Topical erythritol combined with L-ascorbyl-2-phosphate inhibits staphylococcal growth and alleviates staphylococcal overgrowth in skin lesions of canine superficial pyoderma.

Erythritol (ERT) and L-ascorbyl-2-phosphate (APS) are bacteriostatic, but their effects on staphylococcal skin infections remain unknown. We aimed to determine whether ERT combined with APS inhibits the growth of staphylococci that are commonly isolated from pyoderma skin lesions in dogs. We investigated the individual and combined effects of ERT and APS on the growth of Staphylococcus pseudintermedius, S. schleiferi, and S. aureus using turbidity assays in vitro. Skin lesions from 10 dogs with superficial pyoderma were topically treated with 5% ERT and 0.1% APS for 28 days, and swabbed skin samples were then analyzed using 16S rRNA amplicon sequencing and quantitative real-time PCR (qPCR). Results showed that ERT inhibited S. pseudintermedius growth regardless of harboring the mecA gene, and APS increased the inhibitory effects of ERT against S. pseudintermedius, S. schleiferi, and S. aureus in vitro. Moreover, combined ERT and APS decreased the prevalence of staphylococci on canine skin lesions at the genus level. The combination slightly increased the α-diversity but did not affect the ß-diversity of the microbiota. The qPCR results revealed that the combination significantly decreased S. pseudintermedius and S. schleiferi in skin lesions. Topical administration of EPS combined with APS can prevent staphylococcal colonization on the surface of mammalian skin. The results of this study may provide an alternative to systemic antibiotics for treating superficial pyoderma on mammalian skin surfaces.

Immune response towards the amino-terminus of desmoglein 1 prevails across different activity stages in nonendemic pemphigus foliaceus.

BACKGROUND: Pemphigus foliaceus (PF) is a blistering skin disease mediated by antibodies to desmoglein (Dsg) 1. The two major subtypes are nonendemic and endemic PF. A previous study in endemic PF demonstrated that changes in antibody epitope could modulate disease relapse and remission. OBJECTIVES: To characterize the frequency of immunoreactivity to various Dsg1 extracellular (EC) domains in nonendemic PF and to study if there is any change in epitope profile across various activity stages. METHODS: Sera from 34 patients with nonendemic PF were selected. To map the conformational epitopes by immunoprecipitation-immunoblotting, we constructed five Dsg1/Dsg2 domain-swapped molecules, with each molecule representing one EC domain of Dsg1 on a backbone of Dsg2. RESULTS: Dsg1 EC1, EC2, EC3, EC4 and EC5 domains were recognized by 88%, 50%, 13%, 22% and 0% of active PF sera, respectively. Immunoreactivity to EC3 or EC4 often cosegregated with that to either EC1 or EC2. Longitudinal follow-up of 21 patients with PF for a median of 16 months revealed that, in most cases, immunoreactivity to the amino-terminus of Dsg1 persisted across various activity stages; only two patients lost their EC1 reactivity upon remission and changed their major epitope(s) to EC2 ± EC3. CONCLUSIONS: Most of the anti-Dsg1 antibodies in nonendemic PF bind to the amino-terminus of Dsg1, a region critical for intercellular adhesion of cadherins, and this skewed amino-terminal immunoreactivity prevails across various activity stages in most patients, even upon remission. These findings are valuable for understanding the biology of Dsg-mediated cellular adhesion as well as for the development of epitope-based monitoring and therapeutic strategies.

Development of an in vitro submerged culture system to synthesize epidermal ceramides in canine keratinocytes.

