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.

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.

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.

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.

Differentiation of Human Adipose-Derived Mesenchymal Stromal/Stem Cells into Insulin-Producing Cells with A Single Tet-Off Lentiviral Vector System.

OBJECTIVE: Human adipose-derived mesenchymal stromal/stem cells (hASC) constitute an attractive source of stem cells for cell-based therapies in regenerative medicine and tissue engineering as they are easy to acquire from lipoaspirate, expansion, and genetic modification ex vivo. The combination of Pdx-1, MafA, and NeuroD1 has been indicated to possess the ability to reprogram various types of cells into insulin-producing cells. The aim of this study is to investigate whether MafA and NeuroD1 would cooperate with Pdx-1 in the differentiation of hASC into insulin-producing cells. MATERIALS AND METHODS: In this experimental study, we generated polycistronic expression vectors expressing Pdx1 and MafA/NeuroD1 with a reporter from a human EF-1α promoter using 2A peptides in a single tet-off lentiviral vector system. Briefly, hASC were transduced with the lentiviral vectors and allowed to differentiate into insulin-producing cells in vitro and in vivo. Thereafter, RNA expression, dithizone staining, and immunofluorescent analysis were conducted. RESULTS: Cleaved transcriptional factors from a single tet-off lentiviral vector were functionally equivalent to their native proteins and strictly regulated by doxycycline (Dox). Insulin gene expression in hASC transduced with Pdx1, Pdx1/ MafA, and Pdx1/NeuroD1 in differentiation medium were successfully increased by 1.89 ± 0.39, 4.81 ± 0.98, 5.51 ± 0.63, respectively, compared to venus-transduced, control hASC. These cells could form dithizone-positive cell clusters in vitro and were found to express insulin in vivo. CONCLUSION: Using our single tet-off lentiviral vector system, Pdx-1 and MafA/NeuroD1 could be simultaneously expressed in the absence of Dox. Further, this system allowed the differentiation of hASC into insulin-producing cells.

Mathematical modelling for antimicrobial photodynamic therapy mediated by 5-aminolaevulinic acid: An in vitro study.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) using aminolaevulinic acid (ALA) is a promising alternative to antibiotic therapy. ALA administration induces protoporphyrin IX (PpIX) accumulation in bacteria, and light excitation of the accumulated PpIX generates singlet oxygen to bacterial toxicity. Several factors, including drug administration and light irradiation conditions, contribute to the antibiotic effect. Such multiple parameters should be determined moderately for effective aPDT in clinical practice. METHODS: A mathematical model to predict bacterial dynamics in ALA-aPDT following clinical conditions was constructed. Applying a pharmacokineticspharmacodynamics (PK-PD) approach, which is widely used in antimicrobial drug evaluation, viable bacteria count by defining the bactericidal rate as the concentration of singlet oxygen produced when PpIX in bacteria is irradiated by light. RESULTS: The in vitro experimental results of ALA-aPDT for Pseudomonas aeruginosa demonstrated the PK-PD model validity. The killing rate has an upper limit, and the lower power density for a long irradiation time can suppress the viable bacteria number when the light dosages are the same. CONCLUSIONS: This study proposed a model of bacterial viability change in ALA-aPDT based on the PK-PD model and confirmed, by in vitro experiments using PA, that the variation of bacterial viability with light-sensitive substance concentration and light irradiation power densities could be expressed. Further validation of the PK-PD model with other gram negative and gram positive strains will be needed.

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.

Open study of photodynamic therapy for skin ulcers infected with MRSA and Pseudomonas aeruginosa.

BACKGROUND: Drug-resistant bacterial infections are a global problem. Novel treatment methods that simultaneously control infection and promote wound healing without leading to new resistant bacteria are needed. Photodynamic therapy (PDT) is a useful antibiotic-free treatment approach. Our previous studies have shown that PDT for skin ulcers infected with methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA) can achieve infection control and promoting wound healing in vitro and in vivo murine model. Here, we investigated the safety and effectiveness of PDT with 5-aminolevulinic acid (ALA-PDT) for human skin ulcers infected with MRSA and PA. METHODS: ALA-PDT with macrogol ointment containing 0.5% ALA-HCl and 0.005% EDTA-2Na (wavelength 410 nm, 10 J/cm2) was performed on consecutive days in patients aged ≥20 years who had skin ulcers infected with MRSA and PA. RESULTS: Six of our seven patients showed a clear tendency for ulcer area reduction to ≤60% of that measured at baseline. ALA-PDT was judged to be completely safe in all patients; only one patient had an increase in bacterial count. CONCLUSIONS: ALA-PDT is safe and effective for MRSA and PA infected skin ulcers to control and heal wound.

