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Synopsis Objective Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation. In addition, HA has been shown to have different biological activities depending on its molecular weight. It has been reported that HA-mediated CD44 activation regulates keratinocyte differentiation. Therefore, the aim of this study was to investigate the influence of HA tetrasaccharides (HA4) on the regulation of keratinocyte differentiation, CD44 gene expression and CD44-phosphorylated protein in human keratinocytes, and compare HA4 with high molecular weight HA. Methods Normal human epidermal keratinocytes (NHEKs) were treated at doses of 1 μg mL−1 HA or HA oligosaccharides (HA4). After treatment, cell viability was checked using an MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each differentiation marker and CD44 mRNA expression was detected by real-time PCR. Each differentiation marker and CD44-phosphorylated protein was assessed by Western blotting. Results Hyaluronan and HA4 showed no cytotoxicity up to a dose of 1 μg mL−1. On day 3 after HA4 treatment, each differentiation marker mRNA and K10 protein level was higher than that of the control. On day 9, late differentiation marker mRNA and protein levels were increased with HA and HA4 treatment. In addition, HA4 treatment increased the expression of CD44 mRNA, CD44-phosphorylated protein and intracellular calcium concentrations. HA4 enhanced keratinocyte differentiation and increased CD44-phosphorylated protein levels. Conclusion HA4 may induce epidermal differentiation through phosphorylation of CD44. Resume Objectif L'acide hyaluronique joue un role dans la proliferation des keratinocytes et la differenciation. En outre, l'acide hyaluronique a ete montre d' avoir des activites biologiques differentes en fonction de son poids moleculaire. Il a ete rapporte que l'activation du CD44 mediee par l'acide hyaluronique regule la differenciation des keratinocytes. Par consequent, le but de la presente etude etait d'etudier l'influence de tetrasaccharides de l'acide hyaluronique (HA4) sur la regulation de la differenciation des keratinocytes , l'expression du gene CD44 et la proteine CD44-phosphorylee dans les keratinocytes humains , et de comparer HA4 avec un acide hyaluronique de haut poids moleculaire (HA). Methodes des keratinocytes epidermiques humains normaux (NHEK) ont ete traites a des doses de 1 pg mL−1 HA ou HA4. Apres le traitement, la viabilite cellulaire a ete verifiee par un test MTT. L'expression de l'ARNm de chaque marqueur de differenciation et du CD44 a ete detectee par PCR en temps reel. Chaque marqueur de differenciation et la proteine CD44-phosphorylee ont ete evalues par western blot. Resultats HA et HA4 n'ont montre aucune cytotoxicite jusqu'a une dose de 1 pg mL−1. Le 3e jour apres traitement au HA4, chaque ARNm des marqueurs de differenciation et le niveau de la proteine K10 etaient superieurs a ceux du temoin. Au jour 9, l'ARNm des marqueurs de differenciation tardifs et de proteines ont ete augmentes avec le traitement au HA et HA4. En outre, l'HA4 traitement a augmente l'expression de l'ARNm du CD44, de la proteine CD44-phosphorylee, et des concentrations de calcium intracellulaire. HA4 a ameliore la differenciation des keratinocytes et conduit a des niveaux accrus de la proteine CD44-phosphorylee. Conclusion HA4 peut induire la differenciation epidermique par la phosphorylation de CD44.

BACKGROUND The quality-switched ruby laser (QSRL) has been widely used for the treatment of pigmented lesions, but clinical evaluations in most studies have been conducted on macroscopic skin color observation comparing the laser-treated skin with its nontreated surrounding area. A few investigations examined skin changes after laser therapy at a cellular level, but almost none did so noninvasively. OBJECTIVE To elucidate the dynamic changes after QSRL irradiation of facial solar lentigo using noninvasive optical techniques. MATERIALS AND METHODS Time-sequential imaging of Japanese female patients with a clinical diagnosis of solar lentigo was performed using ultraviolet photography, high-magnification videomicroscopy, and reflectance-mode confocal microscopy to examine pigmentary change after QSRL irradiation. RESULTS The present study showed that remaining melanocytes were visible in the solar lentigo of all subjects when crusts peeled off, despite hardly observable skin pigmentation to the naked eye. Moreover, noninvasive confocal imaging revealed that pigmented melanocytes varied in each solar lentigo after QSRL treatment, as indicated by melanin reflection level. CONCLUSIONS Optical techniques facilitate the evaluation of the in vivo dynamics of epidermal-melanocytic changes in solar lentigo after QSRL therapy and may be useful for monitoring outcomes after laser irradiation.

Intense pulsed light (IPL) therapy is reported to be effective for pigment removal from pigmented lesions. However, the dynamic mechanism of pigment removal by IPL therapy is not completely understood. We investigated the mechanism of IPL therapy for the removal of pigmented skin lesions through non-invasive observation of the epidermis. Subjects with solar lentigines on the face were treated with three sessions of IPL therapy. The solar lentigines were observed on consecutive days after the treatments using reflectance-mode confocal microscopy (RCM) and optical coherence tomography (OCT). In addition, desquamated microcrusts that formed after the treatment were investigated by transmission electron microscopy (TEM). The images of RCM and OCT showed that the melanosomes in the epidermal basal layer rapidly migrated to the skin surface. The TEM images of the extruded microcrusts revealed numerous melanosomes together with cell debris. It was also found that the IPL irradiated melanocytes in the lesions seemed to be left intact and resumed their high activity after treatment. We conclude that IPL therapy effectively removed the dense melanosomes in the epidermal-basal layer. However, additional application of suppressive drugs such as hydroquinone or Q-switched laser irradiation is necessary to suppress the remaining active melanocytes.

