Toshiroh Iwasaki

Alteration of stratum corneum ceramide profiles in spontaneous canine model of atopic dermatitis

Abstract:  Ceramides (CERs) in the stratum corneum (SC) are thought to play a key role in cutaneous barrier function. It has been reported that human SC contains 11 free CER classes and that their profiles are altered in humans with atopic dermatitis (AD). Although decreased proportions of free CERs or quantities of protein‐bound CERs in the SC have been reported in dogs with AD, the overall profile of CERs in the canine SC has not been fully elucidated. The aim of this study was thus to investigate the profile of free CERs in the canine SC and to identify alterations in the CER profiles in dogs with AD. Normal‐phase liquid chromatography–electrospray ionization–mass spectrometry indicated 11 clusters of peaks for free CER classes, similar to those recognized in the human SC. The fractions of free SC CER in dogs with AD and in breed‐ and age‐matched healthy dogs were quantitatively compared using high‐performance thin‐layer chromatography. CER[EOS], CER[EOP] and CER[NP], which are known to be decreased in the skin of humans with AD, were also decreased in the skin of dogs with AD. These findings highlight canine AD as a spontaneous animal model for investigating the disruption of CER‐associated cutaneous barrier functions in the corresponding human disease.

Critical role for constitutive type I interferon signaling in the prevention of cellular transformation

Interferons‐α/β, which are produced upon viral infection, are key soluble factors for the establishment of an antiviral state, but are also produced at low levels in the absence of infection. Herein, we demonstrate that a weak signal by these constitutively produced IFN‐α/β show a preventive role in cellular transformation. Ifnar1‐deficient (Ifnar1−/–) MEF, which are devoid of IFN‐α/β signal, undergo a spontaneous transformation during long‐term cell culture. Similar to Irf1−/– MEF, primary Ifnar1−/– MEF become tumorigenic in nude mice by the expression of activated c‐Ha‐Ras oncoprotein. However, Ifnar1−/– MEF do not show any abnormal growth properties. A similar observation is made in Ifnb−/– MEF that fail to produce constitutive IFN‐α/β, whereas such a transforming property is not found in MEF that lack any of the IFN receptor downstream molecules including Stat1, IRF9 and IRF1. Furthermore, Ifnar1−/– mice develop chemically‐induced skin papilloma more severely than wild‐type mice. In addition, the expression levels of IFNAR1 mRNA are significantly decreased in human gastric cancer tissues. These results suggest a cell‐intrinsic role of the weak signal by constitutively produced IFN‐α/β to prevent cells from transformation, which may be mediated by a hitherto‐unknown pathway(s) downstream of the IFN‐α/β receptor. (Cancer Sci 2009; 100: 449–456)

Canine Follicle Stem Cell Candidates Reside in the Bulge and Share Characteristic Features with Human Bulge Cells

The hair follicle bulge has attracted great interest as a stem cell repository. Previous studies have focused on rodent or human bulge stem cells, and our understanding of those in other species is limited. In this study, we attempted to localize and characterize stem cell candidates in canine hair follicles. The canine skin xenografting study located label-retaining cells in the outer root sheath around the insertion point of the arrector pili muscle, where the immunoreactivity of human bulge markers, keratin 15 and follistatin, were detected. Canine bulge cell-enriched keratinocytes up-regulated human bulge biomarkers CD200 and DIO2, and conserved key cell regulators of bulge stem cells, such as SOX9 and LHX2. Importantly, canine bulge-derived keratinocytes were highly proliferative in vitro and, when combined with trichogenic dermal cells, reconstituted pilosebaceous structures as well as the epidermis in vivo. Successful detection of canine specific DNA sequences suggested that the regenerated tissue was of canine origin. In addition, canine specific bulge cell and sebocyte lineage markers were expressed in reconstituted pilosebaceous units, implying the multipotency of canine bulge cells. Our findings demonstrate a unique strategy utilizing canine bulge cells to investigate human stem cell biology and intractable hair disorders that involve the bulge region.

Identification of a novel Staphylococcus pseudintermedius exfoliative toxin gene and its prevalence in isolates from canines with pyoderma and healthy dogs.

