Crystal structure of exfoliative toxin D from Staphylococcus aureus.

Staphylococcus aureus exfoliative toxins (ETs) are serine proteases responsible for staphylococcal scalded skin syndrome. Four ETs, ETA, ETB, ETD, and ETE, have been identified, all of which cleave desmoglein-1. This study presents the crystal structure of ETD at 1.75 Å resolution. The protein exhibits a structure composed of two ß-barrels and two α-helices as described in previous studies of ETs. A predicted model of ETD in complex with Ile380-Glu381-Gly382-Pro383 (IEGP), a segment of human desmoglein-1 (hDsg1), was constructed. Glu381 of hDsg1 was predicted to interact with as many as six amino acid residues in ETD, whereas two amino acid residues in ETD primarily constituted subsite S1', and a space near subsite S1' was noted. It is likely that polypeptide chains located near the IEGP segment in the predicted structure of hDsg1 bind to this space. The structure of loop D, which was predicted to participate in subsite S2', in ETD was markedly different from those in other ETs.

Identification and characterization of the genome of a papillomavirus from skin lesions of four-toed hedgehogs (Atelerix albiventris).

The present study describes the clinical and pathological characteristics of skin lesions in two four-toed hedgehogs (Atelerix albiventris). We performed inverse PCR to identify the genome of papillomavirus (PV) in the skin lesions and subsequently sequenced the full genome of the virus, which was tentatively named Atelerix albiventris papillomavirus 1 (AalbPV1). The overall sequences of the viral genomes of both four-toed hedgehogs were identical. This study first identified the presence of a novel PV in Japanese four-toed hedgehogs and provided genetic information about this virus.

Erythritol alters gene transcriptome signatures, cell growth, and biofilm formation in Staphylococcus pseudintermedius.

BACKGROUND: Erythritol was found to inhibit the growth of microorganisms. The present study aimed to demonstrate the growth inhibition of Staphylococcus pseudintermedius by erythritol and to define the changes in gene transcription signatures induced by erythritol. Changes in the gene transcription profiles were analysed by RNA sequencing and quantitative reverse transcription PCR. Gene ontology analysis was performed to assign functional descriptions to the genes. RESULTS: Erythritol inhibited S. pseudintermedius growth in a dose-dependent manner. We then performed a transcriptome analysis of S. pseudintermedius with and without 5% (w/w) erythritol exposure to validate the mechanism of growth inhibition. We revealed that erythritol induced up-regulation of three genes (ptsG, ppdK, and ppdkR) that are related to the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Glucose supplementation restored the up-regulation of the PTS-related genes in response to erythritol. In addition, erythritol down-regulated eleven genes that are located in a single pur-operon and inhibited biofilm formation of S. pseudintermedius. CONCLUSIONS: These findings indicated that erythritol antagonistically inhibits PTS-mediated glucose uptake, thereby exerting a growth inhibitory effect on S. pseudintermedius. Moreover, erythritol inhibits the 'de novo' IMP biosynthetic pathway that may contribute to biofilm synthesis in S. pseudintermedius.

A preliminary investigation of the impact of artificially carbonated water bathing on canine skin physiological and barrier function.

BACKGROUND: The effect of carbon dioxide (CO2 )-rich water bathing on the skin has been studied extensively in humans. However, there have been few studies evaluating the impact of CO2 -rich water bathing on canine skin physiology and barrier functions. OBJECTIVES: To evaluate the impact of artificially carbonated water (ACW) bathing on skin parameters in healthy beagles. ANIMALS: Six healthy beagles with no history of skin disease. MATERIALS AND METHODS: Body temperature, skin temperature, transepidermal water loss (TEWL), skin hydration and skin blood flow were evaluated before and after single ACW bathing (37°C, 20 min) with a CO2 concentration of >1000 ppm. RESULTS: After ACW bathing, skin blood flow significantly increased (p < 0.0001), yet there were no significant changes in body temperature (p = 0.3124), skin temperature (p = 0.4911), TEWL (p = 0.5167) or skin hydration (p = 0.3084). There were no adverse events during the trials. CONCLUSIONS AND CLINICAL IMPORTANCE: Artificially carbonated water water bathing could potentially increase skin blood flow without affecting skin temperature, body temperature and skin barrier function in dogs, similar to its effects in humans.

Validation of laser doppler flowmetry to measure dermal blood flow of the pinnae in dogs with pinnal alopecia.

