Safety Assessment of Keratin and Keratin-Derived Ingredients as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of 8 keratin-derived ingredients, which function mainly as skin and hair conditioning agents in personal care products. The Panel reviewed relevant data provided in this safety assessment and concluded that the 8 keratin-derived ingredients are safe in the present practices of use and concentration described in this safety assessment.

Keratin-Based Nanoparticles with Tumor-Targeting and Cascade Catalytic Capabilities for the Combinational Oxidation Phototherapy of Breast Cancer.

Photodynamic therapy (PDT) holds tantalizing prospects of a prominent cancer treatment strategy. However, its efficacy remains limited by virtue of the hypoxic tumor microenvironment and the inadequate tumor-targeted delivery of photosensitizers, and these can be further exacerbated by the lack of development of a well-controlled nitric oxide (NO) release system at the target site. Inspired by Chinese medicine, we propose a revealing new keratin application. Keratin has garnered attention as an NO generator; however, its oncological use has rarely been investigated. We hypothesized that the incorporation of a phenylboronic acid (PBA) targeting ligand/methylene blue (MB) photosensitizer with a keratin NO donor would facilitate precise tumor delivery, enhancing PDT. Herein, we demonstrated that MB@keratin/PBA/d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) nanoparticles (MB@KPTNPs) specifically targeted breast cancer cells and effectively suppressed their growth. Through MB-mediated biometabolism, the endocytic MB@KPTNPs produced a sufficient amount of intracellular NO that reduced the glutathione level while boosting the efficiency of PDT. A therapeutic combination of NO/PDT was therefore achieved, resulting in significant inhibition of both in vivo tumor growth and lung metastasis. These findings underscore the importance of utilizing keratin-based nanoparticles that simultaneously combine targeting of the tumor and self-generating NO with a cascading catalytic ability as a novel oxidation therapeutic strategy for enhancing PDT.

Effects of the Blending Ratio on the Design of Keratin/Poly(butylene succinate) Nanofibers for Drug Delivery Applications.

In recent years there has been a growing interest in the use of proteins as biocompatible and environmentally friendly biomolecules for the design of wound healing and drug delivery systems. Keratin is a fascinating protein, obtainable from several keratinous biomasses such as wool, hair or nails, with intrinsic bioactive properties including stimulatory effects on wound repair and excellent carrier capability. In this work keratin/poly(butylene succinate) blend solutions with functional properties tunable by manipulating the polymer blending ratios were prepared by using 1,1,1,3,3,3-hexafluoroisopropanol as common solvent. Afterwards, these solutions doped with rhodamine B (RhB), were electrospun into blend mats and the drug release mechanism and kinetics as a function of blend composition was studied, in order to understand the potential of such membranes as drug delivery systems. The electrophoresis analysis carried out on keratin revealed that the solvent used does not degrade the protein. Moreover, all the blend solutions showed a non-Newtonian behavior, among which the Keratin/PBS 70/30 and 30/70 ones showed an amplified orientation ability of the polymer chains when subjected to a shear stress. Therefore, the resulting nanofibers showed thinner mean diameters and narrower diameter distributions compared to the Keratin/PBS 50/50 blend solution. The thermal stability and the mechanical properties of the blend electrospun mats improved by increasing the PBS content. Finally, the RhB release rate increased by increasing the keratin content of the mats and the drug diffused as drug-protein complex.

Intrinsically water-stable keratin nanoparticles and their in vivo biodistribution for targeted delivery.

Highly water-stable nanoparticles of around 70 nm and capable of distributing with high uptake in certain organs of mice were developed from feather keratin. Nanoparticles could provide novel veterinary diagnostics and therapeutics to boost efficiency in identification and treatment of livestock diseases to improve protein supply and ensure safety and quality of food. Nanoparticles could penetrate easily into cells and small capillaries, surpass detection of the immune system, and reach targeted organs because of their nanoscale sizes. Proteins with positive and negative charges and hydrophobic domains enable loading of various types of drugs and, hence, are advantageous over synthetic polymers and carbohydrates for drug delivery. In this research, the highly cross-linked keratin was processed into nanoparticles with diameters of 70 nm under mild conditions. Keratin nanoparticles were found supportive to cell growth via an in vitro study and highly stable after stored in physiological environments for up to 7 days. At 4 days after injection, up to 18% of the cells in kidneys and 4% of the cells in liver of mice were penetrated by the keratin nanoparticles.

Understanding the nano-topography changes and cellular influences resulting from the surface adsorption of human hair keratins.

