Effect of photodynamic therapy with 5-aminolevulinic acid and EDTA-2Na against mixed infection of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

BACKGROUND: The increasing abundance of drug-resistant bacteria is a global threat. Photodynamic therapy is an entirely new, non-invasive method for treating infections caused by antibiotic-resistant strains. We previously described the bactericidal effect of photodynamic therapy on infections caused by a single type of bacterium. We showed that gram-positive and gram-negative bacteria could be killed with 5-aminolevulic acid and 410 nm light, respectively. However, clinically, mixed infections are common and difficult to treat. OBJECTIVE: We investigated the bactericidal effects of photodynamic therapy on mixed infections of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. METHODS: We compared bacterial growth with and without photodynamic therapy in vitro. Then, in vivo, we studied mixed infections in a mouse skin ulcer model. We evaluated the rates of ulcer area reduction and transitions to healing in treated and untreated mice. In addition, a comparison was made between PDT and existing topical drugs. RESULTS: We found that photodynamic therapy markedly reduced the growth of both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, in culture, and it reduced the skin ulcer areas in mice. PDT was also more effective than existing topical medicines. CONCLUSION: This study showed that photodynamic therapy had antibacterial effects against a mixed infection of gram-positive and gram-negative bacteria, and it promoted skin ulcer healing. These results suggested that photodynamic therapy could be effective in both single- and mixed-bacterial infections.

In Silico Evaluation of Nanosecond Laser Treatment of Pigmented Lesions Based on Skin Optical Properties Using a Model of Melanosome Disruption Threshold Fluence.

OBJECTIVES: This study aimed to evaluate the efficacy and safety of nanosecond laser treatment of pigmented lesions in silico using a model of melanosome disruption threshold fluence (MDTF) based on skin optical properties. METHODS: Particle size analysis and scanning electron microscopy were performed to determine the threshold fluence for melanosome disruption using a nanosecond laser. By inputting the obtained threshold fluence into the MDTF model and considering the variability in skin optical properties, irradiation parameters were calculated and compared with the results from clinical studies. RESULTS: The threshold fluences for 532 and 755 nm nanosecond laser irradiation were determined to be 3.0 and 15.0 J/cm2, respectively. In silico analysis showed that the incident fluence for moderately pigmented skin should be 1.2 times that for lightly pigmented skin, whereas it should be 50% lower than that for lightly pigmented skin to achieve the same level of energy deposition. Clinically applied fluences for moderately pigmented skin are at the low end of the calculated range of values, suggesting that the clinical fluence is chosen to minimize energy deposition in normal tissues. CONCLUSIONS: Our results showed that the MDTF model can be used to evaluate nanosecond laser treatments and provide clinical guidance on fluence settings based on laser-tissue interactions in moderately pigmented skin. The in silico method can, therefore, provide a robust and quantitative retrospective evaluation of the treatment effects that accounts for variation in irradiation parameters among patients by combining the MDTF model with the in vivo optical properties of individual skin types.

Wavelength-dependent threshold fluences for melanosome disruption to evaluate the treatment of pigmented lesions with 532-, 730-, 755-, 785-, and 1064-nm picosecond lasers.

BACKGROUND AND OBJECTIVES: A threshold fluence for melanosome disruption has the potential to provide a robust numerical indicator for establishing clinical endpoints for pigmented lesion treatment using a picosecond laser. Although the thresholds for a 755-nm picosecond laser were previously reported, the wavelength dependence has not been investigated. In this study, wavelength-dependent threshold fluences for melanosome disruption were determined. Using a mathematical model based on the thresholds, irradiation parameters for 532-, 730-, 755-, 785-, and 1064-nm picosecond laser treatments were evaluated quantitatively. STUDY DESIGN/MATERIALS AND METHODS: A suspension of melanosomes extracted from porcine eyes was irradiated using picosecond lasers with varying fluence. The mean particle size of the irradiated melanosomes was measured by dynamic light scattering, and their disruption was observed by scanning electron microscopy to determine the disruption thresholds. A mathematical model was developed, combined with the threshold obtained and Monte Carlo light transport to calculate irradiation parameters required to disrupt melanosomes within the skin tissue. RESULTS: The threshold fluences were determined to be 0.95, 2.25, 2.75, and 6.50 J/cm² for 532-, 730-, 785-, and 1064-nm picosecond lasers, respectively. The numerical results quantitatively revealed the relationship between irradiation wavelength, incident fluence, and spot size required to disrupt melanosomes distributed at different depths in the skin tissue. The calculated irradiation parameters were consistent with clinical parameters that showed high efficacy with a low incidence of complications. CONCLUSION: The wavelength-dependent thresholds for melanosome disruption were determined. The results of the evaluation of irradiation parameters from the threshold-based analysis provided numerical indicators for setting the clinical endpoints for 532-, 730-, 755-, 785-, and 1064-nm picosecond lasers.

