Transdermal Delivery of Functional Collagen \u3cem\u3eVia\u3c/em\u3e Polyvinylpyrrolidone Microneedles

Collagen makes up a large proportion of the human body, particularly the skin. As the body ages, collagen content decreases, resulting in wrinkled skin and decreased wound healing capabilities. This paper presents a method of delivering type I collagen into porcine and human skin utilizing a polyvinylpyrrolidone microneedle delivery system. The microneedle patches were made with concentrations of 1, 2, 4, and 8% type I collagen (w/w). Microneedle structures and the distribution of collagen were characterized using scanning electron microscopy and confocal microscopy. Patches were then applied on the porcine and human skin, and their effectiveness was examined using fluorescence microscopy. The results illustrate that this microneedle delivery system is effective in delivering collagen I into the epidermis and dermis of porcine and human skin. Since the technique presented in this paper is quick, safe, effective and easy, it can be considered as a new collagen delivery method for cosmetic and therapeutic applications

Laminin-511 and integrin beta-1 in hair follicle development and basal cell carcinoma formation

<p>Abstract</p> <p>Background</p> <p>Initiation of the hair follicle placode and its subsequent growth, maturation and cycling in post-natal skin requires signaling interactions between epithelial cells and adjacent dermal cells and involves Shh signaling via the primary cilium. Previous reports have implicated laminins in hair follicle epithelial invagination.</p> <p>Results</p> <p>Here we use a human BCC model system and mouse mutants to re-evaluate the role of laminin-511 in epithelial invagination in the skin. Blocking laminin 511 and 332 in BCCs maintains primary cilia and Shh signalling, but prevents invagination. Similarly, in laminin-511 and dermal beta-1 integrin mutants, dermal papilla development and primary cilia formation are normal. Dermal beta-1 integrin mutants have normal hair follicle development.</p> <p>Conclusions</p> <p>Our data provides support for a primary role of laminin-511 promoting hair follicle epithelial downgrowth without affecting dermal primary cilia and Shh target gene induction.</p

NC1 Domain of Type VII Collagen Binds to the β3 Chain of Laminin 5 Via a Unique Subdomain Within the Fibronectin-Like Repeats

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, globular domains, NC1 and NC2. Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC1 binds to various extracellular matrix components including a complex of laminin 5 and laminin 6 (Chen et al. 1997a). In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments). Both authentic and purified recombinant NC1 bound to human and rat laminin 5 as measured by enzyme-linked immunosorbant assay and by binding of 125I-radiolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. NC1 bound predominantly to the β3 chain of laminin 5, but also to the γ2 chain when examined by a protein overlay assay. The binding of 125I-NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-glycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclonal antibody to the β3 chain. In contrast, the NC1–laminin 5 interaction was not affected by a monoclonal antibody to the α3 chain. Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760–1045) subdomain of NC1 was identified as the binding site for laminin 5. IgG from an epidermolysis bullosa acquisita serum containing autoantibodies to epitopes within NC1 that colocalized with the laminin 5 binding site inhibited the binding of NC1 to laminin 5. Thus, perturbation of the NC1–laminin 5 interaction may contribute to the pathogenesis of epidermolysis bullosa acquisita

Autocrine laminin-5 ligates α6β4 integrin and activates RAC and NFκB to mediate anchorage-independent survival of mammary tumors

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express α6β4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail–truncated α6β4 integrin. α6β4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of β4 integrin is necessary for basal and epidermal growth factor–induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional β1 and β4 integrin through activation of NFκB, and overexpression of NFκB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5–α6β4 integrin–RAC–NFκB signaling

Laminin-6 and Laminin-5 Are Recognized by Autoantibodies in a Subset of Cicatricial Pemphigoid

We characterized basement membrane zone (BMZ) autoantigens targeted by autoantibodies (AAb) from patients with cicatricial pemphigoid. Serum from a patient with severe oral cicatricial pemphigoid contained IgG anti-BMZ AAb. The AAb labeled a lower BMZ component on salt-split skin and localized to the lower lamina lucida/lamina densa by direct and indirect immunoelectron microscopy (HEM) but did not label blood vessels. The AAb did not react with EHS laminin-1 and type IV collagen, pepsinized human type IV collagen, recombinant entactin, or NC1 domain of type VII collagen by dot blotting and western blotting. We focused our studies on the laminin family, as laminin-5 was identified as an autoantigen in cicatricial pemphigoid. Culture-conditioned media from normal keratinocytes (containing laminin-6 and laminin-5) and JEB keratinocytes (containing laminin-6 but not laminin-5) were studied by western blotting. Under nonreducing conditions, the patient's AAb recognized a 600-kDa protein (laminin-6) intensely and a 400-kDa protein (laminin-5) weakly in normal keratinocyte medium even though abundant laminin-5 was present. In JEB keratinocyte medium, however, the 600-kDa protein (laminin-6) alone was recognized by the patient's AAb. The AAb also immunolabeled BMZ of JEB skin that lacked laminin-5. The AAb from this patient and two other patients with anti-laminin-5 cicatricial pemphigoid immunoprecipitated both laminin-6 an4 laminin-5. Taken together, the results of IEM, non-reducing western blotting, immunoprecipitation, and JEB skin BMZ immunolabeling indicate that laminin-6, as well as laminin-5, is identified by the AAb from a subset of cicatricial pemphigoid patients. We propose the name “anti-laminin cicatricial pemphigoid” for this subset