Measurement of absorption and reduced scattering coefficients in Asian human epidermis, dermis, and subcutaneous fat tissues in the 400- to 1100-nm wavelength range for optical penetration depth and energy deposition analysis (Errata).

The errata correct errors that appeared in Table 2 of the published article.

Glucose-6-phosphate dehydrogenase correlates with tumor immune activity and programmed death ligand-1 expression in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare and highly malignant skin cancer. Some cases have a good prognosis and spontaneous regression can occur. Reported prognostic markers, such as Merkel cell polyoma virus infection or programmed death ligand-1 (PD-L1) expression, remain insufficient for precisely estimating the vastly different patient outcomes. We performed RNA sequencing to evaluate the immune response and comprehensively estimate prognostic values of immunogenic factors in patients with MCC. METHODS: We collected 90 specimens from 71 patients and 53 blood serum samples from 21 patients with MCC at 10 facilities. The mRNA was extracted from formalin-fixed paraffin-embedded tissues. Next-generation sequencing, immunohistochemical staining and blood serum tests were performed. RESULTS: Next-generation sequencing results classified MCC samples into two types: the 'immune active type' was associated with better clinical outcomes than the 'cell division type'. Expression of the glucose-6-phosphate dehydrogenase (G6PD) gene was highly significantly upregulated in the 'cell division type'. Among 395 genes, G6PD expression correlated with the presence of lymph node or distant metastases during the disease course and significantly negatively correlated with PD-L1 expression. Immunohistochemical staining of G6PD also correlated with disease-specific survival and exhibited less heterogeneity compared with PD-L1 expression. G6PD activity could be measured by a blood serum test. The detection values significantly increased as the cancer stage progressed and significantly decreased after treatment. CONCLUSIONS: G6PD expression was an immunohistochemically and serum-detectable prognostic marker that negatively correlated with immune activity and PD-L1 levels, and could be used to predict the immunotherapy response.

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.

Measurement of absorption and reduced scattering coefficients in Asian human epidermis, dermis, and subcutaneous fat tissues in the 400- to 1100-nm wavelength range for optical penetration depth and energy deposition analysis.

SIGNIFICANCE: In laser therapy and diagnosis of skin diseases, the irradiated light distribution, which is determined by the absorption coefficient µa and reduced scattering coefficient µs' of the epidermis, dermis, and subcutaneous fat, affects the treatment outcome and diagnosis accuracy. Although values for µa and µs' have been reported, detailed analysis for Asian skin tissues is still lacking. AIM: We present µa and µs' measurements of Asian skin tissues in the 400- to 1100-nm wavelength range for evaluating optical penetration depth and energy deposition. APPROACH: The measurements with Asian human skin samples are performed employing a double integrating sphere spectrometric system and an inverse Monte Carlo technique. Using the measured parameters, the optical penetration depth and energy deposition are quantitatively analyzed. RESULTS: The µa of the epidermis layer varies among different ethnic groups, while the µa of the other layers and the µs' of all of the layers exhibit almost no differences. The analysis reveals that the optical penetration depth and the energy deposition affect the photodynamic therapy treatment depth and the heat production in skin tissue, respectively. CONCLUSIONS: The experimentally measured values of µa and µs' for Asian skin tissues are presented, and the light behavior in Asian skin tissues is analyzed using a layered tissue model.

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.

Adipose-derived stromal/stem cells improve epidermal homeostasis.

Wound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

Relationship between dermoscopy and pathology in a case of clonal-type pigmented Bowen's disease: Observation with vertical-view dermoscopy.

Pigmented Bowen's disease (pBD) is a subtype of Bowen's disease, which presents clinically as a well-circumscribed, hyperpigmented plaque. Its clinical manifestations are not fully characterized, and differential diagnoses include various pigmented skin lesions. Dermoscopy could be useful for the diagnosis, although nothing has been reported on the dermoscopic features of clonal-type pBD. We herein report a first case of clonal-type pBD on the sole and its dermoscopic features. Dermoscopy showed brown to blue-gray dots/globules and focally anastomosing lines on the non-weight-bearing area, while the weight-bearing area had a brown to blue-gray fibrillar-like pattern. To investigate the relationship between dermoscopy and histopathology, we focused on the melanin distribution in the horny layer of the epidermis, and used vertical dermoscopy observation. We investigated the relationship between dermoscopy and pathology by melanin depth estimation using a color lightness value.