Recent interest in the use of human hair keratins as a biomaterial has grown, fuelled by improvements in keratin extraction methods and better understanding of keratin bioactivity. The use of keratins as a bioactive coating for in vitro cell culture studies is an attractive proposition. In this light, the surface adsorption of human hair keratins onto tissue culture polystyrene surfaces has been investigated. Keratin density, nano-topography and hydrophobicity of keratin coated surfaces were characterized. To understand the cellular influence of these coated surfaces, murine L929 fibroblasts were cultured on them and evaluated for cytotoxicity, proliferation, metabolic activity and detachment behaviors compared to collagen type 1 coated surfaces. Keratins were deposited up to a density of 650 ng/cm(2) when a coating concentration of 80 µg/ml or higher was used. The surface features formed by adsorbed keratins also changed in a coating concentration dependent manner. These surfaces improved L929 mouse fibroblast adhesion and proliferation in comparison to uncoated and collagen type 1 coated tissue culture polystyrene. Furthermore, the expression of fibronectin was accelerated on surfaces coated with solutions of higher keratin concentrations. These results suggest that human hair keratins can be used as a viable surface coating material to enhance substrate compliance for culturing cells.

Cultivo in vitro con colágeno y fibroblastos humanos de un equivalente de mucosa oral de espesor total / In vitro culture with collagen and human fibroblasts of a full-thickness oral mucosa equivalent

Objetivos. El presente trabajo tiene por objetivo obtener, mediantecultivo in vitro, láminas de tejido oral en las que se pueda identificar lasestructuras de una mucosa oral completa. La aplicación clínica del presenteestudio permitiría, en determinados casos, la sustitución del empleo de injertoslibres de piel o autólogos de mucosa oral por esta técnica. Material y Método.A partir de pequeñas biopsias de mucosa oral se hicieron cultivos primariosde queratinocitos. A partir de estos cultivos primarios se realizaron cultivossecundarios sobre una submucosa artificial constituida por colágeno y fibroblastoshumanos. Se analizaron histológicamente sus características in vitro, yulteriormente se procedió a la realización de injertos en ratones atímicospara conocer su comportamiento in vivo. Resultados. Los cultivos primariosfueron confluentes en un plazo mínimo de 10 días y máximo de 12 días, periodosimilar al observado para la confluencia de los cultivos secundarios. El tiempotranscurrido desde la toma de la muestra hasta la obtención de una mucosaartificial completa osciló entre los 20 y los 22 días, mostrando las característicashistológicas de una mucosa normal. Tras 17 días de injerto en ratonesinmunoincompetentes, sin ningún tipo de contingencia clínica, la caracterizaciónhistológica e inmunohistoquímica (citoqueratinas 13 y 19, colágenoIV y laminina) confirmó la similitud de la mucosa in vitro con la mucosa oralsana. Conclusión. Es posible mediante técnicas de cultivo in vitro la obtenciónde un equivalente de mucosa oral completa con colágeno y fibroblastos. Sibien esta mucosa muestra un importante grado de retracción, su manejo clínicoes muy favorable(AU)

Objectives. The objective of this study was to obtain,by in vitro culture, sheets of oral tissue in which complete oral mucosastructures can be identified. Clinical application of the findings ofthis study will allow the replacement of free skin grafts or autologousoral mucosa grafts by this technique in certain cases.Material and Method. Primary keratinocyte cultures were preparedfrom small biopsy samples of oral mucosa. Secondary cultures wereprepared from these primary cultures on an artificial submucosaconstituted by collagen and human fibroblasts. The cell cultureswere analyzed histologically in vitro and then used for graft implantsin athymic mice to study their behavior in vivo.Results. The primary cultures were confluent within a minimumperiod of 10 days and maximum of 12 days, which is similar to theperiod that the secondary cultures required to reach confluence.The time from sampling to achieving a complete artificial mucosaranged from 20 to 22 days. The artificial mucosa showed histologiccharacteristics of a normal mucosa. After 17 days of graftimplantation in immunoincompetent mice without any clinicalcontingency, histologic and immunohistochemical characterization(cytokeratins 19 and 13, collagen IV, and laminin) confirmed thesimilarity of the mucosa in vitro to healthy oral mucosa.Conclusion. A complete oral mucosa equivalent can be preparedwith collagen and fibroblasts using in vitro culture techniques.Although this mucosa shows considerable retraction, its clinicalhandling is very favorable(AU)

Altered expression of epithelial cell surface glycoconjugates and intermediate filaments at the margins of mucosal wounds.

