Inhibitory Effects of Bilirubin on Colonization and Migration of A431 and SK-MEL-3 Skin Cancer Cells Compared with Human Dermal Fibroblasts (HDF).

This study evaluated the inhibitory effects of bilirubin on colony formation and cell migration of melanoma and non-melanoma skin cancer cell lines SK-MEL-3 and A431, compared with normal human dermal fibroblasts (HDF). The IC50 obtained from the MTT assay was 125, 100, and 75 µM bilirubin for HDF, A431, and SK-MEL-3 cells, respectively. The colony formation and cell migration of cancer cells, treated with 100 µM bilirubin, were reduced significantly (p < 0.05). Bilirubin decreased cell adhesion and inhibited cell colonization via inducing apoptosis and cell death. Also by interaction with migration main factors, bilirubin caused inhibition the cell migration.

[Fundamentals and principles of grafts and flaps]. / Fundamentos y principios de los injertos y colgajos.

Reconstructive surgery of large urethral stenosis and the management of congenital anomalies such as hypospadias and epispadias require covering large cutaneous and mucosal defects with different techniques. The objective of this work is to define the main differences between tissues to be transferred and to study the principles that must govern the management of the various flaps and grafts used for these techniques. We analyze the anatomical and physiological features that may be key to understand the success and possible failures of these procedures, and we review technical details that must accompany in every case, not only during the operation, but also during the preoperative and postoperative period. We conclude stating that grafts (mainly oral and preputial mucosa) and flaps are increasingly used for the repair of urethral stenosis. Grafts must be prepared adequately in the back table and thinned to the maximum, and also be fixed properly, to guarantee their immobility until neovascularization is assured.

PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK).

Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism.

Melanocyte spheroids are formed by repetitive long-term trypsinization.

BACKGROUND: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. OBJECTIVE: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. METHODS: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. RESULTS: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. CONCLUSION: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

Immunohistochemical and ultrastructural features of Langerhans cells in condyloma acuminatum.

BACKGROUND: There are few studies on the abnormal morphology of Langerhans cells (LCs) in condyloma acuminatum (CA) lesions and the essence of the abnormal morphology of LCs in CA lesions is still not well elucidated. The aim of this study was to further investigate the morphological features of LCs in CA lesions. METHODS: CD1a(+) LCs in 13 CA lesions and in 13 normal controls were labeled using immunohistochemistry and examined by light microscopy. Ultrastructural investigation on LCs in six CA lesions and in six normal controls was performed by electron microscopy. RESULTS: Compared with those in normal controls, most CD1a(+) LCs in CA lesions exhibited dysplastic dendrities and abnormal distribution. The number of CD1a(+) LCs in CA lesions (26.31 +/- 18.84) was statistically lower (p < 0.001) than that in normal controls (72.00 +/- 27.40). Electron microscopy showed that the number of Birbeck granules within lesional LCs (4.00 +/- 2.94) was significantly decreased (p < 0.001) than that within normal LCs (10.80 +/- 4.78). The ultrastructures of most lesional LCs displayed degenerative changes. CONCLUSIONS: The morphology of most LCs in CA lesions shows degenerative changes, which suggest that these LCs have been functionally impaired.

Why human prepuce is a valuable resource in science? Histological, ultrastructural, immunohistochemical and statistical analysis / ¿Por qué el prepucio humano es un recurso valioso en la ciencia? Análisis histológicos, ultraestructurales, inmunohistoquímicos y estadísticos

SUMMARY: This research was to examine the histological and ultrastructural characteristics of prepuce samples, as well as vimentin and S100 protein localization and statistical analysis. Urologists have long struggled with the prepuce, which is used to treat a variety of urethral problems. Skin biopsies were collected from the prepuce at the moment of circumcision and processed for light microscopy, electron microscope examination, immunohistochemical techniques, and statistical analysis in a total of six boys. Histologically, the prepuce epidermis displayed focal spiky ridges, which are saw-toothed interspersed with sulci, slight hyperpigmentation, looser connective tissue and plentiful vascular components. Immunohistochemically, the existence of melanocytes and Langerhans cells in the epidermis, as well as smooth muscles in the dermis, was stained positively for vimentin. Also, there was a positive reactivity of the Langerhans cells in the epidermis and around Meissner's corpuscles in the dermis for S100 protein staining. Ultrastructurally, the prepuce's intercellular gaps were widened, melanocytes rested on a folded basement membrane, and desmosomal content was reduced, with a prominent active euchromatic nucleus. Cytoplasmic projections were distended and elongated, and the interstitial blood vessels were surrounded by endothelial cells and rested on a basement membrane. There were also minimal collagen fibers in the interstitium. The prepuce's histological and ultrastructural features, as well as immunohistological studies using vimentin and S100 protein as intermediate filaments and statistical analysis, all demonstrated that it is a useful scientific resource.