A Newly Identified 105-kD Lower Lamina Lucida Autoantigen Is an Acidic Protein Distinct from the 105-kD γ2 Chain of Laminin-5

A 105-kD lower lamina lucida antigen (p105) has been detected by autoantibodies (anti-p105) from patients with a novel immunobullous disease. To distinguish p105 from other known lamina lucida components, we performed comparative irnmunoblotting on purified human amniotic laminin-5 (kalinin), 804G matrix (enriched in laminin-5), and keratinocyte and fibroblast proteins using anti-804G matrix antibody 0-18) and anti-p105. J-18 labeled the truncated laminin-5 γ2 chain in amniotic laminin-5, 804G matrix, and keratinocyte conditioned medium, but did not label fibroblast cytosol. Conversely, anti-p105 did not label amniotic laminin-5 or 804G matrix, but did label p105 in both keratinocyte conditioned medium and fibroblast cytosol. J-18 labeled the 105-kD laminin-5 γ2 chain in reduced keratinocyte proteins and a 400-kD laminin-5 complex under non-reducing conditions. In contrast, anti-p105 labeled p105 under both reducing and non-reducing conditions but did not label a 400-kD protein complex. Similarly, comparative immunoblotting on keratinocyte proteins using anti-p105 and anti-laminin-1 revealed no commonly labeled protein bands. Electrophoretic fractionations by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of these fractions revealed that the peak fractions of keratinocyte proteins reactive with anti-p105 are different from those reactive with J-18. Furthermore, keratinocyte proteins fractionated by Mono Q anionexchange chromatography revealed fractions immunoreactive with anti-p105, whereas J-18 showed no reactivity with these fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-p105 revealed p105 to be an acidic protein with isoelectric points between 5.7 and 6.3, distinct from the isoelectric points of laminin-5 γ2 chain. We conclude that p105 is an acidic protein located in the lamina lucida and distinct from the truncated laminin-5 γ2 chain and the laminin-1 family

Subepidermal blistering induced by human autoantibodies to BP180 requires innate immune players in a humanized bullous pemphigoid mouse model

Bullous pemphigoid (BP) is a cutaneous autoimmune inflammatory disease associated with subepidermal blistering and autoantibodies against BP180, a transmembrane collagen and major component of the hemidesmosome. Numerous inflammatory cells infiltrate the upper dermis in BP. IgG autoantibodies in BP fix complement and target multiple BP180 epitopes that are highly clustered within a non-collagen linker domain, termed NC16A. Anti-BP180 antibodies induce BP in mice. In this study, we generated a humanized mouse strain, in which the murine BP180NC14A is replaced with the homologous human BP180NC16A epitope cluster region. We show that the humanized NC16A (NC16A+/+) mice injected with anti-BP180NC16A autoantibodies develop BP-like subepidermal blisters. The F(ab′)2 fragments of pathogenic IgG fail to activate complement cascade and are no longer pathogenic. The NC16A+/+ mice pretreated with mast cell activation blocker or depleting of complement or neutrophils become resistant to BP. These findings suggest that the humoral response in BP critically depends on innate immune system players

Cells from discarded dressings differentiate chronic from acute wounds in patients with Epidermolysis Bullosa

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application

Definitions and outcome measures for mucous membrane pemphigoid: Recommendations of an international panel of experts

Mucous membrane pemphigoid encompasses a group of autoimmune bullous diseases with a similar phenotype characterized by subepithelial blisters, erosions, and scarring of mucous membranes, skin, or both. Although knowledge about autoimmune bullous disease is increasing, there is often a lack of clear definitions of disease, outcome measures, and therapeutic end points. With clearer definitions and outcome measures, it is possible to directly compare the results and data from various studies using meta-analyses. This consensus statement provides accurate and reproducible definitions for disease extent, activity, outcome measures, end points, and therapeutic response for mucous membrane pemphigoid and proposes a disease extent score, the Mucous Membrane Pemphigoid Disease Area Index