BACKGROUND: Laser Doppler flowmetry (LDF) is a noninvasive method of measuring regional blood flow in humans. However, this method has not been widely applied to measure blood flow in dogs. HYPOTHESIS/OBJECTIVES: We hypothesised that LDF can measure changes in blood flow in canine pinnae accurately. The objectives were to determine whether LDF could accurately detect dermal blood flow changes in canine pinnae caused by haemodynamic drugs and characterize the dermal blood flow in dogs with pinnal alopecia. ANIMALS: Sixteen laboratory-owned healthy dogs, 25 client-owned healthy control dogs and six dogs with pinnal alopecia suspected to be secondary to ischaemic dermatoses. MATERIALS AND METHODS: Clinical doses of the haemodynamic drugs atropine, medetomidine and dibutyryl cyclic adenosine monophosphate (dBcAMP), as well as topical dBcAMP, were administered to healthy beagles. Subsequently, an LDF apparatus was attached to the pinnae to analyse changes in dermal blood flow. Finally, LDF was used to measure auricular dermal blood flow in dogs with pinnal alopecia compared to healthy dogs. RESULTS: Dermal blood flow increased after atropine injection, during dBcAMP infusion and after topical dBcAMP ointment application, and decreased after medetomidine injection. Auricular dermal blood flow (in mL/min/100 g tissue) was significantly (p < 0.05) lower in dogs with pinnal alopecia than in healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Laser Doppler flowmetry is useful for measuring dermal blood flow in canine pinnae; it can be a noninvasive method to monitor ischaemic conditions of dog skin.

Clinical efficacy of artificially carbonated water bathing on superficial bacterial folliculitis in dogs.

BACKGROUND: Bathing with artificially carbonated water is reported to be a valuable therapeutic option for various human skin disorders. OBJECTIVE: To evaluate the clinical efficacy of artificially carbonated water bathing on superficial bacterial folliculitis (SBF) caused by Staphylococcus pseudintermedius (SP) in dogs. ANIMALS: Nineteen dogs with SBF from whom SP was isolated from skin lesions were enrolled. METHODS AND MATERIALS: Dogs with SBF were allocated randomly to either the artificially carbonated water bathing group or the control group bathed with tap water. The dogs were bathed with the designated water type on day (D)0, D7 and D14. Clinical scores and skin surface pH were evaluated on D0 and D21. Colony forming unit (cfu) assays were performed in vitro to investigate whether the artificially carbonated water affected growth of clinical SP isolates. RESULTS: The mean rate of improvement in the clinical scores was significantly higher in the carbonated water group than in the control group. Dogs bathed with carbonated water exhibited significant decreases in their skin surface pH after bathing; dogs bathed with tap water did not. No dogs experienced significant adverse events. The cfus of SP incubated in vitro with artificially carbonated water did not significantly differ from those incubated with tap water. CONCLUSION: Bathing with artificially carbonated water might be an effective and safe adjunctive therapy for canine SP-induced SBF.

Natto extract, a Japanese fermented soybean food, directly inhibits viral infections including SARS-CoV-2 in vitro.

Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.

Narrow-band ultraviolet B therapy attenuates cutaneous T-cell responses in hapten-induced, experimental contact dermatitis in beagles.

BACKGROUND: In human medicine, narrow-band ultraviolet B (NB-UVB) phototherapy has been used to treat various T-cell-mediated skin diseases. However, the effect of NB-UVB on inflamed canine skin remains uncertain. OBJECTIVES: To investigate the effect of NB-UVB phototherapy on the skin of dogs with hapten-induced contact dermatitis. ANIMALS: Seven healthy beagles without skin problems. METHODS AND MATERIALS: Dogs were irradiated with varying doses of NB-UVB to determine the minimal erythema dose (MED). After determining the MEDs of six dogs (excluding one of the seven whose skin did not show a visible reaction), we investigated the effect of NB-UVB on their inflamed skin by topically applying 2,4-dinitrochlorobenzene (DNCB), which causes type 1 helper T cell (Th1)- and cytotoxic T-cell (Tc)1-induced skin inflammation. We then irradiated the skin with NB-UVB. We analysed the treated skin samples via histopathological and immunohistochemical methods, and TdT-mediated dUTP nick-end labelling (TUNEL) to demonstrate apoptotic cells. We also analysed the cytokine gene transcription via real-time quantitative reverse transcription PCR. RESULTS: The NB-UVB MEDs caused mild inflammatory changes yet no severe epidermal exfoliations in the irradiated skin. In DNCB-treated skin irradiated by the NB-UVB MEDs, TUNEL-positive dermal apoptotic cells were increased significantly compared with those of DNCB-treated, nonirradiated skin. INF-γ and TNF-α transcription levels in DNCB-treated, irradiated skin were significantly lower than those in the DNCB-treated, nonirradiated skin. CONCLUSION AND CLINICAL RELEVANCE: Phototherapy using NB-UVB MEDs attenuated cutaneous Th1 and Tc1 cytokine responses with minimal skin damage in a canine model of hapten-induced contact dermatitis.