RESUMEN: El presente trabajo de investigación se realizó para examinar las características histológicas y ultraestructurales de las muestras de prepucio, así como la localización y el análisis estadístico de la vimentina y la proteína S100. Los urólogos han intentado trabajar durante mucho tiempo con el prepucio, que se usa para tratar una variedad de problemas uretrales. Se recolectaron biopsias de piel del prepucio de seis niños en el momento de la circuncisión y se procesaron para microscopía óptica, examen con microscopio electrónico, técnicas inmunohistoquímicas y análisis estadístico. Histológicamente, la epidermis del prepucio mostraba crestas puntiagudas focales, intercaladas con surcos, hiperpigmentación leve, tejido conectivo más laxo y abundantes componentes vasculares. Inmunohistoquímicamente, la existencia de melanocitos y células dendríticas epidérmicas (células de Langerhans), así como músculo liso en la dermis, se tiñeron positivamente para vimentina. Además, hubo una reactividad positiva de las células dendríticas epidérmicas en la epidermis y alrededor de los corpúsculos del tacto (de Meissner) en la dermis para la tinción de la proteína S100. Ultraestructuralmente, los espacios intercelulares del prepucio se ensancharon, los melanocitos descansaban sobre una membrana basal plegada y el contenido desmosómico se redujo, con un núcleo eucromático activo prominente. Las proyecciones citoplasmáticas estaban distendidas y alargadas, y los vasos sanguíneos intersticiales estaban rodeados por células endoteliales y descansaban sobre una membrana basal. También había fibras de colágeno mínimas en el intersticio. Las características histológicas y ultraestructurales del prepucio, así como los estudios inmunohistológicos utilizando vimentina y proteína S100 como filamentos intermedios y el análisis estadístico, demostraron que es un recurso científico útil.

Ultrastructural Analysis of the Foreskin in Patients With True Phimosis Treated or Not Treated With Topical Betamethasone and Hyaluronidase Ointment.

OBJECTIVE: To evaluate, by using scanning electron microscopy (SEM), the possible alterations of the foreskin connective tissue in patients with true phimosis submitted to topical treatment with betamethasone and hyaluronidase ointment. MATERIALS AND METHODS: We studied 15 patients (mean 5.3 years old) submitted or not to topical application of betamethasone 0.2% and hyaluronidase cream. For qualitative analysis of the connective tissue, we studied 5 samples from each foreskin, with 2 mm length. The samples were submitted to fixation for SEM by immersing tissue fragments in a modified Karnovsky solution for 48 hours at 4°C. The obtained acellular preparations were then processed for high-vacuum SEM, and observations were performed on a LEO 435 (Zeiss, Oberkochen, Germany) scanning electron microscope with an acceleration voltage of 15 to 20 kV. RESULTS: Parents of 7 children did not agree with the clinical treatment and opted for circumcision directly. These patients served as the control group (nontreated). Eight patients submitted to topical treatment could not expose the glans and were referred for circumcision. In SEM, with a magnification of 5000×, we observed important differences in the organization of the collagen and elastic system fibers when comparing treated and nontreated patients with betamethasone and hyaluronidase. Treated patients presented a different organization of collagen with a clear decrease in the elastic system fibers. CONCLUSION: The treatment of phimosis with betamethasone + hyaluronidase showed changes in the structure of the foreskin with a decrease of elastic system fibers, which is characteristic of the healing processes.

Non-invasive topical delivery of plasmid DNA to the skin using a peptide carrier.

Topical delivery to skin is an essential step in non-invasive application of nucleic acid therapeutics for cutaneous disorders. The barrier posed by different layers of the skin - stratum corneum on top followed by the viable epidermis below - makes it extremely challenging for large hydrophilic molecules like nucleic acids to efficiently enter the uncompromised skin. We report an amphipathic peptide Mgpe9 (CRRLRHLRHHYRRRWHRFRC) that can penetrate the uncompromised skin, enter skin cells and deliver plasmid DNA efficiently as nanocomplexes in vitro and in vivo without any additional physical or chemical interventions prevalent currently. We observe efficient gene expression up to the highly proliferating basal layer of the skin without observable adverse reactions or toxic effects after delivery of reporter plasmids. The entry mechanism of nanocomplexes possibly involves reversible modulation of junction proteins accompanied by transient changes in skin structure. This peptide holds potential to be used as an efficient transporter of therapeutic nucleic acids to the skin.

The effects of calcipotriol on the dendritic morphology of human melanocytes under oxidative stress and a possible mechanism: is it a mitochondrial protector?