Alterations in cell to cell adhesion are necessary to enable the type of cell movements that are associated with epithelial wound healing and malignant invasion. Several studies of transformed cells have related epithelial cell movement to changes in the cell surface expression of the carbohydrate structures represented by the ABO blood group antigens and, in particular, by Lewis antigens and their biosynthetic precursors. To study further the relationship between cell surface carbohydrates and keratinocyte cell movement, experimental wounds were created in human oral mucosa and examined by immunohistochemical methods for their expression of selected cytokeratins (K5, K16, K19), basement membrane components (laminin alpha5 and gamma2-chains, BP180, collagen IV and collagen VII), and blood group antigen precursor structures Le(x), sialosyl-Le(x), Le(y), H antigen, N-acetyllactosamine, and sialosyl-T antigen. The changes induced by wounding in the expression of collagen IV, laminin gamma2-chain (laminin-5), and laminin alpha5-chain were similar to those found in skin wounds and served to define the region of epithelial movement. This region was found to show a marked increase in staining for both Lewis antigen Y (Le(y)) and H blood group antigen, and decreased staining of Le(x), thus indicating an upregulation in wounded epithelium of the fucosyltransferases responsible for the synthesis of the H antigen. The changes in carbohydrate expression extended beyond the wound margin into the nonwounded epithelium, a pattern of expression similar to K16, which was also strongly upregulated in both the outgrowth and the adjacent nonwounded epithelium. These findings provide further support for an influence of such carbohydrate structures on the migratory behavior of epithelial cells.

[Effect of keratin on the efficacy of fluconazole]. / Einfluss von Keratin auf die Wirksamkeit von Fluconazol.

In this study we have investigated the influence of keratin on the efficacy and pharmacokinetics of fluconazole and additional azole antifungal agents. It is well known that the penetration and distribution of oral antifungals is strongly influenced by plasma protein binding. Especially, itraconazole and ketoconazole show a high binding affinity to plasma proteins, whereas fluconazole binds only with 12%. All of these antifungals, however, are accumulated in the stratum corneum of the skin. These observations have stimulated interest, if structure proteins of the skin like keratin interact with antifungals may explain of the accumulation process. Therefore we have measured the binding kinetics between keratin and the azoles. Keratin from sheep wool was degreased, purified and incubated with azole antifungals. After defined incubation periods the azoles were extracted and assayed by thin layer chromatography following UV-detection. There was a specific binding between keratin and all of the used substances. Interestingly, the binding affinity of fluconazole to keratin was much higher than to plasma proteins. Thus our observations indicate that the accumulation in the stratum corneum is a consequence of the interaction between keratin and azole derivatives.

The physicochemical and biopharmaceutical properties of fragmented keratin as a new drug carrier.

Two types of fragmented keratin were prepared from buffalo horn and hoof using savinase and Na2S, and their physicochemical and biopharmaceutical properties were examined in mice. The number-average molecular weight of enzymatically fragmented keratin (E-FK), chemically fragmented keratin (C-FK), and fragmented gelatin (FG) were 8000, 33,000, and 6600, respectively. The systematic acute toxicity of FKs was significantly low. Moreover, the immunogenicity of FKs was significantly lower than that of superoxide dismutase. FKs and FG were partially hydrolyzed by trypsin. FKs were digested easily by alpha-chymotrypsin, but FG underwent less hydrolysis under the same conditions. FKs were bound to plasma proteins, including albumin, and also to some proteins in liver and kidney homogenates. In plasma, E-FK was hydrolyzed slowly, but in liver and kidney homogenates it showed slightly faster hydrolysis. In contrast, FG was not hydrolyzed in any of the media used here. After intravenous administration of FKs and FG to mice, these molecules were rapidly eliminated from the plasma. E-FK and C-FK were taken up into the kidneys (CLuptake, kidney; 10,400, 11,600 microliters/h/g), and then gradually excreted in urine. FG was excreted rapidly into urine (CLurine; 6360 microliters/h). Interestingly, C-FK was also taken up into the liver (CLliver; 4820 microliters/h). These results indicated that fragmented keratins are biodegradable materials and might be used as new types of liver- and kidney-specific targeting carriers.

Estudo imuno-histoquímico do carcinoma adenóide cístico de glândula salivar menor / Immunohistochemical study of minor salivary gland's adenoid cystic carcinoma

A expressäo de um painel de anticorpos, constituídos pelas citoqueratinas 7, 8, 10, 13, 14, 18 e 19, vimentina e HHF-35, foi estudada em 21 casos de carcinomas adenóide císticos de glândula salivar menor, de diferentes sub-tipos histológicos, utilizando-se a técnica da imunohistoquímica, pelo método da estreptpavidina-biotina. Os resultados mostraram que nos tumores cribriformes, as células luminas das estruturas ductais foram positivas para as citoqueratinas 7, 8, 14, 18 e 19 e negativas para a vimentina e HHF-35, enquanto as células externas dos ductos, as células que revestiam os espaços pseudo-císticos e as células periféricas dos cilindros foram negativas para as citoqueratinas testadas e positivas para a vimentina e HHF-35. Nos tumores tubulares, enquanto as células internas dos ductos mostraram-se positivas para as citoqueratinas 7, 8, 14, 18 e 19 e negativas para vimentina e HHF-35, as células periféricas das estruturas ductais, apresentaram-se sempre positivas para a vimentina e HHF-35 e ocasionalmente positivas para a citoqueratina 14. Nas neoplasias do subtipo sólido, observou-se positividade esporádica das células, especialmente para as citoqueratinas 7 e 14, bem como para a vimentina. Os nossos achados mostraram que o carcinoma adenóide cístico, de um modo geral, mimetiza, em seu perfil imuno-histoquímico, o segmento de ducto intercalado da glândula salivar normal, apresentando variaçöes que sugerem diferentes graus de diferenciaçäo para os seus três subtipos histológicos. Os tumores tubulares representam a variante mais diferenciada, os tumores sólidos, o subtipo menos diferenciado, e os tumores cribiformes, neoplasias de um grau intermediário de diferenciaçäo entre aquele visto nas outras duas variantes histológicas

