Hypertension-Associated Genes in the Mesenteric Artery of Three Spontaneously Hypertensive Rat Substrains Identified Using a DNA Array Method.

BACKGROUND: Although the mesenteric artery plays a key role in regulating peripheral blood pressure, the molecular mechanisms that underlie the development of essential hypertension are not yet fully understood. MATERIALS AND METHODS: We explored candidate genes for hypertension using three related strains of spontaneously hypertensive rats (SHRs) that mimic human essential hypertension. In this study we used DNA microarrays, a powerful tool for studying genetic diseases, to compare gene expression in the mesenteric artery of three SHR substrains: SHR, stroke-prone SHR (SHRSP), and malignant SHRSP (M-SHRSP). RESULTS: Compared to normotensive 6-week old Wistar Kyoto rats (WKY), higher blood pressure correlated with overexpression of 31 genes and with down regulation of 24 genes. Adam23, which negatively regulates potassium current, and the potassium channel genes, Kcnc2 and Kcnq5, were associated with the onset of hypertension. In addition, Spock2 and Agtrap were identified as strengtheners of hypertension by analyzing up and down regulated genes at 9-weeks of age. CONCLUSIONS: Adam23, Kcnc2 and Kcnq5 appear to be factors for the onset of hypertension, while Spock2 and Agtrap are as factors that strengthen hypertension. These findings contribute to our understanding of the pathophysiology of hypertension and to the development of treatment for this condition.

The "Enseki" sandbath: A novel, safe and effective far-infrared bathing procedure for health.

BACKGROUND: Far-infrared (FIR) is well known with various therapeutic benefits. Recently, we have developed a novel FIR bathing system called the Enseki sandbath. In this regard, we focused on physical nature of ceramic to radiate FIR rays when heated adequately. METHODS: A bathtub was filled with ceramic beads and was equipped with computerized system which enabled to supply hot water over the ceramic beads and to drain out when beads were sufficiently heated. This system was used like sandbathing. Healthy volunteers were laid in bathtubs, covered in heated ceramic beads and were bathed for 15 minutes. Microbiological analysis was done in samples obtained from the skin surface, ceramic beads, or drained water. Furthermore, various physiological parameters were monitored, including blood pressure, heart rates, oral temperature, body weight, and blood viscosity. Blood samples were simultaneously collected and subjected to biochemical analysis, including blood glucose, HbA1c, uric acid, lactate, fatty acid, and others. RESULTS: All data showed no physiological overload for tested individuals, and any biochemical analysis did not present abnormal score. Bacteriological culture grew no pathogens. Results of questionnaires demonstrated that 90% of the participants answered the comfort and wished to further repeat the bathing. LIMITATIONS: This is a nonrandomized prospective case study. CONCLUSION: We concluded that the Enseki method is a safe and well-tolerated FIR bathing procedure for regeneration and relaxation.

Antagonism of IL-4 signaling by a phosphodiesterase-4 inhibitor, rolipram, in human T cells.

BACKGROUND: Phosphodiesterase (PDE4) inhibitors prevent breakdown of cAMP and affect the increase in cellular levels of cAMP, which is known to regulate immune cell functions. Because IL-4 plays a causal role in the pathogenesis of allergic disorders, we were interested to study the modulatory mechanisms of a PDE4 inhibitor, rolipram, in IL-4-mediated signaling in T cells. METHODS: Human peripheral T cells were stimulated with IL-4 in combination with rolipram, and RT-PCR was performed using primers specific for IL-5. To monitor activation of transcription factors, immunostaining was employed. RESULTS: Rolipram or a cAMP-analogue, 8-Br-cAMP, significantly downregulated IL-4-induced expression of IL-5 mRNA. The rolipram-induced inhibition of IL-5 mRNA was mediated by activation of protein kinase A (PKA), because rolipram-downregulated mRNA expression of IL-5 was restored by PKA inhibitors. Immunostaining revealed that rolipram interfered with IL-4-induced nuclear translocation of activator protein (AP)-1 components. CONCLUSIONS: This is the first demonstration of suppression of IL-4 signaling by PDE4 inhibitors via prevention of nuclear translocation of AP-1.

The presence of tryptase-positive and bikunin-negative mast cells in psoriatic skin lesions.