Ceramides (CERs) in the stratum corneum (SC) are known to play a crucial role in determining skin barrier function in dogs. We aimed to develop an in vitro culture system that synthesized epidermal CER classes to better understand the synthesis of CER classes in canine SC-. Canine keratinocyte cells (MSCEK) at appropriate confluency were incubated with high Ca2+ (1.8 mM CaCl2) supplemented serum-free medium. Eight days post Ca2+ application, the surface of cultured MSCEK was broadly stained with anti-loricrin antibody implying that the keratinocytes had stratified into stratum granulosum. MSCEK cells synthesized heterogenous epidermal CERs, similar to those seen during the stratification of canine keratinocytes. CER fractions obtained from MSCEK cells were comparable to those from canine SC, including CER[EOS] (combination of ω-hydroxy fatty acids and sphingosines), CER[NP] (combination of non-hydroxy fatty acids and phytosphingosines), and CER[EOP] (combination of ω-hydroxy fatty acids and phytosphingosines), all of which are lowered in the SC during canine atopic dermatitis. Thus, the present study provides a simple culture system as a tool for in-depth analysis of CER production in canine keratinocytes.

Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus.

The development of experimental models of active autoimmune diseases can be difficult due to tolerance of autoantigens, but knockout mice, which fail to acquire tolerance to the defective gene product, provide a useful tool for this purpose. Using knockout mice lacking desmoglein 3 (Dsg3), the target antigen of pemphigus vulgaris (PV), we have generated an active disease model for this autoantibody-mediated disease. Dsg3(-/-) mice, but not Dsg3(+/-) littermates, produced anti-Dsg3 IgG that binds native Dsg3, when immunized with recombinant mouse Dsg3. Splenocytes from the immunized Dsg3(-/-) mice were then adoptively transferred into Rag-2(-/-) immunodeficient mice expressing Dsg3. Anti-Dsg3 IgG was stably produced in the recipient mice for more than 6 months without further boosting. This IgG bound to Dsg3 in vivo and disrupted the cell-cell adhesion of keratinocytes. Consequently, the recipient mice developed erosions in their oral mucous membranes with typical histologic findings of PV. In addition, the recipient mice showed telogen hair loss, as found in Dsg3(-/-) mice. Collectively, the recipient mice developed the phenotype of PV due to the pathogenic anti-Dsg3 IgG. This model will be valuable for developing novel therapeutic strategies. Furthermore, our approach can be applied broadly for the development of various autoimmune disease models.

Detection of antigen-specific B cells in patients with pemphigus vulgaris by enzyme-linked immunospot assay: requirement of T cell collaboration for autoantibody production.

Patients with pemphigus vulgaris have circulating IgG autoantibodies against desmoglein 3, which inhibit cell-cell adhesion of keratinocytes and cause blister formation in the skin and mucous membrane. To examine cellular mechanisms underlying the autoantibody production in pemphigus vulgaris patients, we have successfully developed an enzyme-linked immunospot assay which was able to detect desmoglein 3-specific autoimmune B cells quantitatively. Circulating B cells producing anti-desmoglein 3 antibodies were detected exclusively in three patients with severe disease (1.3-2.3/105 peripheral blood mononuclear cells), but not in 10 patients with mild disease or in remission or in seven healthy individuals. When this enzyme-linked immunospot assay was combined with in vitro stimulation of peripheral blood mononuclear cells with pokeweed mitogen and recombinant-desmoglein 3, we could detect circulating desmoglein 3-specific memory B cells in nine of 14 patients (6.3-84. 0/105 peripheral blood mononuclear cells), but in none of 10 healthy individuals. We further analyzed the role of CD4 + T cells in promoting anti-desmoglein 3 antibody production. The in vitro anti-desmoglein 3 antibody production was abolished when CD4 + cells were depleted or when anti-HLA-DR or anti-HLA-DQ monoclonal antibody was added to the cultures. Our results demonstrated the quantitative detection of circulating "activated" and "memory" desmoglein 3-specific B cells and suggested the important part of HLA class II-restricted CD4 + T cells in the autoantibody production in pemphigus vulgaris. In addition, the enzyme-linked immunospot assay in combination with in vitro stimulation of B cells could be broadly applied to study mechanisms for autoantibody production in various autoimmune diseases.

Use of domain-swapped molecules for conformational epitope mapping of desmoglein 3 in pemphigus vulgaris.