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG), which specifically acts on the stratum corneum, suppresses the development of skin tumors in UVB-irradiated hairless mice. To elucidate the mechanism through which chemical peeling with SA-PEG suppresses skin tumor development, the effects of chemical peeling on photodamaged keratinocytes and cornified envelopes (CEs) were evaluated in vivo. Among UVB-irradiated hairless mice, the structural atypia and expression of p53 protein in keratinocytes induced by UVB irradiation were intensely suppressed in the SA-PEG-treated mice 28 days after the start of weekly SA-PEG treatments when compared to that in the control UVB-irradiated mice. Incomplete expression of filaggrin and loricrin in keratinocytes from the control mice was also improved in keratinocytes from the SA-PEG-treated mice. In photo-exposed human facial skin, immature CEs were replaced with mature CEs 4 weeks after treatment with SA-PEG. Restoration of photodamaged stratum corneum by treatment with SA-PEG, which may affect remodeling of the structural environment of the keratinocytes, involved the normalization of keratinocyte differentiation and suppression of skin tumor development. These results suggest that the stratum corneum plays a protective role against carcinogenesis, and provide a novel strategy for the prevention of photo-induced skin tumors.

Humidity is 1 of the environmental factors which regulate skin conditions. Effects of humidity on the cutaneous immune reaction were examined. Contact hypersensitivity to 2,4,6-trinitrochlorobenzene was elicited in C57BL/6 mice. The reaction was greater in mice housed under low humidity conditions (about 10%) for 2 days, at either the induction or elicitation phase, than in mice housed under rather high humidity conditions (80%). After housing under controlled humidity for 2 days, the number of I-A positive cells was 16% higher in the epidermis exposed to the dry condition. The increased population of FITC-positive cells were in regional lymph nodes after painting of FITC during housing under lower humidity. Our study demonstrated that the cutaneous immune reaction is regulated by environmental humidity and suggested 2 possible mechanisms, i.e., increase in Langerhans cells and increased penetration of allergen with low humidity.

Ampiroxicam (APX), a prodrug of piroxicam (PXM), has been reported to induce photosensitivity. Antigenic characterization of these photosensitivities, however, is still insufficient. The purpose of the present study was to elucidate further mechanism of photosenstivity induced by APX and PXM using an in vivo model of contact hypersensitivity in guinea pigs. Animals sensitized with ultraviolet-A (UVA)-irradiated 1% APX showed positive reaction in the patch testing to UVA-irradiated 1% APX and 1% thiosalicylate (TOS), while they were negative in challenge with UVA-irradiated 1% PXM, non-irradiated APX and PXM, whereas none of UVA-irradiated or non-irradiated APX and PXM showed positive patch test reaction in animals sensitized with UVA-irradiated 1% PXM or control vehicles. Animals sensitized with 1% TOS were successfully challenged by 1% TOS and cross-reacted with UVA-irradiated 1% APX; however, they failed to react with UVA-irradiated PXM, non-irradiated APX and PXM. Indeed, the in vitro study revealed that the concentration of APX was easily reduced by the increase of UVA irradiation dose, as compared with that of PXM. Interestingly, absorption spectrum of UVA-irradiated APX was similar to that of TOS, which is thought to be an active hapten of PXM. In the present study, we succeeded in the development of a novel animal model reflecting the clinical observations. Furthermore, these results suggested that contact hypersensitivity induced by UVA-irradiated APX is developed by photoproducts of APX itself, but not by the biotransformation of APX to PXM.

Gene expression can be evaluated quantitatively by conventional RT-PCR or Northern blotting with the aid of a correction based on the expression of an internal control gene. However, this approach is not suitable for quantitating gene expression in a group of heterogeneous cell subsets, because the internal control gene expression may vary among the subsets. Therefore, we developed a new method for quantitative PCR using rat poly(A)+ RNA as an external control. We used this method to investigate cytokine gene expression in lymph node cells from mice during the induction of contact hypersensitivity. Expression of the murine glyceraldehydephosphate dehydrogenase (GAPDH) gene, a candidate internal control, was not constant in cells from trinitrochlorobenzene- and vehicle-applied animals, suggesting that GAPDH gene expression changes in heterogeneous lymph node-cell subsets during induction of contact hypersensitivity. Therefore, we decided to use rat GAPDH mRNA as an external control. Cytokine gene expression was measured by quantitative PCR and was corrected based on external rat GAPDH cDNA. The reliability of this quantitative PCR was superior to that of the conventional method with an internal control.