IgE reactivity to fish allergens from Pacific cod (Gadus macrocephalus) in atopic dogs.

BACKGROUND: IgE reactivity to fish allergens in atopic dogs, which are used as models for food allergy, has not been elucidated to date. We investigated IgE reactivity to crude extracts and purified allergens derived from the Pacific cod (Gadus macrocephalus) in atopic dogs to identify the allergenic proteins of cod. RESULTS: The levels of specific IgE to crude cod extracts were measured in the sera of 179 atopic dogs, including 27 dogs with cod allergy, using enzyme-linked immunosorbent assay (ELISA). Specific IgE to crude cod extracts were present in 36 (20%) of the 179 atopic dogs and in 12 (44%) of the 27 dogs with cod allergy. The allergens in crude cod extracts were analyzed by ELISA, immunoblotting, and liquid chromatography-tandem mass spectrometry. In allergen component analysis, IgE reactivity to tropomyosin and enolase was observed in the sera of dogs with cod allergy. IgE reactivity to parvalbumin, collagen, and tropomyosin was evaluated using the sera of atopic dogs that tested positive for specific IgE to crude cod extracts. Among the 36 dogs with IgE reactivity to crude cod extracts, 9 (25%), 14 (39%), and 18 (50%) dogs tested positive for specific IgE to parvalbumin, collagen, and tropomyosin, respectively. CONCLUSIONS: The IgE reactivity to cod allergens observed in dogs was similar to that in humans, and this finding further supports the use of atopic dogs with fish allergy as a model for fish allergy in humans.

Supplementation with eicosapentaenoic acid and linoleic acid increases the production of epidermal ceramides in in vitro canine keratinocytes.

BACKGROUND: Few studies have investigated the effects of essential fatty acids on the production of epidermal ceramide (CER) in canine keratinocytes. HYPOTHESIS/OBJECTIVES: To investigate the effects of eicosapentaenoic acid (EPA) and linoleic acid (LA) supplementation on the production of CERs using an in vitro canine keratinocyte culture system. METHODS AND MATERIALS: Canine keratinocyte cells (MSCEK) were incubated with high Ca2+ [1.8 mM calcium chloride (CaCl2 )] serum-free medium, supplemented with 3 µM EPA and 15 µM LA when the cells showed confluency. On Day 8 of application, lipid analysis using high-performance thin layer chromatography and real-time PCR for detecting glucosylceramide synthase and ceramidase were performed. RESULTS: It was revealed that the amounts of CER (EOS) (combination of ω-hydroxy fatty acids and sphingosines), CER [EOP] (combination of ω-hydroxy fatty acids and phytosphingosines) and a mixture of CER [NS] (combination of nonhydroxy fatty acids and sphingosines) and [NDS] (combination of nonhydroxy fatty acids and dihydrosphingosines), as well as total CERs, were significantly increased in cells incubated with EPA and LA compared to those of the vehicle, with increased mRNA expression of glucosylceramide synthase. CONCLUSIONS AND CLINICAL IMPORTANCE: These findings suggest that EPA and LA can potentially alter the CER profile of the skin and this may contribute to its epidermal barrier function in canine skin.

Progenitor cells expressing nestin, a neural crest stem cell marker, differentiate into outer root sheath keratinocytes.