Pemphigus vulgaris is an autoimmune blistering disease caused by autoantibodies against desmoglein 3, a member of the desmosomal cadherin family. These autoantibodies recognize conformation-dependent epitopes on desmoglein 3. In this study we attempted to map the conformational epitopes of desmoglein 3 in pemphigus vulgaris using recombinant desmoglein 3 produced by the baculovirus expression system. We developed a series of domain-swapped molecules between desmoglein 3 and desmoglein 1, which have similar structures but distinct epitopes. These were developed by substituting deleted segmental regions of desmoglein 3 by the corresponding desmoglein 1. Thus domain-swapped molecules containing desmoglein 3 residues 1-403, 1-161, 163-566, and 405-566 were constructed and used as competitors for competition enzyme-linked immunosorbent assay against the entire extracellular domain of desmoglein 3 with 25 pemphigus vulgaris sera. Considering more than 50% absorption as significant, residues 1-403 and 1-161 showed significant absorption in 24 out of 25 (96%) and 18 out of 25 (72%) pemphigus vulgaris sera, respectively, whereas only one serum and no sera showed significant absorption by residues 163-566 and 405-566, respectively. Furthermore, no apparent change in their major epitopes was seen during the time course in four pemphigus vulgaris cases tested. These findings indicate that the domain-swapping approach is useful for conformational epitope mapping in pemphigus and that amino-terminal residues 1-161, which are considered to include a region essential for cell-cell adhesion in cadherins, contain the critical residues of the conformational epitope of desmoglein 3 recognized by the autoantibodies in pemphigus vulgaris sera.

Solubilization and reconstitution of membrane proteins of Escherichia coli using alkanoyl-N-methylglucamides.

Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.

Nitro reaction in mice injected with pyrene during exposure to nitrogen dioxide.

We have previously reported that the beta-glucuronidase-treated urine of mice injected intraperitoneally with pyrene during exposure to NO2 contained highly mutagenic compounds such as nitropyrene metabolites when tested by the Ames assay using Salmonella typhimurium strain TA98. In the present study, we found that the formation of these mutagens was dose-dependent between 10 and 200 mg of pyrene per kg of body weight at 5 and 10 ppm of NO2. Further, to elucidate the substrate of nitration in vivo, we injected 1-hydroxypyrene, which is the metabolite of pyrene, to mice intraperitoneally during exposure to NO2. Since the results were the same as those obtained by injection with pyrene, we suggest that the pyrene was not nitrated directly but after its hydroxylation.

Detection of circulating autoantibodies using living keratinocyte staining on MCA-B1 method in dogs with pemphigus foliaceus.

In this study, we compared the sensitivity and specificity of three immunofluorescence techniques used to detect circulating autoantibodies in dogs with pemphigus foliaceus (PF); living keratinocyte staining on a canine keratinocyte cell line, MCA-B1, indirect immunofluorescence (IIF) on canine lip and IIF on bovine esophagus. Sera from canine PF cases were positive in four out of 27 dogs (14.8%) using living keratinocyte staining on MCA-B1 cells method, and five (18.5%) and eight sera (29.6%) using IIF on canine lip and bovine esophagus methods, respectively. By contrast, none of the 31 sera from dogs with non-pemphigus dermatoses reacted with MCA-B1 cells, whereas two (6.5%) as well as five sera (16.1%) obtained from those dogs showed positive reactivity with IIF on canine lip and bovine esophagus, respectively. Our results suggest that, although it exhibits the least sensitivity, the positive reactivity obtained by living keratinocyte staining on MCA-B1 cells can support the diagnosis of canine PF.

Comparison of response to immunotherapy by intradermal skin test and antigen-specific IgE in canine atopy.

