Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.

Functional Optical Coherence Tomography: In Vivo Applications in Dermatology and Neuroscience

Thesis (Ph.D.)--University of Washington, 2016-06

Transdermal delivery of a tetrapeptide: Evaluation of passive diffusion

Skin penetration of the tetrapeptide Ac-Ala-Ala-Pro-Val-NH2 was assessed. This peptide sequence fits the P-P-1 subsites of elastase and inhibits human neutrophil elastase competitively. Consequently this peptide may be therapeutically useful in a variety of inflammatory disorders, including psoriasis. in which elevated levels of human neutrophil elastase have been reported. Peptide penetration was assessed across whole human skin, whole skin with the stratum corneum removed by tape stripping and epidermis, which had been removed from the dermis by heat separation. The influence of 75% aqueous ethanol as a potential penetration enhancer of the tetrapeptide across epidermis was also assessed. The tetrapeptide did not penetrate whole human skin or epidermis, even under the influence of 75% aqueous ethanol. However, when the stratum corneum was removed tetrapeptide flux of 73.39 mug cm(-2) h(-1) was achieved. The study demonstrates that the stratum corneum is the main barrier to tetrapeptide skin penetration and must be overcome if therapeutically relevant amounts of tetrapeptide are to be delivered to the skin.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: An international multicenter study using quasi-standardized methods and materials

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. Standardized calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 mug cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 mug cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intra-laboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required. (C) 2005 Wiley-Liss, Inc.

Inhibition of S/G(2) phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.

Gas and particle dynamics of a contoured shock tube for pre-clinical microparticle drug delivery

We investigate the gas-particle dynamics of a device designed for biological pre-clinical experiments. The device uses transonic/supersonic gas flow to accelerate microparticles such that they penetrate the outer skin layers. By using a shock tube coupled to a correctly expanded nozzle, a quasi-one-dimensional, quasi-steady flow (QSF) is produced to uniformly accelerate the microparticles. The system utilises a microparticle cassette (a diaphragm sealed container) that incorporates a jet mixing mechanism to stir the particles prior to diaphragm rupture. Pressure measurements reveal that a QSF exit period - suitable for uniformly accelerating microparticles - exists between 155 and 220 mus after diaphragm rupture. Immediately preceding the QSF period, a starting process secondary shock was shown to form with its (x,t) trajectory comparing well to theoretical estimates. To characterise the microparticle, flow particle image velocimetry experiments were conducted at the nozzle exit, using particle payloads with varying diameter (2.7-48 mu m), density (600-16,800 kg/m(3)) and mass (0.25-10 mg). The resultant microparticle velocities were temporally uniform. The experiments also show that the starting process does not significantly influence the microparticle nozzle exit velocities. The velocity distribution across the nozzle exit was also uniform for the majority of microparticle types tested. For payload masses typically used in pre-clinical drug and vaccine applications (

Engineering of needle-free physical methods to target epidermal cells for DNA vaccination

A challenge in epidermal DNA vaccination is the efficient and targeted delivery of polynucleotides to immunologically sensitive Langerhans cells. This paper investigates this particular challenge for physical delivery approaches. The skin immunology and material properties are examined in the context of the physical cell targeting requirements of the viable epidermis. Selected current physical cell targeting technologies engineered to meet these needs are examined: needle and syringe; diffusion patches; liquid jet injectors; microneedle arrays/patches; and biolistic particle injection. The operating methods and relative performance of these approaches are discussed, with a comment on potential future developments and technologies. (c) 2005 Elsevier Ltd. All rights reserved.

Formulation of topical non-steroidal anti-inflammatory agents

Reversed-pahse high-performance liquid chromatographic (HPLC) methods were developed for the assay of indomethacin, its decomposition products, ibuprofen and its (tetrahydro-2-furanyl)methyl-, (tetrahydro-2-(2H)pyranyl)methyl- and cyclohexylmethyl esters. The development and application of these HPLC systems were studied. A number of physico-chemical parameters that affect percutaneous absorption were investigated. The pKa values of indomethacin and ibuprofen were determined using the solubility method. Potentiometric titration and the Taft equation were also used for ibuprofen. The incorporation of ethanol or propylene glycol in the solvent resulted in an improvement in the aqueous solubility of these compounds. The partition coefficients were evaluated in order to establish the affinity of these drugs towards the stratum corneum. The stability of indomethacin and of ibuprofen esters were investigated and the effect of temperature and pH on the decomposition rates were studied. The effect of cetyltrimethylammonium bromide on the alkaline degradation of indomethacin was also followed. In the presence of alcohol, indomethacin alcoholysis was observed and the kinetics of decomposition were subjected to non-linear regression analysis and the rate constants for the various pathways were quantified. The non-isothermal, sufactant non-isoconcentration and non-isopH degradation of indomethacin were investigated. The analysis of the data was undertaken using NONISO, a BASIC computer program. The degradation profiles obtained from both non-iso and iso-kinetic studies show that there is close concordance in the results. The metabolic biotransformation of ibuprofen esters was followed using esterases from hog liver and rat skin homogenates. The results showed that the esters were very labile under these conditions. The presence of propylene glycol affected the rates of enzymic hydrolysis of the ester. The hydrolysis is modelled using an equation involving the dielectric constant of the medium. The percutaneous absorption of indomethacin and of ibuprofen and its esters was followed from solutions using an in vitro excised human skin model. The absorption profiles followed first order kinetics. The diffusion process was related to their solubility and to the human skin/solvent partition coefficient. The percutaneous absorption of two ibuprofen esters from suspensions in 20% propylene glycol-water were also followed through rat skin with only ibuprofen being detected in the receiver phase. The sensitivity of ibuprofen esters to enzymic hydrolysis compared to the chemical hydrolysis may prove valuable in the formulation of topical delivery systems.