Staphylococcal exfoliative toxins are involved in some cutaneous infections in mammals by targeting desmoglein 1 (Dsg1), a desmosomal cell-cell adhesion molecule. Recently, an exfoliative toxin gene (exi) was identified in Staphylococcus pseudintermedius isolated from canine pyoderma. The aim of this study was to identify novel exfoliative toxin genes in S. pseudintermedius. Here, we describe a novel orf in the genome of S. pseudintermedius isolated from canine impetigo, whose deduced amino acid sequence was homologous to that of the SHETB exfoliative toxin from Staphylococcus hyicus (70.4%). The ORF recombinant protein caused skin exfoliation and abolished cell surface staining of Dsg1 in canine skin. Moreover, the ORF protein degraded the recombinant extracellular domains of canine Dsg1, but not Dsg3, in vitro. PCR analysis revealed that the orf was present in 23.2% (23/99) of S. pseudintermedius isolates from dogs with superficial pyoderma exhibiting various clinical phenotypes, while the occurrence in S. pseudintermedius isolates from healthy dogs was 6.1% (3/49). In summary, this newly found orf in S. pseudintermedius encodes a novel exfoliative toxin, which targets a cell-cell adhesion molecule in canine epidermis and might be involved in a broad spectrum of canine pyoderma.

Canine Bullous Pemphigoid (BP): Identification of the 180-kd Canine BP Antigen by Circulating Autoantibodies

Human bullous pemphigoid (BP) is an immune-mediated blistering disease characterized by autoantibodies against BP antigens (230/180 kd), which are constitutive glycoproteins of hemidesmosomes found in basal keratinocytes. Blistering diseases similar to human BP have been reported in dogs. IgG deposits at the basement membrane zone (BMZ) are a common feature of canine BP. Although circulating anti-BMZ IgG autoantibodies have been demonstrated in some cases of canine BP, the specific skin protein targeted by these autoantibodies has not been identified. In this study, we characterized the antigenic target of the autoantibodies in the serum from a 3-year-old castrated male Pit Bull Terrier with BP. Direct immunofluorescence of the patients skin demonstrated IgG deposits at the dermal-epidermal junction. Indirect immunofluorescence demonstrated autoantibodies in the patients serum that stained the epidermal roof of salt-split canine skin and left the dermal floor unstained. These serum autoantibodies did not stain normal intact dog skin but labeled intact bovine tongue. Direct immunoelectron microscopy of the dogs skin revealed IgG deposits within the hemidesmosomes of the basal keratinocytes. Western immunoblotting experiments showed that canine keratinocytes express both the 230-kd and 180-kd bullous pemphigoid antigens, and the autoantibodies from the patients serum recognized the 180-kd bullous pemphigoid antigen in proteins extracted from canine and human keratinocytes. Canine BP has many parallel features with human BP including similar immune deposition of IgG within hemidesmosomes and a hemidesmosome-associated 180-kd glycoprotein target for circulating autoantibodies.

Canine hair-follicle keratinocytes enriched with bulge cells have the highly proliferative characteristic of stem cells

Homeostasis of the epidermis and skin appendages is maintained by tissue-specific stem cells. In mice and humans, two populations of epithelial stem cells have been identified: one in the basal layer of the interfollicular epidermis and another in the bulge area of hair follicles. However, our understanding of canine epithelial stem cells is extremely limited. In this study, in vitro colony-forming assays were performed to locate highly proliferative keratinocytes in canine skin. Their phenotypic resemblance to epithelial stem cells in other species was also assessed. When equal numbers of epidermal or hair-follicle keratinocytes were cultured, colonies derived from follicular keratinocytes were significantly larger both in total numbers and size, than those derived from epidermal keratinocytes. In addition, immunoreactivity for CD34, a putative bulge stem-cell marker in the mouse, was predominantly detected in follicular keratinocytes. Thus in dogs, follicular keratinocytes were distinct from epidermal keratinocytes in proliferative capacity and CD34 expression. Using microdissection, highly proliferative keratinocytes were located within the middle portion of hair follicles, including the bulge area. Immunohistochemical study revealed that keratin 15, an established marker of bulge stem cells in mice and humans, was also predominantly expressed in the canine bulge area. Flow cytometry analysis revealed high numbers of keratin-15-positive cells in the highly proliferative keratinocyte compartment. Of note, keratin 15(high) cells possessed the phenotypic characteristics of putative stem cells. This study represents the first in vitro identification and isolation of highly proliferative canine keratinocytes, which represent candidate epithelial stem cells.

Staphylococcus pseudintermedius exfoliative toxin EXI selectively digests canine desmoglein 1 and causes subcorneal clefts in canine epidermis.

Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell-cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus-secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1β and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo.

