The ultraviolet A-activated photosensitizer 2-(4-aminophenyl)-7-methoxybenzothiazole suppresses proliferation and induces apoptosis of keloid fibroblasts: a potential adjunctive therapy for keloids.

BACKGROUND: Photodynamic therapy (PDT), a therapeutic approach employing a photosensitizer and a specific wavelength of light, is an emerging option for treating neoplastic and nonneoplastic diseases. Keloids are fibroproliferative dermal lesions characterized by the proliferation of fibroblasts. Recently, PDT has been demonstrated as a potential treatment for keloids. AIM: To investigate the effects of our newly synthesized photosensitizer 2-(4-aminophenyl)-7-methoxybenzothiazole (6d) plus ultraviolet (UV)A irradiation (6d-UVA) on proliferation and apoptosis in keloid fibroblasts (KFs). METHODS: Fibroblasts cultured from normal skin and keloids were treated with 6d-UVA. Relevant assays including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-bromo-2'-deoxyuridine incorporation assay, immunofluorescence assay and flow cytometry analysis were performed. RESULTS: The combination of 6d (2.0 or 5.0 µmol/L) and UVA 0.5 J/cm(2) significantly decreased the viability and proliferation of KFs but not normal fibroblasts (NFs). Cell cycle analyses showed significant G0/G1 arrest and increased sub-G1 distribution in NFs induced by UVA-activated 6d at 5.0 µmol/L (hereafter referred to as 6d-UVA). This treatment also significantly induced generation of intracellular reactive oxygen species (ROS), loss of mitochondrial membrane potential (ΔΨm), and increased expression of active caspase-3. Pretreatment with N-acetyl-L-cysteine (aROS scavenger) reversed the increased active caspase-3 expression induced by 6d-UVA, indicating the involvement of ROS in 6d-UVA-induced apoptosis. CONCLUSIONS: This study indicates that 6d-UVA treatment exerts antiproliferative and pro-apoptotic effects in KFs. We propose that 6d-UVA could be a potentially usefull ancillary method for keloid treatment.

Clinical characteristics and outcomes of patients with pleural infections due to Stenotrophomonas maltophilia at a medical center in Taiwan, 2004-2012.

Stenotrophomonas maltophilia can cause various clinical diseases; however, pleural infections due to S. maltophilia are rare. We evaluated the clinical characteristics and outcomes of patients with pleural infections (complicated parapneumonic effusion or empyema) due to S. maltophilia who were treated at a medical center in Taiwan from 2004 to 2012. During the study period, 40 patients were treated for pleural infections due to S. maltophilia. The incidence of S. maltophilia pleural infections ranged from 2.66 per 1,000,000 patient-days in 2009 to 12.44 per 1,000,000 patient-days in 2011. Most of the patients with S. maltophilia pleural infections were immunocompromised male adults and all of the infections were acquired in healthcare settings. The majority of patients had polymicrobial pleural infections (n = 31, 77.5 %) and the most common pathogen was Pseudomonas aeruginosa (n = 12). The causes of pleural infections due to S. maltophilia were pneumonia due to S. maltophilia in two patients (5 %), post-surgical/tube thoracostomy in 26 (65 %) patients, and fistula (bronchopleural, esophagopleural and biliopleural) in 12 (30 %) patients. The 14-day and 30-day mortality rates were 32.5 % and 42.5 %, respectively. Pleural infections due to S. maltophilia are most commonly the result of surgical procedures, thoracostomy, and underlying fistulas. These infections are associated with a high mortality rate, especially among immunocompromised patients.

High-glucose environment reduces human ß-defensin-2 expression in human keratinocytes: implications for poor diabetic wound healing.