A ubiquitin-like protein encoded by the "noncoding" RNA TINCR promotes keratinocyte proliferation and wound healing.

Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal differentiation-induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL.

Possible Involvement of Dermal Fibroblasts in Modulating Nrf2 Signaling in Epidermal Keratinocytes.

Epidermal keratinocytes protect themselves by cooperating with neighboring cells against internal and external stresses, which leads not only to the maintenance of cell homeostasis but also to the prevention of skin aging. Although it is known that nuclear factor (NF)-E2-related factor 2 (Nrf2) signaling plays a pivotal role in ameliorating oxidative stress and inflammatory responses under stress situations, it is unclear whether Nrf2 signaling in keratinocytes cooperates with neighboring cells such as dermal fibroblasts. Thus, this study was conducted to examine the influence of dermal fibroblasts on Nrf2 signaling in epidermal keratinocytes using a co-culture system. The results show that expression levels of Nrf2-regulated antioxidant factors, such as glutathione and heme oxygenase-1, in HaCaT keratinocytes (HaCaT KCs) are up-regulated in the presence of normal human dermal fibroblasts (NHDFs). In addition, the secretion of pro-inflammatory molecules, including interleukin-1α (IL-1α) and prostaglandin E2 (PGE2), is suppressed in co-cultures of NHDFs and UVB-irradiated HaCaT KCs. Interestingly, the localization of Nrf2 protein in HaCaT KCs was immediately translocated from the cytoplasm to the nucleus after the co-culture with NHDFs. These results suggest the possibility that Nrf2 signaling in keratinocytes is regulated in cooperation with dermal fibroblasts.

Nonlinear absorption-based analysis of energy deposition in melanosomes for 532-nm short-pulsed laser skin treatment.

BACKGROUND AND OBJECTIVES: The clinical use of 532-nm short-pulsed lasers has provided effective treatment of epidermal pigmented lesions. However, the detection of significant differences in treatment effects between picosecond and nanosecond lasers has still varied among clinical studies. For robust evaluation of the differences based on the treatment mechanism, this study presents a nonlinear absorption-based analysis of energy deposition in melanosomes for 532-nm short-pulsed laser treatment. STUDY DESIGN/MATERIALS AND METHODS: Nonlinear absorption by melanin is modeled based on sequential two-photon absorption. Absorption cross-sections and nonradiative lifetimes of melanin, which are necessary for the nonlinear absorption-based analysis, are determined from transmittance measurement. Using the model and parameters, energy deposition in melanosomes was calculated with varying fluence and pulse width settings, including actual clinical parameters. RESULTS: The energy deposition in melanosomes increased with shorter laser pulses, and subnanosecond laser pulses were found to be most efficient. The comparison of energy deposition calculated using clinical parameters demonstrated the differences in treatment effects between picosecond and nanosecond lasers reported in clinical studies. CONCLUSION: The nonlinear absorption-based analysis provides quantitative evidence for the safety and efficacy evaluation of short-pulsed laser treatments, which may lead to the establishment of numerical indices for determining treatment conditions. Future studies considering the effects of the surrounding tissue on energy deposition in melanosomes will be needed.

Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705-SH2 through C-terminal tail modulation.

Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705-SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT-CTT intermolecular and the CTT-SH2 intramolecular interactions that support pY705-SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705-SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705-SH2 interaction. Thus, pS727 triggers pY705-SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation-inactivation for proper function of STAT3.

Dermal Fibroblasts Internalize Phosphatidylserine-Exposed Secretory Melanosome Clusters and Apoptotic Melanocytes.