Effect of Substrate on Indirect Immunofluorescence Test for Canine Pemphigus Foliaceus

The effect of substrate on indirect immunofluorescence (IIF) tests for the detection of circulating autoantibodies was studied by examining sera from 14 canine pemphigus foliaceus patients, six sera with non-pemphigus dermatoses and ten normal dog sera against five different substrates from three species. These substrates included bovine esophagus, bovine nose, bovine tongue, monkey esophagus, and canine nose skin. Nine out of 14 (64.3%) sera from patients with canine pemphigus foliaceus showed intercellular space staining by indirect immunofluorescence using bovine esophagus as substrate. However, sera from nonpemphigus dermatoses and normal dog did not react with bovine esophagus. In other substrates, only bovine tongue showed 1/8 (12.5%) positive reaction at the intercellular space by sera from canine pemphigus foliaceus. Dog nose skin showed the intercellular space staining against ten of ten (100%) normal dog serum. Monkey esophagus showed the fluorescent deposit at the intercellular space in four of nine (44.4%) of pemphigus foliacues dog sera, however, four of ten (40%) of normal dog sera revealed nonspecific intercellular staining. These results indicate that the sensitivity and the specifity of IIF test in canine pemphigus foliaceus depend on the substrate. The best substrate for detecting circulating autoantibody in canine pemphigus foliaceus patients among five different substrates was bovine esophagus because of its sensitivy and high specifity. The diagnosis of canine pemphigus foliaceus should be made on the basis of a combination of clinical signs, histopathology, direct immunofluorescence, and the detection of circulating autoantibody.

Lamininα3 LG4 Module Induces Keratinocyte Migration: Involvement of Matrix Metalloproteinase-9

Abstract Laminin α 3 chain, a functionally key subunit of laminin-5, contains a large globular module (G module) which consists of a tandem repeat of five homologous LG modules (LG1∼ 5). We previously demonstrated that the LG4 module of laminin α 3 chain (α 3 LG4) induces a matrix metalloproteinase-1 (MMP-1) expression through the interaction with syndecans leading to MAPK activation/IL-1β expression signaling loop (Utani et al., J. Biol. Chem. 278, 34483–34490, 2003). Here, we show that a recombinant α 3 LG4 and synthetic peptides containing syndecan binding motif induced a cell motility and a MMP-9 expression in ketarinocytes. The synthetic peptide (A3G756)-induced cell migration and MMP-9 upregulation were inhibited by each application of a heparin and an IL-1 receptor antagonist (IL-1RA), suggesting the involvement of syndecans and IL-1β autocrine. Furthermore, the A3G756-induced cell motility was inhibited by an MMP-9 inhibitor and a neutralizing antibody of MMP-9, indicating induced cell motility was dependent on an MMP-9 activity. Taken these together, laminin-5 α 3 LG4 module may play an important role in re-epithelialization at tissue remodeling.

Removal of amino-terminal extracellular domains of desmoglein 1 by staphylococcal exfoliative toxin is sufficient to initiate epidermal blister formation

BACKGROUND In both bullous impetigo and staphylococcal scalded-skin syndrome (SSSS), exfoliative toxins (ETs) produced by Staphylococcus aureus cause superficial intraepidermal blisters. ETs are known to cleave specifically a single peptide bond in the extracellular domains 3 and 4 of desmoglein (Dsg) 1. However, the precise mechanisms underlying ET-induced epidermal blister formation remain poorly understood. OBJECTIVE To determine whether cleavage of Dsg1 by an ET is sufficient to induce blister formation in vivo or if the subsequent internalization of cleaved Dsg1 or other desmosomal components is required. METHODS Skin samples obtained from neonatal mice injected with ETA were analyzed by time-lapse immunofluorescence and transmission electron microscopy for desmosomal components. RESULTS Epidermal blister formation was observed as early as 60 min after ETA treatment. At this time, the amino-terminal extracellular domains of Dsg1 disappeared from the surface of keratinocytes, while the cleaved carboxy-terminal domain of Dsg1 (Dsg1-C) as well as the extracellular domains of desmocollin 1 (Dsc1-N) remained on the cell surface. Half-split desmosomes with intracytoplasmic dense plaques and attached tonofilaments were recognized ultrastructurally on the split surface of keratinocytes at 60 min. Subsequent to this, Dsg1-C and Dsc1-N gradually disappeared from the surface layer of keratinocytes. CONCLUSION Our findings suggest that the removal of amino-terminal extracellular domains of Dsg1 by ETs is sufficient to initiate epidermal blister formation in bullous impetigo and SSSS.