Human mast cells are well known to produce a serine protease, tryptase, which appears to play a pathogenic role in various skin inflammations. It was previously reported that a rat homologue of bikunin may inhibit tryptase activity. Various type of cells (i.e. keratinocytes) are able to produce this protein inhibitor, it still remains unclear if bikunin is present in dermal inflammatory milieu, in which mast cells, through secretion of tryptase, play an inflammatory role. Therefore, the purpose of the present study was to exploit expression and production of bikunin in dermis and dermal constituents. We first compared the dermal mast cells in psoriatic lesions with those in lesional skin of atopic dermatitis or of chronic eczema by use of immunoelectron microscopy and immunohistochemical analyses using antibodies to bikunin and tryptase. Then, we tested what kinds of cytokines may regulate the de novo synthesis of bikunin. To do so, RNA was extracted from a human mastocytic cell line, HMC-1, reverse-transcribed, and semiquantitative RT-PCR was performed using primers specific for bikunin. With immunoelectron microscopy, bikunin was found to localize on the cell membrane, while tryptase was in the secretary granules of the mast cells. In psoriatic lesions, around 70% of dermal mast cells were positive for both tryptase and bikunin, and the remaining was mostly positive for tryptase, but the expression of bikunin was under the detection limit of the experimental setting. This observation was seen in only psoriatic lesions, even in almost cured lesions, while in atopic dermatitis or chronic eczema only mast cells doubly positive for bikunin and tryptase were seen. In HMC-1, bikunin was constitutively expressed at an mRNA level, which was upregulated by stimulation with interleukine-4, but was suppressed by interferon-gamma. Bearing in mind the concept that in psoriasis local cytokine milieu is shifted toward a Th1 pattern (predominant secretion of interferon-gamma), tryptase-positive, bikunin-negative mast cells may be induced.

Itraconazole suppresses an elicitation phase of a contact hypersensitivity reaction.

Contact dermatitis is caused by epicutaneous exposure to environmentally and/or industrially derived allergens. As the exposure is unavoidable in many instances, therapeutic suppression of allergic inflammation appears to be of clinical relevance. It was recently reported that itraconazole (ITZ), an anti-fungal agent, may be of therapeutic importance in allergic skin diseases. Therefore, we were interested in the effect of ITZ on contact hypersensitivity (CHS). Mice (C3H/HeN or Balb/c) were administered with ITZ orally before sensitization or challenged with haptens (dinitrofluorobenzene or oxazolone). Consequently, the administration of ITZ before challenge, but not before sensitization, significantly suppressed the reaction. Intriguingly, ITZ failed to suppress the irritant dermatitis induced by croton oil or benzalkonium chloride, suggesting that it may affect molecule(s) rather selectively involved in the elicitation of CHS. To further analyze mechanisms involved, splenic T cells obtained from sensitized or naive mice were stimulated with plate-bound anti-CD3 in the presence or absence of ITZ and release of cytokines was tested by ELISA. T cells from hapten-immunized mice produced a significant amount of IFN-gamma, which was markedly suppressed by ITZ. Our study demonstrates that ITZ selectively suppresses the elicitation phase of CHS possibly via downmodulation of IFN-gamma.

The receptor for erythropoietin is present on cutaneous mast cells.

Skin samples from patients with extra-mammary Paget disease, Bowen's disease, atopic dermatitis, psoriasis and non-lesional skin of nevus pigmentosus were immunohistochemically examined with an anti-soluble erythropoietin receptor antibody (anti-sEPOR antibody), and only the dermal mast cells positively stained in all skin samples were examined. These positively stained dermal cells were proved to be mast cells by double staining with anti-sEPOR antibody and either with anti-bikunin antibody or anti-tryptase antibody. Immunoelectron microscopically these EPOR were found in the secretory granules of the dermal mast cells. Further, EPOR in the mast cells may be consisting of only the extracellular domain of erythropoietin receptor molecule as the mast cells were immunohistochemically not reacted with an antibody to the C-terminal peptide of EPOR. Human mast cell line, HMC-1 cells has immunohistochemically the erythropoietin receptor, which was consisting of a 43 kDa major protein and a 20 kDa minor protein in the immunoelectrophoresis. These data may indicate that EPOR in the mast cells may not be the whole molecule, but probably the soluble one of EPOR.

Intravenous infusion of syngeneic apoptotic cells by photopheresis induces antigen-specific regulatory T cells.

The basis of extracorporeal photopheresis is the reinfusion of leukocytes previously exposed to 8-methoxypsoralen (8-MOP) and UVA radiation. It has been approved for the palliative treatment of cutaneous T cell lymphoma and has reported benefits in autoimmune diseases, transplant rejection, and graft-vs-host disease. However, the underlying mechanism of photopheresis remains unresolved. Because UVB radiation can cause immune tolerance via induction of regulatory T cells, we studied whether photopheresis exerts a similar effect extracorporeally. Therefore, we established a model of photopheresis using a murine model of contact hypersensitivity. Splenocytes and lymph node cells of mice that were sensitized with dinitrofluorobenzene were exposed to 8-MOP plus UVA in vitro. Intravenous injection of these cells into naive mice caused inhibition of a hapten immune response, which was lost upon depletion of CD11c(+) cells but not T cells. Mice that received untreated cells or cells exposed to UVA or 8-MOP alone were not affected. Inhibition was cell-mediated and Ag-specific as demonstrated by transfer of tolerance from the primary recipients into naive animals, which could, however, properly respond to the unrelated hapten oxazolone. Transfer activity was lost when cells were depleted of CD4(+) or CD25(+) subpopulations. These data suggest that photopheresis exerts its immunomodulatory effects via the induction of Ag-specific regulatory T cells.