BACKGROUND: Nestin, which was originally described as a neural crest stem cell marker, is known to be expressed in bulge follicle cells of human, canine and murine anagen hairs. However, the capacity of nestin-expressing cells to differentiate into the components of the hair follicle or the epidermis has been insufficiently investigated. HYPOTHESIS/OBJECTIVES: To determine whether nestin-expressing cells are capable of differentiating into keratinocytes. ANIMALS/MATERIALS: A double-transgenic mouse line Nes-Cre/CAG-CAT-EGFP, in which enhanced green fluorescent protein (EGFP) is expressed upon Cre-based recombination driven by the nestin promoter. METHODS AND MATERIALS: The tissue distribution of EGFP+ and nestin+ cells in the skin of the mouse line was analysed by immunofluorescence and immunohistochemical analyses. RESULTS: EGFP+ cells were recognized in the outer epithelial cell layers of anagen and telogen hair follicles, but rarely seen in the interfollicular epidermis. The EGFP+ cells in the outer layers of the hair follicles coexpressed keratin 14, a marker of the outer root sheath (ORS) keratinocytes, but not trichohyalin granules, an inner root sheath keratinocyte cell marker. Immunostaining for nestin failed to detect its expression in the majority of hair follicle epithelial cells, suggesting that the EGFP+ cells in the ORS were derived from nestin-expressing progenitor cells that had become further committed along the epithelial cell lineage, where nestin is no longer expressed. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest that progenitor cells that differentiate into ORS keratinocytes are distinct from those for other hair follicle or epidermal components and provide implications for regenerative medicine and the molecular classification of hair follicle tumours.

Piperacillin and ceftazidime produce the strongest synergistic phage-antibiotic effect in Pseudomonas aeruginosa.

The combined use of phage and antibiotics can show synergistic antimicrobial effects, so-called phage-antibiotic synergy (PAS). Here, we screened and examined PAS against Pseudomonas aeruginosa in vitro. Testing four different phages infecting P. aeruginosa, phage KPP22 classified within the family Myoviridae genus Pbunavirus showed PAS with the widest range of antibiotics, and showed PAS with anti-Pseudomonas drugs such as piperacillin and ceftazidime. Thus, evidence suggests that the combined use of phage and antibiotics is a promising therapeutic strategy against P. aeruginosa infections, with consideration needed regarding the optimal selection and adequate application timing of these phages and antibiotics.

Staphylococcus aureus penetrate the interkeratinocyte spaces created by skin-infiltrating neutrophils in a mouse model of impetigo.

BACKGROUND: Impetigo is a bacterial skin disease characterized by intraepidermal neutrophilic pustules. Previous studies have demonstrated that exfoliative toxin producing staphylococci are isolated in the cutaneous lesions of human and canine impetigo. However, the mechanisms of intraepidermal splitting in impetigo remain poorly understood. OBJECTIVE: To determine how staphylococci penetrate the living epidermis and create intraepidermal pustules in vivo using a mouse model of impetigo. METHODS: Three Staphylococcus aureus strains harbouring the etb gene and three et gene negative strains were epicutaneously inoculated onto tape-stripped mouse skin. The skin samples were subjected to time course histopathological and immunofluorescence analyses to detect intraepidermal neutrophils and infiltrating staphylococci. To determine the role of neutrophils on intraepidermal bacterial invasion, cyclophosphamide (CPA) was injected intraperitoneally into the mice to cause leucopenia before the inoculation of etb gene positive strains. RESULTS: In mice inoculated with etb gene positive S. aureus, intraepidermal pustules resembling impetigo were detected as early as 4 h post-inoculation (hpi). Neutrophils in the epidermis were detected from 4 hpi, whereas intraepidermal staphylococci was detected from 6 hpi. The dimensions of the intraepidermal clefts created in mice inoculated with etb gene positive strains at 6 hpi were significantly larger than those in mice inoculated with et gene negative strains. In CPA treated mice, staphylococci or neutrophils were not detected in the deep epidermis until 6 hpi. CONCLUSION: Our findings indicate that intraepidermal neutrophils play an important role in S. aureus invasion into the living epidermis in a mouse model of impetigo.

Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus).

UNLABELLED: Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. IMPORTANCE: Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

The stratum corneum: the rampart of the mammalian body.