The intradermal skin test (IDST) and serologic allergy test (SAT) has been developed for confirming a diagnosis of canine atopy and determining allergens for immunotherapy. To determine the prevalence of causative allergens for canine atopic dermatitis in Japan, IDST and SAT were performed with the CMG Immunodot strips on 95 atopic dogs using 9 allergens. In addition, we compared agreement rate, sensitivity and specificity between them (using IDST as the standard). The allergen most commonly positive in both tests was house dust mites (IDST: 69.5%, SAT: 48.4%). Moreover, Japanese cedar, mugwort and grass mix were detected as attendant causative allergens. Agreement rates between the two tests ranged from 67.4% to 96.8%; the overall mean agreement rate were 81%. SAT was shown to have sensitivity to IDST ranging from 16.7 to 68.2%. The specificities were very high for all allergens, on the order of 94.9-100% (median=98.7%). Finally, the efficacy of immunotherapy was evaluated on 27 atopic dogs based on IDST (15 dogs) and SAT (12 dogs) results. Overall, 60% (9/15) of the IDST group and 66.8% (8/12) of the SAT group experienced a 50% to 100% reduction in their symptomatology. No significant differences were found in response to immunotherapy during the follow-up period between allergen selection methods. These results indicate the value of serologic tests as an aid to identifying an allergen solution for immunotherapy.

A non-joint tissue biphasic synovial sarcoma in a dog.

A subcutaneous tumour was identified in the malar region of a 14-year-old neutered female mixed breed dog. The dog was humanely destroyed and necropsy examination was performed. The tumour did not invade neighbouring tissues and no metastasis was found. Microscopically, the tumour showed a range of features including the presence of multinucleated giant cells, chondrocyte differentiation and cystic or slit-like structures. All of these features are consistent with previously reported descriptions of synovial sarcomas in dogs. Mesenchymal cells accounted for the majority of the tumour, but cytokeratin-positive epithelioid components were also confirmed by immunohistochemistry. The tumour was diagnosed as a biphasic type of synovial sarcoma. Synovial sarcoma in man may develop in tissues unrelated to joints and this is the first report of a non-joint synovial sarcoma in a dog.

Multi-system progressive angiomatosis in a dog resembling blue rubber bleb nevus syndrome in humans.

A six-year-old, neutered, female golden retriever was presented with generalised, dark purple to black cutaneous nodules and gastrointestinal haemorrhage. Histopathologically, all cutaneous nodules were diagnosed as benign cavernous haemangiomas. Endoscopic analysis revealed similar nodules in the oesophagus, stomach and duodenum. At laparotomy, similar nodules were seen on the visceral peritoneal lining of abdominal organs. Metastatic haemangiosarcoma was ruled out based on histological features and lack of primary tumour in spleen, liver or heart ultrasonographically. Blood loss associated with gastrointestinal haemorrhage was managed with blood transfusion. To the authors' knowledge, this is the first canine case of multi-system progressive angiomatosis resembling blue rubber bleb nevus syndrome in humans.

Canine hyperplastic intraepidermal pustular and suprabasal acantholytic dermatosis with features of human pemphigus vegetans.

Pemphigus vegetans is a rare autoimmune blistering acantholytic dermatosis of humans that combines unusually hyperplastic and verrucous pustular skin lesions and mucosal erosions. We report herein the clinical, histopathologic, and immunologic findings in a dog whose lesions resembled, but were not identical to, those of human pemphigus vegetans. A 4-year-old male Greater Swiss Mountain Dog presented with multifocal cutaneous verrucous and crusted papules and pustules, as well as skin and mucosal erosions and ulcers. Microscopic lesions consisted of exophytic papillated epidermal hyperplasia, superficial and deep intraepidermal acantholytic neutrophilic and eosinophilic pustules, and suprabasal epidermal clefts leaving rounded basal keratinocytes at the bottom of the vesicles. Direct and indirect immunofluorescence revealed antikeratinocyte IgG autoantibodies. Immunoprecipitation immunoblotting and immunoabsorption experiments with recombinant canine desmogleins confirmed that autoantibodies recognized desmoglein-1. In this dog, clinical and histopathologic features resembled those of human pemphigus vegetans, while circulating autoantibodies against canine desmoglein-1 were solely identified. This antigen target is different from that of the human disease in which antidesmoglein-3 autoantibodies are detected most commonly.

Interdigital involvement in a case of primary cutaneous canine histoplasmosis in Japan.