Inducible Overexpression of Endothelin-3 in a Transgenic Mouse Leads to Hyperpigmentation That Decreases Over Time

Endothelin-3 (Edn3) has been shown to be an essential environmental cue in melanocyte development. Edn3 and its receptor, EdnrB, are allelic to mouse mutations occurring at the lethal spotting and piebald loci, respectively; these mutations result in hypopigmentation phenotypes. Mutations in the genes for both Edn3 and EdnrB are implicated in human pigmentation disorders such as Waardenburg-Shah syndrome, which is characterized by pigmentation defects, deafness, and megacolon. In this study, a tetracycline-inducible transgenic mouse model that overexpresses Edn3 under the control of the Keratin 5 promoter was shown to produce a hyperpigmentation phenotype that decreases over time. The expression pattern of transgenic Edn3 and its effects on the melanocyte population were examined in transgenic embryos, postnatal skin, and the skin of adult mice that exhibit faded hyperpigmentation. These studies suggest that overexpression of Edn3 in this model is restricted primarily to the roof plate of the neural tube and surface ectoderm in the developing embryo and to keratinocytes in the epidermis of postnatal mice. A decline in transgenic expression and a reduction in the dermal melanocytes and free melanin that characterize the phenotype in juvenile mice were shown to correlate with the fading of the hyperpigmentation phenotype. Transgenic mice in which transgenic expression was repressed (resulting in the disappearance of the hyperpigmentation phenotype) also exhibited a decrease in the dermal melanocyte population. The Edn3-overexpressing mice used in this study might be helpful m understanding human skin conditions characterized by dermal melanocytosis.

UV-induced melanoma mouse model dependent on endothelin 3 over-expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.^

Uma interface para o controle de robôs móveis por intermédio de gestos

The main objective of this work was to enable the recognition of human gestures through the development of a computer program. The program created captures the gestures executed by the user through a camera attached to the computer and sends it to the robot command referring to the gesture. They were interpreted in total ve gestures made by human hand. The software (developed in C ++) widely used the computer vision concepts and open source library OpenCV that directly impact the overall e ciency of the control of mobile robots. The computer vision concepts take into account the use of lters to smooth/blur the image noise reduction, color space to better suit the developer's desktop as well as useful information for manipulating digital images. The OpenCV library was essential in creating the project because it was possible to use various functions/procedures for complete control lters, image borders, image area, the geometric center of borders, exchange of color spaces, convex hull and convexity defect, plus all the necessary means for the characterization of imaged features. During the development of the software was the appearance of several problems, as false positives (noise), underperforming the insertion of various lters with sizes oversized masks, as well as problems arising from the choice of color space for processing human skin tones. However, after the development of seven versions of the control software, it was possible to minimize the occurrence of false positives due to a better use of lters combined with a well-dimensioned mask size (tested at run time) all associated with a programming logic that has been perfected over the construction of the seven versions. After all the development is managed software that met the established requirements. After the completion of the control software, it was observed that the overall e ectiveness of the various programs, highlighting in particular the V programs: 84.75 %, with VI: 93.00 % and VII with: 94.67 % showed that the nal program performed well in interpreting gestures, proving that it was possible the mobile robot control through human gestures without the need for external accessories to give it a better mobility and cost savings for maintain such a system. The great merit of the program was to assist capacity in demystifying the man set/machine therefore uses an easy and intuitive interface for control of mobile robots. Another important feature observed is that to control the mobile robot is not necessary to be close to the same, as to control the equipment is necessary to receive only the address that the Robotino passes to the program via network or Wi-Fi.

Bioengineered Approaches to Prevent Hypertrophic Scar Contraction

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

UV-Induced Melanoma Mouse Model Dependent on Endothelin 3 Over-Expression

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.

On The Dirichlet Distribution

The Dirichlet distribution is a multivariate generalization of the Beta distribution. It is an important multivariate continuous distribution in probability and statistics. In this report, we review the Dirichlet distribution and study its properties, including statistical and information-theoretic quantities involving this distribution. Also, relationships between the Dirichlet distribution and other distributions are discussed. There are some different ways to think about generating random variables with a Dirichlet distribution. The stick-breaking approach and the Pólya urn method are discussed. In Bayesian statistics, the Dirichlet distribution and the generalized Dirichlet distribution can both be a conjugate prior for the Multinomial distribution. The Dirichlet distribution has many applications in different fields. We focus on the unsupervised learning of a finite mixture model based on the Dirichlet distribution. The Initialization Algorithm and Dirichlet Mixture Estimation Algorithm are both reviewed for estimating the parameters of a Dirichlet mixture. Three experimental results are shown for the estimation of artificial histograms, summarization of image databases and human skin detection.