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

Anti-BP180-type mucous membrane pemphigoid immunoglobulin G shows heterogeneity of internalization of BP180/collagen XVII into keratinocyte cytoplasm.

Anti-BP180-type mucous membrane pemphigoid (BP180-MMP) is a rare autoimmune subepidermal blistering disease that targets the C terminus of BP180/collagen XVII. Currently, the pathomechanism of BP180-MMP is not well understood. We reported previously that immunoglobulin G (IgG) from patients with bullous pemphigoid (BP) can induce internalization of BP180 via a macropinocytic pathway, which depletes BP180 and weakens epidermal cell-matrix integrity. The purpose of the present study was to elucidate the pathomechanism of BP180-MMP. Immunohistochemistry of biopsy specimens from two patients with BP180-MMP revealed that one patient had BP180 internalization, but the other did not. In live-cell imaging using IgG from patients with BP180-MMP on several keratinocyte cell lines, IgG from only three out of the seven patients was associated with BP180 internalization into the cytoplasm. Our results suggest that IgG from patients with BP180-MMP shows heterogeneity of internalization of BP180. This variability in BP180 internalization in patients with BP or BP180-MMP may lead to differences in clinical presentation.

Intraoperative adjustment of eyelid level in aponeurotic blepharoptosis surgery.

There have been numerous reports on the use of aponeurotic surgery to correct involutional blepharoptosis. However, it is still difficult to determine optimal eyelid level during operation. Here we present our new method to adjust eyelid level intraoperatively. After the aponeurosis was temporally sutured to the tarsus, while still in the supine position, the patient was asked to look up, and the position of the eyelid margin was confirmed. The margin should be located above the pupil but within the cornea while the patient gazes up. And it is ideal if the eyelid position is located in the upper half of this range. Although 3 of 29 patients were reoperated on in the follow-up period, only 1 patient required readjustment in the perioperative period. Our method is simple, easy and reduces operative time, because it is not necessary to change patient position during the operation.

Bullous pemphigoid IgG induces BP180 internalization via a macropinocytic pathway.

Bullous pemphigoid (BP) is an autoimmune blistering skin disease induced by pathogenic autoantibodies against a type II transmembrane protein (BP180, collagen type XVII, or BPAG2). In animal models, BP180 autoantibody-antigen interaction appears insufficient to develop blisters, but involvement of complement and neutrophils is required. However, cultured keratinocytes treated with BP-IgG exhibit a reduction in the adhesive strength and a loss of expression of BP180, suggesting that the autoantibodies directly affect epidermal cell-extracellular matrix integrity. In this study, we explored the consequences of two distinct epithelial cells treated with BP-IgG, particularly the fate of BP180. First, we followed the distribution of green fluorescent protein-tagged BP180 in an epithelial cell line, 804G, and normal human epidermal keratinocytes after autoantibody clustering. After BP-IgG treatment, the adhesive strength of the cells to their substrate was decreased, and BP180 was internalized in both cell types, together with the early endosomal antigen-1. By using various endocytosis inhibitors and a fluid-uptake assay, we demonstrated that BP-IgG-induced BP180 internalization is mediated via a macropinocytic pathway. Moreover, a macropinocytosis inhibitor rescued a BP-IgG-induced reduction in the adhesive strength of the cells from their substrate. The results of this study suggest that BP180 internalization induced by BP-IgG plays an important role in the initiation of disease pathogenesis.

The discovery of epidermal tight junctions.

It was previously thought that the skin barrier is composed singly by the stratum corneum. However, this concept was overturned by the report of Tsukita's group in 2002. They convinced us that tight junctions exist in the stratum granulosum of the epidermis, with the constituent proteins being occludin, claudin-1 and claudin-4. However, more than 30 years before this, Hashimoto et al. described the possible existence of tight junctions in the epidermis in 'Intercellular spaces of the human epidermis as demonstrated with lanthanum' in 1971. Dr. Hashimoto observed lanthanum nitrate-injected human skin by electron microscopy. He discovered that the injected lanthanum penetrated the intercellular spaces of the basal and spinous layers of the epidermis and then moved towards the skin surface until penetration was halted in the granular cell layer near the stratum corneum. He described the cell-to-cell adhesion structures that blocked the movement of lanthanum as 'truly tight junctions'. Thus, this was the first description of the existence of tight junctions in the epidermis. However, the presence of these structures was denied by others and was forgotten. Thanks to the discovery of claudin, the existence of tight junctions between epidermal keratinocytes was finally confirmed. It is interesting that Hashimoto's finding was eventually proved to be correct three decades later as a result of progress in molecular biology. This article encourages us to recognize the importance of careful observation in the molecular biology era.