A 5-year-old male Siberian husky bred outdoor in Tokyo had a swollen paw with interdigital granulomatous lesions in the left hindlimb. The dog had no apparent pulmonary or gastrointestinal involvement. Histopathological analysis of the skin lesions demonstrated yeast-like organisms predominantly within macrophages. Sequence analysis of fungal ribosome RNA gene isolated from a paraffin sample revealed a 100% homology with the teleomorph of Histoplasma capsulatum. The present case may support the concept of primary cutaneous canine histoplasmosis as an endemic phenotype recognized in Japan.

Biliary excretion of glutathione conjugates of 4,5-epoxy-4,5-dihydro-1- nitropyrene and 9,10-epoxy-9,10-dihydro-1-nitropyrene in rats administered 1-nitropyrene orally and their further metabolism in the intestinal tract.

1-Nitropyrene (1-NP), a mutagenic and carcinogenic substance that occurs in the environment, is metabolized by reductive and oxidative pathways. 4,5-Epoxy,4,5-dihydro-1-NP (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-NP (1-NP 9,10-oxide) are oxidatively activated metabolites of 1-NP, and react with glutathione. HPLC analysis of biliary metabolites from rats administered [3H]1-NP orally showed the presence of glutathione conjugates of 1-NP 4,5-oxide and 1-NP 9,10-oxide and their metabolites, cysteinylglycine and cysteine conjugates. During the 48 h following [3H]1-NP administration, 21.4% of the biliary metabolites were excreted as glutathione, cysteinylglycine and cysteine conjugates of 1-NP oxides. The proportions of glutathione conjugates of 1-NP 4,5-oxide and 1-NP 9,10-oxide were 2.6 and 10.4% respectively. Bile and pancreatic juice collected from normal rats metabolized glutathione conjugates of 1-NP oxides and produced the corresponding cysteinylglycine and cysteine conjugates. In particular, the gamma-glutamyltransferase activity of pancreatic juice was markedly high. When glutathione conjugates of 1-NP oxides were incubated with a cell-free extract of small intestinal contents, they decreased rapidly and cysteine conjugates increased via cysteinylglycine conjugates, indicating that pancreatic juice plays an important role in the small intestine for the metabolism of glutathione conjugates to corresponding cysteine conjugates. Although degradation activity of small intestinal contents for cysteine conjugates was very low, degradation activity of the contents of the cecum and large intestine was high and was inhibited by an inhibitor of cysteine conjugate beta-lyase (beta-lyase) activity, aminooxyacetic acid. Furthermore, the intestinal anaerobic bacteria Peptostreptococcus magnus and Eubacterium limosum showed high beta-lyase activity, suggesting that the cysteine conjugates might be further metabolized by beta-lyase of the normal bacterial flora in the lower intestine to the reactivated metabolites and reabsorbed.

In vitro intestinal microflora-mediated metabolism of biliary metabolites from 1-nitropyrene-treated rats.

To investigate the modifying role of the intestinal microflora in the metabolism of 1-nitropyrene (1-NP) via enterohepatic circulation, we collected bile from male Wistar rats administered [3H]1-NP orally. The bile was mixed with the intestinal contents (IC) prepared from untreated rats and the mixture was incubated anaerobically under an atmosphere of nitrogen at 37 C. Samples of the reaction mixture were removed at intervals to assay their mutagenic potential, to determine the radioactivity bound to the IC, and for analysis of the biliary metabolites. The binding of the radioactivity to the IC increased linearly as a function of time during the 1-hr incubation. The time-dependent binding does not occur with heat-treated IC and the binding was inhibited by addition of D-saccharic acid 1,4-lacton, a beta-glucuronidase inhibitor. The mutagenicity (for Salmonella typhimurium strain TA98 without S9 mix) of the bile increased early in the incubation period and then decreased very rapidly. The mutagenicity of the bile was also enhanced by treatment with a sonicated IC extract or beta-glucuronidase, but not with a heat-treated IC or aryl-sulfatase. The metabolites produced after the bile was incubated for short periods with the IC were mainly nitrohydroxypyrenes; at later times nitroreduction occurred. The level of acetylaminohydroxypyrenes, which were formed by deconjugation, did not change during the incubation. To determine the degree of contribution of the IC to the total acetylating capacity, we measured acetyltransferase activity of the IC and various organs in Wistar rats. The liver had the highest N-acetyltransferase activity among the seventeen organs examined. Considerable activity was also detected in the kidney, small intestine, lung, and testis, but the IC showed very low activity. The acetylating capacity of the IC was 0.27% of the total capacity in rats, and that of the liver was more than 80%. These results suggest that the nitrohydroxypyrenes formed from 1-NP in the liver were conjugated to glucuronic acid and excreted via the bile duct into intestine. Hydrolysis of these glucuronide conjugates by bacterial beta-glucuronidase liberated into intestine, free nitrohydroxypyrenes, which were direct-acting mutagens. The released aglycons were then rapidly nitro-reduced by intestinal microflora, but contribution of the intestinal microflora to acetylation of the reduced metabolites is very low.