BACKGROUND: The stratum corneum (SC) is the outermost region of the epidermis and plays key roles in cutaneous barrier function in mammals. The SC is composed of 'bricks', represented by flattened, protein-enriched corneocytes, and 'mortar', represented by intercellular lipid-enriched layers. As a result of this 'bricks and mortar' structure, the SC can be considered as a 'rampart' that encloses water and solutes essential for physiological homeostasis and that protects mammals from physical, chemical and biological assaults. STRUCTURES AND FUNCTIONS: The corneocyte cytoskeleton contains tight bundles of keratin intermediate filaments aggregated with filaggrin monomers, which are subsequently degraded into natural moisturizing compounds by various proteases, including caspase 14. A cornified cell envelope is formed on the inner surface of the corneocyte plasma membrane by transglutaminase-catalysed cross-linking of involucrin and loricrin. Ceramides form a lipid envelope by covalently binding to the cornified cell envelope, and extracellular lamellar lipids play an important role in permeability barrier function. Corneodesmosomes are the main adhesive structures in the SC and are degraded by certain serine proteases, such as kallikreins, during desquamation. CLINICAL RELEVANCE: The roles of the different SC components, including the structural proteins in corneocytes, extracellular lipids and some proteins associated with lipid metabolism, have been investigated in genetically engineered mice and in naturally occurring hereditary skin diseases, such as ichthyosis, ichthyosis syndrome and atopic dermatitis in humans, cattle and dogs.

Skin lipid profiling in normal and seborrhoeic shih tzu dogs.

BACKGROUND: Seborrhoea is a clinical condition resulting in excessive lipid and/or scale on the skin and is a common and important skin disease of dogs. However, there is little information on the skin surface lipid composition of dogs with seborrhoea. HYPOTHESIS/OBJECTIVES: To compare skin surface lipid profiles in normal and seborrhoeic shih tzu dogs. METHODS: Fourteen client-owned dogs (seven seborrhoeic and seven normal) were investigated. Lipids in sebaceous glands (SGs) were extracted from homogenized tissues of SG hyperplasia. Surface lipid was collected by tape stripping [stratum corneum (SC)-enriched fraction] and acetone-wetted cotton swab (acetone-extracted fraction). Lipids in SGs, SC-enriched fractions and acetone-extracted fractions were evaluated by high-performance thin-layer chromatography. RESULTS: Lipids in SGs mainly consisted of cholesterol esters, wax esters and triglycerides, whereas lipids in the SC-enriched fraction mainly consisted of ceramides. The acetone-extracted fraction contained a mixture of lipid classes recognized in SG- and SC-enriched fractions. In seborrhoeic dogs, concentrations of wax esters and triglycerides in the acetone-extracted fraction were significantly higher than in control dogs (P = 0.0285). Amounts of total ceramides (in micrograms) per milligram of SC were not significantly different between the two groups (P = 0.5204). Interestingly, two unknown ceramide fractions, which accounted for 20% of the total ceramides, were recognized exclusively in seborrhoeic dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: These results provide evidence that the skin surface lipid profiles are altered in shih tzu dogs with seborrhoea.

Usefulness of cefovecin disk-diffusion test for predicting mecA gene-containing strains of Staphylococcus pseudintermedius and clinical efficacy of cefovecin in dogs with superficial pyoderma.

BACKGROUND: Cefovecin has been widely used to treat skin infections in dogs. The relationship of the cefovecin disk-diffusion test results to the presence of the mecA gene and the clinical efficacy of cefovecin have not been fully evaluated. HYPOTHESIS/OBJECTIVES: To determine the usefulness of an in vitro cefovecin disk-diffusion test in predicting the presence of the mecA gene in Staphylococcus pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma. METHODS: Twenty-six S. pseudintermedius strains isolated from 22 dogs with pyoderma were used. In vitro disk-diffusion test results of cefovecin were compared with agar-dilution test results, the presence of the mecA gene, and the improvement in clinical scores of dogs with superficial pyoderma at 14 days post treatment. RESULTS: There was a significant linear correlation (r = -0.83) between the diameter of the obvious zone of inhibition by disk diffusion and the minimal inhibitory concentration for cefovecin (P < 0.0001). Receiver operating characteristic analysis revealed that zone diameters between 25 and 27 mm exhibited better sensitivity (92.9%) and specificity (100.0%) for detection of strains carrying the mecA gene. The mean improvement in clinical scores in dogs carrying cefovecin-resistant strains was significantly lower than in dogs carrying cefovecin-susceptible strains (P < 0.01). CONCLUSIONS AND CLINICAL IMPORTANCE: The cefovecin disk-diffusion test with a cut-off value estimated in this study was valuable for predicting mecA gene carriage in S. pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius.

Characterization of canine filaggrin: gene structure and protein expression in dog skin.

