐UVB phototherapy on recalcitrant nodular prurigo. NB‐UVB phototherapy was performed once a week on 10 patients with recalcitrant nodular prurigo. The initial dose was 0.4 J/cm2, and the dose was increased by 0.1 J/cm2 for each treatment. The treatment was performed until the eruption was almost clear. In each patient, a mean cumulative dose of 23.88 J/cm2 was applied over a mean of 24.3 irradiations. The mean maximum daily dose of ultraviolet B was 1.2 ± 0.4 J/cm2. NB‐UVB phototherapy notably improved the eruption of nodular prurigo in all patients. Follow up at 1 year revealed that only one patient had relapsed. The remaining nine patients continued to derive long‐term benefits. NB‐UVB phototherapy appears to be an effective treatment for recalcitrant nodular prurigo, offering long‐term benefits in the majority of those treated.

Stat6-Independent Tissue Inflammation Occurs Selectively on the Ocular Surface and Perioral Skin of IκBζ-/-Mice

PURPOSE IkappaBzeta(-/-) mice have been reported to be affected by allergic dermatitis. This study was conducted to analyze the pathophysiological role of IkappaBzeta and to address the functional relevance of Th2-mediated immune responses in the development of ocular surface inflammation and dermatitis by IkappaBzeta(-/-) mice. METHODS BALB/c background IkappaBzeta(-/-) mice were established without individual differences; IkappaBzeta/Stat6 double-knockout (WKO) mice unable to produce Th2 cytokine were created; and microscopic-, histologic-, and immunochemical studies were performed. In IkappaBzeta(-/-) mice the serum IgE levels were examined by ELISA, and quantitative PCR was used to study the gene expression of IFN-gamma, IL4, IL10, TNFalpha, IL6, IL17alpha, and CCL11 in eyelid tissue. RESULTS IkappaBzeta(-/-) mice exhibited a severe inflammatory phenotype on the ocular surface and perioral skin. The inflammatory infiltrates in the perioral skin consisted primarily of CD4(+) and CD8(+) cells; CD4(+) and CD45R/B220(+) cells were mainly detected in the conjunctiva. In eyelid and perioral skin tissue, the expression of IL-17alpha and of Th1 and Th2 cytokines, but not of CCL11, was augmented. IkappaBzeta(-/-) and IkappaBzeta(+/-) mice did not differ significantly in their serum total IgE levels before, 0 to 4 weeks, and 5 to 9 weeks after disease onset. IkappaBzeta/Stat6 WKO mice showed the same or slightly more severe inflammation than did IkappaBzeta(-/-) mice. CONCLUSIONS IgE and Stat6 are not responsible for the immune pathologic response leading to the development of ocular surface and perioral skin inflammation in IkappaBzeta(-/-) mice. IkappaBzeta(-/-) mice may be a suitable model for Stevens-Johnson syndrome, but not for atopic dermatitis.

Brunsting-Perry cicatricial pemphigoid associated with autoantibodies to the C-terminal domain of BP180.

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Successful treatment of diabetic gangrene with topical application of a mixture of peripheral blood mononuclear cells and basic fibroblast growth factor

Diabetic gangrene is a non‐healing skin ulcer that is often resistant to most common treatments. It is caused by microvascular disorders and an immunocompromised state which are induced by diabetes mellitus. We report a 65‐year‐old man with an aggressive, refractory diabetic gangrene on his left foot. Treatment of his diabetic gangrene with topical application of a mixture of peripheral blood mononuclear cells (PBMC) and basic fibroblast growth factor (bFGF) resulted in a dramatic improvement in a short time. The ulcer was completely closed and, in the past 6 months, no new ulceration has been observed. The patient is able to stand and walk by himself. Topical application of a mixture of PBMC and bFGF appears to be a useful, non‐invasive and convenient method for the treatment of diabetic gangrene.