A case of herpetiform pemphigus with anti-desmoglein 3 IgG autoantibodies.

Herpetiform pemphigus (HP) is a rare variant of pemphigus characterized by a unique clinical phenotype of erythematous or urticarial plaques and vesicles that present in a herpetiform arrangement. Most HP cases have circulating anti-desmoglein 1 (Dsg1) IgG autoantibodies, but some HP cases have anti-desmoglein 3 (Dsg3) IgG. A 92-year-old Japanese woman presented with severely pruritic annular erythema and vesicles in a herpetiform arrangement on the trunk. No oral mucosal lesions were present. Histopathologically, these vesicles showed eosinophilic spongiosis as well as suprabasilar acantholysis. Direct immunofluorescence showed in vivo IgG deposition on keratinocyte cell surfaces, and indirect immunofluorescence showed circulating IgG autoantibodies against keratinocyte cell surfaces at a titer of 1:30. Enzyme-linked immunosorbent assay using recombinant Dsg1 and Dsg3 revealed the presence of anti-Dsg3 IgG but no anti-Dsg1 IgG autoantibodies. The lack of oral mucosal involvement and the unique clinical features favored the diagnosis of HP. It remains to be clarified why the anti-Dsg3 IgG autoantibodies in this patient induced this unique features of HP, rather than the mucosal dominant type of pemphigus vulgaris.

Successful treatment of pulmonary metastasis and local recurrence of angiosarcoma with docetaxel.

Angiosarcoma of the face and scalp of the elderly frequently recurs locally, metastasizes early despite various treatments, and has a poor prognosis. We describe a patient who had angiosarcoma of the scalp with pulmonary metastasis. Local recurrence occurred after excision and local and arterial administration of IL-2. A weekly administration method of docetaxel was therefore selected, resulting in complete remission of the pulmonary metastasis and a partial response of the local recurrence. This favorable clinical outcome in our case suggests that docetaxel therapy may be an option for the treatment of angiosarcoma of the scalp with pulmonary metastasis.

Characterization of Kdap, a protein secreted by keratinocytes.

Using a signal sequence-trap we identified a human gene encoding a polypeptide of 99 amino acids with a putative signal sequence. The gene was identical to keratinocyte differentiation-associated protein (Kdap), which was reported previously by Oomizu et al (Gene 256: 19-27, 2000) to be expressed in embryonal rat epidermis at the mRNA level. In humans, we found Kdap mRNA expression to be restricted to epithelial tissue at high levels. The 12.5 kDa protein was detected in culture supernatant of keratinocytes and those transfected adenovirally with the Kdap gene. In normal skin, Kdap protein was found exclusively within lamellar granules of granular keratinocytes and in the intercellular space of the stratum corneum. By contrast, in lesional skin of patients with psoriasis, Kdap was expressed more widely throughout suprabasal keratinocytes. When induced to differentiate in vitro, keratinocytes showed marked upregulation of Kdap mRNA expression similar to that of involucrin mRNA, but with differing kinetics. Finally, a spliced variant of Kdap mRNA was generated by alternative splicing mechanisms. Our studies indicate that human Kdap resembles rat Kdap with respect to tissue and cell expression at the mRNA level and that Kdap is a low-molecular-weight protein secreted by keratinocytes. Thus Kdap may serve as a soluble regulator of keratinocyte differentiation.

Clinical and pharmacokinetic studies of continuous itraconazole for the treatment of onychomycosis.

Itraconazole, a triazole antifungal agent, has been widely used for onychomycosis with high cure rates. Unchanged itraconazole and a major metabolite hydroxy-itraconazole reach the nail with a strong affinity for keratin. The aim of this study was to elucidate clinical effectiveness and pharmacokinetic profiles of a 6-month continuous itraconazole treatment at a daily dose of 100 mg. Nail growth, the decrease in nail turbidity, and the nail concentrations of unchanged- and hydroxy-itraconazole were investigated. The affected nails we examined demonstrated nail growth proportional to the decrease in turbidity and a quick increase in drug concentration with a long duration of a high concentration after cessation. Our results support the hypothesis that this continuous therapy is a good modality for onychomycosis.

Myoepithelioma possibly originating from the accessory parotid gland.