BACKGROUND: Filaggrin (FLG) is a key protein for skin barrier formation and hydration of the stratum corneum. In humans, a strong association between FLG gene mutations and atopic dermatitis has been reported. Although similar pathogenesis and clinical manifestation have been argued in canine atopic dermatitis, our understanding of canine FLG is limited. HYPOTHESIS/OBJECTIVES: The aim of this study was to determine the structure of the canine FLG gene and to raise anti-dog FLG antibodies, which will be useful to detect FLG protein in dog skin. METHODS: The structure of the canine FLG gene was determined by analysing the publicly available canine genome DNA sequence. Polyclonal anti-dog FLG antibodies were raised based on the canine FLG sequence analysis and used for defining the FLG expression pattern in dog skin by western blotting and immunohistochemistry. RESULTS: Genomic DNA sequence analysis revealed that canine FLG contained four units of repeated sequences corresponding to FLG monomer protein. Western blots probed with anti-dog FLG monomer detected two bands at 59 and 54 kDa, which were estimated sizes. The results of immunohistochemistry showed that canine FLG was expressed in the stratum granulosum of the epidermis as a granular staining pattern in the cytoplasmic region. CONCLUSIONS AND CLINICAL IMPORTANCE: This study revealed the unique gene structure of canine FLG that results in production of FLG monomers larger than those of humans or mice. The anti-dog FLG antibodies raised in this study identified FLG in dog skin. These antibodies will enable us to screen FLG-deficient dogs with canine atopic dermatitis or ichthyosis.

Transcription profile of chemokine receptors, cytokines and cytotoxic markers in peripheral blood of dogs with epitheliotropic cutaneous lymphoma.

BACKGROUND: Mycosis fungoides (MF) is the most common form of canine epitheliotropic cutaneous lymphoma, which is characterized by the accumulation of neoplastic CD8(+) T cells. Given that multifocal skin lesions are commonly seen in MF, neoplastic lymphocytes may actively migrate into the blood circulation. HYPOTHESIS/OBJECTIVES: Cytotoxic T cells with a skin-homing phenotype could be increased in the blood circulation of dogs with MF. ANIMALS: Ten dogs with MF and 10 age-matched healthy dogs were included. METHODS: The transcription levels of chemokine receptors, cytokines and cytotoxic markers in peripheral blood of dogs with MF were quantified by real-time RT-PCR. RESULTS: The dogs with MF had lower transcription levels of chemokine receptors associated with skin homing (CCR4), epitheliotropism (CXCR3), lymph node homing (CCR7), a type-1 cytokine (LT-α) and cytotoxic markers (perforin and granzyme B) in the circulation than healthy control dogs (P < 0.05). CONCLUSIONS AND CLINICAL IMPORTANCE: The present results suggest that the number of peripheral cytotoxic T cells with a skin-homing phenotype could be decreased in the peripheral blood of dogs with MF, which might be due to the sequestration of cytotoxic T cells in the lesional skin.

Epidermal structure created by canine hair follicle keratinocytes enriched with bulge cells in a three-dimensional skin equivalent model in vitro: implications for regenerative therapy of canine epidermis.

BACKGROUND: Keratinocytes in the hair follicle bulge region have a high proliferative capacity, with characteristics of epithelial stem cells. This cell population might thus be an ideal source for generating the interfollicular epidermis in a canine skin equivalent. HYPOTHESIS/OBJECTIVES: This study was designed to determine the ability of canine hair follicle bulge cell-enriched keratinocytes to construct canine living skin equivalents with interfollicular epidermis in vitro. ANIMALS: Four healthy beagle dogs from a research colony. METHODS: Bulge cell-enriched keratinocytes showing keratin 15 immunoreactivity were isolated from canine hair follicles and cultured on dermal equivalent containing canine fibroblasts. Skin equivalents were subjected to histological, immunohistochemical, western blot and RT-PCR analyses after 10-14 days of culture at the air-liquid interface. RESULTS: The keratinocyte sheets showed an interfollicular epidermal structure comprising four to five living cell layers covered with a horny layer. Immunoreactivities for keratin 14 and desmoglein 3 were detected in the basal and immediate suprabasilar layers of the epidermis, while keratin 10 and desmoglein 1 occurred in more superficial layers. Claudin 1 immunoreactivity was seen in the suprabasalar layer of the constructed epidermis, and filaggrin monomers and loricrin were detected in the uppermost layer. Basal keratinocytes in the skin equivalent demonstrated immunoreactivity to antibodies against basement membrane zone molecules. CONCLUSIONS AND CLINICAL IMPORTANCE: A bulge stem cell-enriched population from canine hair follicles formed interfollicular epidermis within 2 weeks in vitro, and thus represents a promising model for regenerative therapy of canine skin.