Treatment of acute temporomandibular joint dislocation using manipulation technique for disk displacement.

Temporomandibular joint dislocation is not frequently encountered, but it is often difficult to reduce the dislocation with conventional methods described in textbooks. The key points to success of reduction depend on the patient's position, route of approach, and timing of reducing each side. We apply a manipulation technique for disk displacement to the reduction that corresponds to these key points. Using our method, temporomandibular joint dislocation can be easily reduced, without using sedative or analgesics. This method is simple, convenient, and worth trying in place of the conventional method.

The role of hemidesmosomes and focal contacts in the skin visualized by dual-color live cell imaging.

Live cell imaging is a powerful tool to elucidate dynamics of protein(s). Our group has concentrated on dynamics of two major cell-matrix adhesion devices, hemidesmosome and focal contact in the keratinocytes. Firstly, we observed the fate of hemidesmosome protein or focal contact protein by single-color live cell imaging in the physiological setting of keratinocytes. Both hemidesmosome proteins and focal contact proteins were highly dynamic. Next, in order to observe the interaction between hemidesmosome protein and focal contact protein, we observed the fate of these proteins at the same time by dual-color live cell imaging in physiological setting and in wound setting of keratinocytes. These hemidesmosome proteins and focal contact proteins showed individual dynamics with minimal overlap expressions in physiological settings. In sharp contrast, both proteins showed highly regulated interaction in wound setting of keratinocytes. Finally, we observed the fate of BP180 protein, which is a major target of autoimmune bullous disease, bullous pemphigoid, and component of hemidesmosome, under the existence of anti-BP180 autoantibody. In results, under such a circumstance, BP180 molecules were internalized and thus keratinocyte showed weakened adhesion to the cell matrix. Our work has elucidated dynamic aspects of cell-matrix adhesion devices under both physiological and pathological conditions.

Ultralow radiant exposure of a short-pulsed laser to disrupt melanosomes with localized thermal damage through a turbid medium.

Short-pulsed lasers can treat dermal pigmented lesions through selective photothermolysis. The irradiated light experiences multiple scattering by the skin and is absorbed by abnormal melanosomes as well as by normal blood vessels above the target. Because the fluence is extremely high, the absorbed light can cause thermal damage to the adjacent tissue components, leading to complications. To minimize radiant exposure and reduce the risk of burns, a model of the melanosome-disruption threshold fluence (MDTF) has been developed that accounts for the light-propagation efficiency in the skin. However, the light-propagation efficiency is attenuated because of multiple scattering, which limits the extent to which the radiant exposure required for treatment can be reduced. Here, this study demonstrates the principle of melanosome disruption with localized thermal damage through a turbid medium by ultralow radiant exposure of a short-pulsed laser. The MDTF model was combined with a wavefront-shaping technique to design an irradiation condition that can increase the light-propagation efficiency to the target. Under this irradiation condition, melanosomes were disrupted at a radiant exposure 25 times lower than the minimal value used in conventional laser treatments. Furthermore, almost no thermal damage to the skin was confirmed through a numerical simulation. These experimental and numerical results show the potential for noninvasive melanosome disruption and may lead to the improvement of the safety of short-pulsed laser treatment.

Intracellular TAS2Rs act as a gatekeeper for the excretion of harmful substances via ABCB1 in keratinocytes.