Mutagenicity and nitropyrene concentration of indoor air particulates exhausted from a kerosene heater.

The particulates in a room warmed with a radiant kerosene heater were collected, extracted and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA98, TA98NR, TA98/1,8-DNP6 and TA100 in the presence and absence of S9 mix. Room air without the heater showed very low mutagenicity. However, a sample from a room at the beginning of the burning period showed very high mutagenicity (237 His+ revertants/plate/m3 of air in strain TA98 in the absence of S9 mix). In contrast, emissions from the heater after it was burning stably showed low mutagenicity (9 His+ revertants/plate/m3). The crude extract of particulates from the heater at the beginning of the burning period was analyzed by high-pressure liquid chromatography (HPLC) and showed a considerable amount of nitropyrenes (NPs); the concentrations of 1-NP and 1,6-diNP were 1.62 ng and 0.149 ng/m3 of air, respectively, and accounted for 1.2% and 17.6%, respectively, of the mutagenicity in strain TA98 in the absence of S9 mix. In addition, an HPLC-Ames histogram showed that peaks of mutagenicity corresponding to 1-NP and diNPs accounted for 75.7% (1-NP, 4.9%; 1,6-diNP, 17.1%; 1,8-diNP, 46.3%; 1,3-diNP, 7.4%) of the HPLC-recovered mutagenicity for strain TA98 without S9 mix. These results that kerosene heaters, especially immediately after ignition, create mutagenic substances such as NPs.

Mutagenic nitropyrenes in foods.

Identification of mutagenic factors in foods is of concern because they may represent carcinogens to man. Cooked foods, especially their basic fractions containing heterocyclic amines, have high mutagenicity; the neutral fractions containing mutagenic nitropyrenes, however, have not been studied in detail. The mutagenicity of various grilled foods--10 vegetables, 4 fish, and 4 kinds of meat with and without sauce--was studied. The concentration of 1-nitropyrene was measured after reduction by a specific nitroreductase purified from Bacteroides fragilis. 1-Nitropyrene was detected in grilled corn, horse-mackerel, and mackerel, and accounted for less than 10% of the total mutagenicity of the crude extracts in the Salmonella mutation test using strain TA98 in the absence of S9 mix. The mutagenicity of the diethyl ether-soluble basic fractions of meat grilled without a marinating sauce was very high. However, the sauce decreased the mutagenicity of the basic fractions and increased the mutagenicity of the neutral fractions. Moreover, considerable amounts of 1-nitropyrene were detected in pork and yakitori (grilled chicken) grilled with the sauce. The neutral fractions of yakitori grilled for 3, 5, and 7 min contained 3.8, 19, and 43 ng, respectively, of 1-nitropyrene per gram of yakitori, accounting for 3.0, 2.7, and 1.3%, respectively, of the total extract mutagenicity. We conclude that formation of 1-nitropyrene in the yakitori is due to pyrene produced by incomplete combustion of fat in the chicken, its nitration at acidic pH by nitrogen dioxide emitted by burning of cooking gas, and some components of the marinating sauce. Antimutagenic activity of edible mushrooms against 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1) was also studied.