Myoepithelioma originates almost exclusively from myoepithelial cells of the salivary, prostate and mammary glands. The skin is a very rare site where myoepithelioma occurs. We describe a patient with a myoepithelioma on the right cheek seen as a subcutaneous nodule that was separated from the parotid gland at surgical resection. Histopathological findings were consistent with those of a myoepithelioma that had originated from the parotid gland, suggesting that this tumor may have developed from the accessory parotid gland.

Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity.

Epicutaneous application of haptens to UV-exposed skin induces hapten-specific tolerance. This is mediated via regulatory T cells (Tr), as i.v. injection of T cells from UV-tolerized mice into naive animals renders the recipients unresponsive to the respective hapten. However, when UV-induced Tr are injected i.v. into sensitized mice, contact hypersensitivity (CHS) is not suppressed, suggesting that Tr inhibit the induction, but not the elicitation, of CHS and are inferior to T effector cells. As sensitization takes place in the lymph nodes, but elicitation occurs in the area of challenge, we postulated that Tr injected i.v. locate to the lymph nodes and not to the periphery and therefore only suppress the induction, not the elicitation, of CHS. Indeed, i.v. injection of Tr into sensitized mice did not inhibit CHS, although injection of Tr into the ears of sensitized mice suppressed the challenge. Inhibition was hapten specific, as injection of dinitrofluorobenzene (DNFB)-specific Tr into the ears of oxazolone (OXA)-sensitized mice did not affect challenge with OXA. However, when ears of OXA-sensitized mice were injected with DNFB-specific Tr and painted with DNFB before OXA challenge, CHS was suppressed. Inhibition correlated with the local expression of IL-10. Depletion studies and FACS analysis revealed that Tr express the lymph node-homing receptor L-selectin, but not the ligands for the skin-homing receptors E- and P-selectin, suggesting that UV-induced Tr, although able to inhibit T effector cells, do not suppress the elicitation of CHS upon i.v. injection, because they obviously do not migrate into the skin.

Involvement of dectin-2 in ultraviolet radiation-induced tolerance.

Hapten sensitization through UV-exposed skin induces hapten-specific tolerance which can be adoptively transferred by injecting T cells into naive recipients. The exact phenotype of the regulatory T cells responsible for inhibiting the immune response and their mode of action remain largely unclear. Dectin-2 is a C-type lectin receptor expressed on APCs. It was postulated that dectin-2 interacts with its putative ligands on T cells and that the interaction may deliver costimulatory signals in naive T cells. Using a soluble fusion protein of dectin-2 (sDec2) which should inhibit this interaction, we studied the effect on contact hypersensitivity (CHS) and its modulation by UV radiation. Injection of sDec2 affected neither the induction nor the elicitation phase of CHS. In contrast, UV-induced inhibition of the CHS induction was prevented upon injection of sDec2. In addition, hapten-specific tolerance did not develop. Even more importantly, injection of sDec2 into tolerized mice rendered the recipients susceptible to the specific hapten, indicating that sDec2 can break established tolerance. FACS analysis of spleen and lymph node cells revealed a significantly increased portion of sDec2-binding T cells in UV-tolerized mice. Furthermore, transfer of UV-mediated suppression was lost upon depletion of the sDec2-positive T cells. Taken together, these data indicate that dectin-2 and its yet unidentified ligand may play a crucial role in the mediation of UV-induced immunosuppression. Moreover, sDec2-reactive T cells appear to represent the regulatory T cells responsible for mediating UV-induced tolerance.

Terfenadine antagonism against interleukin-4-modulated gene expression of T cell cytokines.

Here, we investigated whether an anti-allergy drug, terfenadine, affects interleukin-4-modulated cytokine expression in peripheral T cells. Peripheral blood T cells were first stimulated with recombinant interleukin-4 and then tested for modulation of the mRNA of a panel of cytokines using the reverse transcription-polymerase chain reaction followed by Southern blot analysis. It was found that T cells constitutively expressed mRNA specific to T helper 1 cytokines (interleukin-2, interferon-gamma, tumor necrosis factor-alpha), which was markedly downregulated upon stimulation with interleukin-4, whereas mRNA for T helper 2 cytokines such as interleukins 4, 5, and 6 was induced in response to interleukin-4. Interestingly, the interleukin-4-induced expression of all T helper 2 cytokines examined was markedly downregulated by terfenadine. Among T helper 1 cytokines, interleukin-4-mediated suppression of tumor necrosis factor-alpha was not affected by terfenadine, which, however, markedly restored mRNA expression of interferon-gamma or interleukin-2. Electrophoretic mobility shift assays using [32P]-labeled synthetic oligonucleotides encoding the consensus binding motif of activator protein-1 demonstrated that interleukin-4-induced binding of activator protein-1 composed of JunB was interfered by terfenadine. This study indicates that terfenadine, at least partially, interferes with interleukin-4-activated signaling, leading to terfenadine antagonism against the modulatory impact of interleukin-4 on T cell cytokines.