BACKGROUND: Wound healing is a dynamic and complicated process in which inflammation, re-epithelialization and angiogenesis play important roles. Intriguingly, all three processes have been found to be defective during diabetic wound healing conditions. One common denominator associated with regulation of these events is human ß-defensin-2 (hBD2). It has been shown that skin wounding induces cutaneous hBD2 expression, and diabetic wounds have been associated with inadequate hBD expression. OBJECTIVES: The current study was launched to explore the effects of a high-glucose environment on cultured human keratinocytes. METHODS: Human keratinocytes were exposed to indicated culture conditions. The mRNA and protein levels of hBD2 were determined, and activation of relevant pathways was evaluated. The small interference RNA approach was used to validate the functional role of the proposed pathway on hBD2 expression. RESULTS: We showed that high-glucose cultivated keratinocytes expressed reduced levels of hBD2 and phosphorylated signal transducer and activator of transcription (pSTAT)-1 constitutively. In addition, pSTAT-1 signalling is critically involved in hBD2 expression. Formation of advanced glycation endproducts, a direct consequence of a high-glucose environment, involves constitutive downregulation of pSTAT-1 and hBD2. The addition of interleukin-1ß, an important cytokine during the cutaneous wound healing process, enabled the upregulation of hBD2 expression of both normal- and high-glucose cultivated keratinocytes, but the absolute levels of hBD2 were still significantly lower in the high-glucose-treated group. CONCLUSIONS: As hBD2 plays multifaceted roles during the wound healing process, the inadequate expression of hBD2 during diabetic conditions contributes to impaired wound healing.

Magnetic resonance imaging of transplanted mouse islets labeled with chitosan-coated superparamagnetic iron oxide nanoparticles.

Although only 10% of islet recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years after transplantation. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect Feridex-labeled islet grafts in rodents. In this study, we used a novel MRI contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles, to monitor mouse islet grafts. Male inbred C57BL/6 mice were used as donors and recipients of islet transplantation. The islet cytotoxicity was evaluated by fluorescein diacetate and propidium iodide staining for RAW cells incubated with CSPIO. After being incubated overnight with and without CSPIO (10 mg/mL), 300 islets were transplanted under the left kidney capsule of each mouse. After transplantation, 3.0-Tesla MRI of the recipients was performed biweekly until 19 weeks. At the end of study, the islet graft was removed for insulin and Prussian blue staining. The cell death rates in RAW cells did not increase with increasing CSPIO concentrations or incubation time. The grafts of CSPIO-labeled islets were visualized on MRI scans as distinct hypointense spots homogeneously located at the upper pole of left kidney. Their MRI signal was 30%-50% that of control islets and was maintained throughout the follow-up period. At 18 weeks, the histology of CSPIO-labeled islet graft revealed the insulin- and iron-stained areas to be almost identical. Our results indicate that isolated mouse islets labeled with CSPIO nanoparticles can be effectively and safely imaged by using MRI as long as 18 weeks after transplantation.

Topical tacrolimus has a limited direct effect on ultraviolet B-irradiated keratinocytes: implications for its photocarcinogenic potential.

BACKGROUND: Topical tacrolimus has shown remarkable clinical efficacy in treating many dermatoses. Combining ultraviolet (UV) B and tacrolimus is an intriguing therapeutic regimen, especially for treatment of vitiligo, for which combination therapy may show greater clinical efficacy than topical tacrolimus alone. The photocarcinogenic potential of such a regimen is unclear, and conflicting results have been reported by different investigators. AIM: To clarify this important clinical issue, we investigated the effects of tacrolimus on UVB-irradiated cultured keratinocytes in terms of apoptosis, differentiation, cell-cycle regulation and DNA damage. METHODS: Cultured keratinocytes were treated with tacrolimus before and after UVB irradiation and the various cellular physiological changes were evaluated using trypan blue exclusion, terminal dUTP nick-end labelling, flow cytometry and Western blotting analyses. RESULTS: Our results showed that treatment of tacrolimus before or after UVB irradiation had no significant effects on cultured keratinocytes in terms of cell apoptosis, transglutaminase-1, involucrin expression, cell-cycle progression and phospho-H(2)AX compared with UVB irradiation alone. CONCLUSION: The direct effect of tacrolimus on UVB-irradiated keratinocytes is small, suggesting that clinical regimens combining UVB and tacrolimus also have a limited direct effect on healthy skin compared with UVB irradiation alone.

Hyperglycaemic conditions hamper keratinocyte locomotion via sequential inhibition of distinct pathways: new insights on poor wound closure in patients with diabetes.