Immunotargeting with monoclonal cytokeratin 8 antibodies of human urothelial cancer transplanted to nude mice.

The possibility of using cytokeratin antibodies for the radioimmunolocalization of urinary bladder cancer was studied. A monoclonal murine IgG antibody was raised against cytokeratin 8 and labelled with iodine-125; normal murine IgG was used for control purposes. The urothelial cancer cell line RT4 was transplanted into immunodeficient nude mice. The anti-cytokeratin 8 antibody was administered intraperitoneally and its uptake in the tumour and other organs was analyzed with a computerized gamma camera. Optimal scintigraphic visualization occurred 11 days after antibody administration. The tumour/blood ratio of the specific antibody was 5.64 (+/- 5.01 SD) on day 11, compared with 0.73 (+/- 0.35 SD) in the control. Autoradiography demonstrated antibody uptake preferentially in viable sections of the tumour. The antibody uptake is presumed to be the result mainly of binding to the released cytokeratin in and around cells lysed during natural cellular death. The monoclonal murine anti-cytokeratin antibody is of potential interest in studies aimed at improving the clinical staging of urinary bladder cancer.

Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties.

We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.

Mesotelioma pleural: estudio inmunohistoquímico con anticuerpos monoclonales contra vimentina y subtipos de citoqueratina / Pleural mesothelioma: immunohistochemical study with monoclonal antibodies against vimentine and cytokeratin subtypes

Se estudiaron retrospectivamente 10 casos de mesotelioma maligno de la pleura con técnica de inmunoperoxidasa y complejos avidina-biotina para determinar la expresión de filamentos intermedios por las células tumorales, a saber subtipos de citoqueratina y vimentina. Se utilizaron anticuerpos monoclonales anti-citoqueratina, subtipos 1,5,6,7,8,10,11 y 19 y antivimentina. Del total, 4 mesoteliomas malignos epiteliales presentaron reacción positiva para todos los subtipos de citoqueratina y sólo 1 para vimentina. De los mesoteliomas malignos mixtos, 6 presentaron reacción positiva para todos los subtipos de citoqueratina y 5 para vimentina. Los resultados muestran que es posible estudiar el espectro de expresión inmunohistoquímica de los filamentos intermedios en casos de mesotelioma maligno de la pleura fijados en formalina e incluídos en parafina; la expresión de citoqueratina por todos los mesoteliomas malignos de la pleura estudiados confirma el carácter epitelial de este tumor, los mesoteliomas malignos mixtos coexpresan frecuentemente citoqueratina y vimentina. En neoplasias pleurales en las que se considera el diagnóstico de mesotelioma o adenocarcinoma, el estudio de los filamentos intermedios con anticuerpos monoclonales puede tener gran valor en el diagnóstico diferencial

[Cytokeratins and transitional epithelium of the bladder. Particular distribution of cytokeratins]. / Cytokératines et épithélium transitionnel de la vessie. Distribution particulière de certaines cytokératines.

The purpose of this study was to determine the tissue distribution of certain cytokeratins in the urothelium. A series of ten cystectomy specimens containing normal tissues was investigated by immunocytochemical labeling with specific antibodies against cytokeratins 19, 18, 8, 7, 10. Monoclonal antibodies were used on frozen sections. Distribution of keratin was correlated with morphologic changes. All specimens were lined by a normal transitional epithelium. Low molecular weight cytokeratins (polypeptides 19, 18, 8, 7) were detected in all cell layers. Anticytokeratin 19 stained epithelium in a rather uniform way. Different antibodies specific for cytokeratin 18 reacted differently on the same tissue. With certain anticytokeratins 18 and 8, a higher staining intensity was found in superficial layers as compared with intermediate cell layers. So-called "umbrella" cells were strongly stained. Anticytokeratin 10 revealed very rare endocrine-like cells. This normal urothelial pattern is in agreement with previous reports. This pattern of expression of cytokeratin polypeptides differs from that of nonkeratinizing squamous epithelium. Therefore, histological differentiation of epithelia is accompanied by pronounced changes in the expression of cytokeratin polypeptides.