BACKGROUND: Vitiligo is an acquired pigmentary disorder of unknown etiology that is clinically characterized by the development of white macules in the skin related to the selective loss of melanocytes in those areas. Evidence shows that mitochondria might be a unifying target of reactive oxygen species (ROS) generation, cytokine production, catecholamine release and/or alteration of Ca(2+) metabolism that leads to melanocyte loss. OBJECTIVE: To assess the protective effect of calcipotriol on mitochondria of human melanocytes by investigating their dendritic morphology under oxidative stress. METHODS: Human melanocytes were treated with 0.05% H2O2 as well as various concentrations of calcipotriol, after which the retraction velocity of melanocyte dendrites was assessed. Detection of malondialdehyde (MDA) and superoxide dismutase (SOD) was performed as were the mitochondrial membrane potential (MMP) and intracellular calcium concentration ([Ca(2+)]i). Ultrastructural changes of mitochondria in melanocytes were observed by transmission electron microscopy. In addition, the expression of Beclin1, microtubule-associated protein 1 light chain 3 (LC3), dynamin related protein 1 (Drp1), mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), which are related to autophagy and mitochondrial dynamics, were analyzed by Western blot. RESULTS: Calcipotriol reduced the retraction velocity of melanocyte dendrites. In addition, calcipotriol, from 20nM to 80nM, decreased the level of MDA, increased the activity of SOD, suppressed the reduction of MMP and recovered Ca(2+) homeostasis by reducing [Ca(2+)]i in a concentration-dependent manner. Observation by transmission electron microscopy suggested that calcipotriol might reduce the injury of mitochondria in melanocytes under oxidative stress. Furthermore, the expression of Beclin1, LC3-II/LC3-I, Mfn2 and Drp1 was higher in the calcipotriol-treated melanocytes than in the control or H2O2-treated melanocytes. The level of Mfn1 was almost unchanged, but was higher at a concentration of 80nM calcipotriol than in any other condition. The expression of Mfn2 and Drp1 decreased with increasing calcipotriol concentration. CONCLUSION: Our study demonstrates the antioxidative effect of calcipotriol on melanocytes against oxidative damage. Moreover, calcipotriol could be a promising drug delivery strategy to protect melanocytes against oxidative damage in vitiligo through autophagy or mitophagy.

PECAM1(+)/Sca1(+)/CD38(+) vascular cells transform into myofibroblast-like cells in skin wound repair.

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1(+)/Sca1(+) vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1(+)/Sca1(+)/CD38(+) cells proliferate upon wounding and could give rise to α-SMA(+) myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1(+)/Sca1(+)/CD38(+) vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1(+)/Sca1(+)/CD38(+) cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.

The reduction of serum aminotransferase levels is proportional to the decline of the glomerular filtration rate in patients with chronic kidney disease

OBJECTIVE: This study sought to determine the serum aminotransferase levels of patients with predialysis chronic kidney disease and establish their relationships with serum creatinine levels and glomerular filtration rate. METHODS: Patients with chronic kidney disease were evaluated between September 2011 and May 2012. Aminotransferase and creatinine serum levels were measured using an automated kinetic method, and glomerular filtration rates were estimated using the Cockroft-Gault and Modification of Diet in Renal Disease formulas to classify patients into chronic kidney disease stages. RESULTS: Exactly 142 patients were evaluated (mean age: 64±16 years). The mean creatinine serum level and glomerular filtration rate were 3.3±1.2 mg/dL and 29.1±13 mL/min/1.73 m2, respectively. Patients were distributed according to their chronic kidney disease stages as follows: 3 (2.1%) patients were Stage 2; 54 (38%) were Stage 3; 70 (49.3%) were Stage 4; and 15 (10.5%) were Stage 5. The mean aspartate aminotransferase and alanine aminotransferase serum levels showed a reduction in proportion to the increase in creatinine levels (p=0.001 and p=0.05, respectively) and the decrease in glomerular filtration rate (p=0.007 and p=0.028, respectively). Alanine aminotransferase and aspartate aminotransferase serum levels tended to be higher among patients classified as stage 2 or 3 compared with those classified as stage 4 or 5 (p=0.08 and p=0.06, respectively). CONCLUSIONS: The aspartate aminotransferase and alanine aminotransferase serum levels of patients with predialysis chronic kidney disease decreased in proportion to the progression of the disease; they were negatively correlated with creatinine levels and directly correlated with glomerular filtration rate. .

Bioreactor maintained living skin matrix.

Numerous reconstructive procedures result in wounds that require skin grafting. Often, the amount of tissue available from donor sites is limited. In vivo tissue expanders have been used clinically to generate larger sections of skin, and other methods exist to cover large wounds, but all have significant limitations. We investigated whether these difficulties could be overcome by increasing the surface area of skin in vitro while maintaining tissue viability. Human foreskin was incrementally expanded in a computer-controlled bioreactor system over 6 days to increase its surface dimensions under culture conditions. Morphological, ultrastructural, and mechanical properties of the foreskin were evaluated before and after expansion using histology, scanning electron microscopy, mercury porosimetry, and tensile testing. The surface area of the tissue was 110.7% +/- 12.2% greater, with maintenance of cell viability and proliferative potential. Histomorphological and ultrastructural analyses showed that dermal structural integrity was preserved. The pore diameter of the expanded skin was 64.49% +/- 32.8% greater. The mechanical properties were not adversely affected. These findings show that expansion of living skin matrices can be achieved using a computer-controlled bioreactor system. This technique provides an opportunity to generate large amounts of skin for reconstructive procedures.