BACKGROUND: Diabetes mellitus (DM) is characterized by impaired insulin signalling, elevated plasma glucose, and predisposition towards complications involving several organs. A major complication of DM is impairment of wound healing. In the re-epithelialization process during wound healing, migration of keratinocytes is a crucial step. Our previous report demonstrated that keratinocytes cultured in hyperglycaemic media showed decreased cell mobility. OBJECTIVES: The current study aimed to explore the effects of high glucose on keratinocyte migration after different treatment durations. METHODS: Keratinocytes were cultivated for indicated time periods under various concentrations of glucose. Relevant assays including Transwell migration and in vitro wound scratch assays, flow cytometric analysis, matrix metalloproteinase-1 (MMP-1) activity assay, determination of mRNA expression and Western blotting were performed. RESULTS: We demonstrated that (i) keratinocyte motility progressively and significantly decreased; (ii) the keratinocyte activation marker K16 was significantly suppressed; (iii) expression of alpha2beta1 integrin and MMP-1, both crucial for keratinocyte locomotion on collagen type I, was significantly downregulated; and (iv) expression of the phosphorylated signal transducer and activator of transcription-1 significantly decreased after hyperglycaemic treatment. More specifically, different pathways become involved after prolonged duration of high glucose cultivation to reduce keratinocyte locomotion further. CONCLUSIONS: We have demonstrated that high glucose treatment results in progressive suppression of keratinocyte locomotion and elucidated the molecular mechanisms involved. These results provide a reasonable explanation for the poor wound healing seen in patients with DM.

Daxx mediates SUMO-dependent transcriptional control and subnuclear compartmentalization.

SUMO (small ubiquitin-related modifier) modification is emerging as an important post-translational control in transcription. In general, SUMO modification is associated with transcriptional repression. Although many SUMO-modified transcription factors and co-activators have been identified, little is known about the mechanism underlying SUMOylation-elicited transcriptional repression. Here, we summarize that SUMO modification of transcription factors such as androgen receptor, glucocorticoid receptor, Smad4 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] co-activator results in the recruitment of a transcriptional co-repressor Daxx, thereby causing transcriptional repression. Such a SUMO-dependent recruitment of Daxx is mediated by the interaction between the SUMO moiety of SUMOylated factors and Daxx SUMO-interacting motif. Interestingly, the transrepression effect of Daxx on these SUMOylated transcription factors can be relieved by SUMOylated PML (promyelocytic leukaemia) via altering Daxx partition from the targeted gene promoter to PML nuclear bodies. Because Daxx SUMO-interacting motif is a common binding site for SUMOylated factors, a model of competition for Daxx recruitment between SUMOylated PML and SUMOylated transcription factors was proposed. Together, our findings strongly suggest that Daxx functions as a SUMO reader in the SUMO-dependent regulation of transcription and subnuclear compartmentalization.

FK506 inhibits tumour necrosis factor-alpha secretion in human keratinocytes via regulation of nuclear factor-kappaB.

BACKGROUND: Tacrolimus (FK506) ointment has been used for treatment of inflammatory dermatoses with remarkable success. Our previous studies have indicated that direct modulation of tacrolimus on keratinocytes (KCs) may have an impact on its therapeutic effect. The use of monoclonal antibody specific for tumour necrosis factor (TNF)-alpha has shown efficacy in treating both psoriasis and Crohn disease. Topical tacrolimus has also been shown to be effective for treating cutaneous manifestations of both diseases. OBJECTIVES: To explore the effects of FK506 on human KCs in terms of TNF-alpha secretion and to investigate the regulatory pathway involved. METHODS: Ultraviolet (UV) B-irradiated cultured KCs were treated with various concentrations of FK506. At indicated time points after UVB irradiation we determined: (i) the TNF-alpha concentrations present in the culture supernatants; (ii) the activation and translocation of nuclear factor (NF)-kappaB in the cell nucleus; and (iii) the protein expressions of IkappaB kinase (IKK) and IkappaB in the cell lysates. In addition, a mouse model was used to corroborate our in vitro findings in vivo. More specifically, topical tacrolimus was applied on to mouse skin unilaterally after UVB irradiation. The effects of FK506 on nuclear NF-kappaB expression of UVB-irradiated mouse skin were determined. RESULTS: Our results showed that FK506 dose-dependently downregulated the secretion of TNF-alpha from UVB-irradiated KCs. The activation and translocation of NF-kappaB in UVB-irradiated KCs were also dose-dependently suppressed by FK506. The degradation of IkappaB induced by UVB was also inhibited by FK506, while no change in IKK expression was noted regardless of UVB and FK506 treatment. Murine skin biopsies showed that nuclear NF-kappaB expression induced by UVB was inhibited by topical tacrolimus treatment. CONCLUSIONS: Our results indicate that FK506 inhibits TNF-alpha secretion in human KCs via direct regulation of NF-kappaB. This modulatory effect of FK506 on KCs offers a possible mechanism for how topical tacrolimus regulates cutaneous inflammatory conditions.