Bitter taste receptors (TAS2Rs) are not only expressed in the oral cavity but also in skin. Extraoral TAS2Rs are thought to be involved in non-taste perception and tissue-specific functions. Keratinocytes that express TAS2Rs in the skin provide a first-line defense against external threats. However, the functional roles of these receptors in host defense remain unclear. Here, we demonstrated the sensory role of intracellularly located TAS2Rs against toxic substances in keratinocytes. Although many G protein-coupled receptors elicit signals from the surface, TAS2Rs were found to localize intracellularly, possibly to the ER, in human keratinocytes and HaCaT cells. TAS2R38, one of the TAS2R members, activated the Gα12/13/RhoA/ROCK/p38 MAP kinase/NF-κB pathway upon stimulation by phenylthiocarbamide (PTC), an agonist for this receptor, leading to the production of ABC transporters, such as ABCB1, in these cells. Notably, treatment with bitter compounds, such as PTC and saccharin, induced the upregulation of ABCB1 in HaCaT cells. Mechanistically, intracellular TAS2R38 and its downstream signaling Gα12/13/RhoA/ROCK/p38 MAP kinase/NF-κB pathway were identified to be responsible for the above effect. Pretreatment with PTC prevented the accumulation of rhodamine 123 because of its excretion via ABCB1. Furthermore, pretreatment with PTC or saccharin counteracted the effect of the toxic compound, diphenhydramine, and pretreated HaCaT cells were found to proliferate faster than untreated cells. This anti-toxic effect was suppressed by treatment with verapamil, an ABCB1 inhibitor, indicating that enhanced ABCB1 helps clear toxic substances. Altogether, harmless activators of TAS2Rs may be promising drugs that enhance the excretion of toxic substances from the human skin.

Preauricular transparotid approach to mandibular condylar fractures without dissecting facial nerves.

Preauricular transparotid approach without dissecting the facial nerve was used for surgical treatment of 15 condylar fractures in 14 patients. The parotid fascia was opened just above the fracture site, and by dissecting the parotid gland and masseter muscle, the fracture was directly exposed. The facial nerve itself was not dissected expressly. All fractures could be reduced accurately and fixed firmly with miniplates. A direct approach just above the fracture site provided good vision of the fracture, avoiding facial nerve palsy caused by strong retraction. Moreover, by not dissecting the facial nerve, the operation time was shortened. This approach was useful for surgical treatment of both condylar neck and subcondylar fractures.

Transient simulation of laser ablation based on Monte Carlo light transport with dynamic optical properties model.

Laser ablation is a minimally invasive therapeutic technique to denature tumors through coagulation and/or vaporization. Computational simulations of laser ablation can evaluate treatment outcomes quantitatively and provide numerical indices to determine treatment conditions, thus accelerating the technique's clinical application. These simulations involve calculations of light transport, thermal diffusion, and the extent of thermal damage. The optical properties of tissue, which govern light transport through the tissue, vary during heating, and this affects the treatment outcomes. Nevertheless, the optical properties in conventional simulations of coagulation and vaporization remain constant. Here, we propose a laser ablation simulation based on Monte Carlo light transport with a dynamic optical properties (DOP) model. The proposed simulation is validated by performing optical properties measurements and laser irradiation experiments on porcine liver tissue. The DOP model showed the replicability of the changes in tissue optical properties during heating. Furthermore, the proposed simulation estimated coagulation areas that were comparable to experimental results at low-power irradiation settings and provided more than 2.5 times higher accuracy when calculating coagulation and vaporization areas than simulations using static optical properties at high-power irradiation settings. Our results demonstrate the proposed simulation's applicability to coagulation and vaporization region calculations in tissue for retrospectively evaluating the treatment effects of laser ablation.

Comparison of pediatric and adult nasal fractures.

Nasal fractures are the most common facial fracture in children and adults. Generally, it is believed that reduction of pediatric nasal fracture is more difficult and should be performed earlier compared with that of adult nasal fracture. However, there has been no article to prove this theory. We investigated 423 patients with acute nasal fractures requiring surgery and divided them into the following 2 groups: patients 12 years and younger (pediatric group) and patients 13 years and older (adult group). We then compared these patients in various aspects. There were no significant differences in the cause of fracture or postoperative conditions. Only the type of fracture and the anesthesia were different between these 2 groups. In the pediatric group, the interval between injury and surgery was arbitrarily divided into 2 groups, but there was no significant difference between these groups in the postoperative conditions. Some reports recommended that pediatric nasal fractures should be reduced within 3 to 5 days, but it cannot be proven. In conclusion, it is not necessary to distinguish treatment of pediatric nasal fracture from that of adult nasal fracture.