The local lymph node assay is an effective prediction method for contact allergenicity, but employs radioisotopes. We investigated whether measurement of interleukin-2 (IL-2) production by lymph node cells could be used instead to predict contact allergenicity of chemicals. Test chemicals were applied for three consecutive days to both ears of Balb/c mice and the auricular lymph nodes were obtained on either the fourth or fifth day after the first application. Both IL-2 concentration in supernatant of the suspension and proliferative activity of lymph node cells were determined after 24-, 48-, 72-h cell culture in RPMI-1640 medium by ELISA and by measuring [3H]methylthymidine incorporation, respectively. These two methods detected allergenicity similarly except in the case of TNCB and oxazolone, which showed excessive proliferation-inducing capacity as compared to IL-2 release-increasing effect. Flow cytometry showed that these two chemicals also increased the percentage of Iad-positive cells in the lymph nodes, suggesting that these chemicals might induce not only cellular immunity but also humoral immunity. We conclude that interleukin-2 assay is a convenient and dependable method for screening strong contact allergens without using radioisotopes.

We have demonstrated previously in guinea pigs that the induction of photocontact sensitivity to piroxicam (PXM) also induces a state of cross-reactive contact hypersensitivity to two compounds having structurally related elements, thimerosal (TMS) and thiosalicylate (TOS). The present study was conducted to determine whether oral administration of TOS would desensitize guinea pigs previously photosensitized with PXM. At the same time, the spectrum of reactivities against these compounds and against tenoxicam (TXM) which resembles only piroxicam was assessed by appropriate sensitizing and eliciting protocols. As expected, animals photosensitized to PXM developed reactivities against all four compounds, PXM and TXM (photosensitivity) and TMS and TOS (contact sensitivity). By contrast, photosensitization with TXM induced cross-reactivity only against PXM. Moreover, the induction of contact sensitivity against TMS or TOS induced photosensitive cross-reactivity to PXM, but not to TXM. Finally, the oral administration of TOS produced a transient desensitization only for TMS and TOS. These results suggest that photosensitization with PXM induces two distinct reactivities. The first reactivity cross-reacts with TMS and TOS and is suppressible with orally administered TOS. The second cross-reacts only with TXM and is not suppressible with oral TOS. We conclude that PXM acquires at least two distinct immunogenic epitopes when exposed to UVA irradiation.

Atopic dermatitis (AD) has been clinically well-known to be frequently exacerbated by psychological and physiological stress. In this study, we examined effects of sedative odorant (modified valerian oil) inhalation on patients with AD. We investigated clinical scores, skin physiological parameters and psychological questionnaire (POMS) every 2 weeks. For first 2 weeks, we arranged non-inhalation period. Results for non-inhalation period were compared with these of 2- or 4-week inhalation. As results, sum of skin clinical scores significantly improved after odorant inhalation. Some patients improved for non-inhalation period, too. However, patients that had not improved for non-inhalation period significantly improved after odorant inhalation. Skin conductance and skin dryness/scaling score also improved after odorant inhalation without improving for non-inhalation period. Psychological parameter (POMS) also tended to improve after odorant inhalation. These results suggest that sedative odorants may be useful as a complementary therapy for AD through psychosomatic stress care.

A case of allergic contact dermatitis due to gold pierced earrings is reported. The patient developed recurring redness and swelling on her earlobes a month after the wearing of pierced-type gold earrings, which was followed by the appearance of reddish nodules around the puncture marks. Patch tests revealed positive reactions to 0.1% mercuric chloride, 1% gold sodium thiomalate and 0.2% chloroauric acid. We also demonstrated that guinea pigs contact-sensitized with a mercuric compound developed positive patch test reactions to both mercuric and gold compounds. These results suggest that there may be correlations between gold and mercury hypersensitivities.

We examined the cross-reaction between photosensitivity to piroxicam (PXM) and contact sensitivity to thiosalicylate (TOS) by a lymphocyte proliferation test (LPT) in guinea pigs. The lymph node cells (LNCs) plus peritoneal exudate cells (PECs) from guinea pigs contact-sensitized with TOS remarkably cross-proliferated to PXM under UVA (4 J/cm 2 ) irradiation. On the other hand, the PXM-photosensitized LNCs + PECs also cross-proliferated to TOS. From these results, the reciprocal cross-reaction between TOS-hypersensitivity and PXM-photosensitivity was reconfirmed by the in vitro LPT, indirectly indicating that the PXM-photosensitivity is a cell (probably T cell)-mediated PXM photoallergy in its nature. The TOS-primed LNCs + PECs did not cross-proliferate to UVA (4 J, 180 J or 500 J/cm 2 )-pretreated PXM (UVA-PXM) although it is supposed to contain several photoproducts of PXM. Furthermore, the TOS-primed LNCs developed a remarkable proliferative cross-response to the PECs pulsed with PXM under UVA (4 J/cm 2 ) irradiation (photo-PXM-modified PECs), but not to the PECs pulsed with PXM or UVA-PXM. Therefore, it is presumed that the cross-reactive molecule, which is easily formed from PXM under UVA irradiation, is unstable, and that the formation of complete antigen by the generation of this molecule and its photobinding needs the coexistense of PECs, PXM and UVA irradiation at the same time in the culture.