Michel's medium as a preservative for immunofluorescent staining of cutaneous biopsy specimens in dogs and cats

Paired 3-mm skin biopsy specimens from 24 dogs and 11 cats with suspected autoimmune dermatopathies were stained with fluorescein-conjugated antisera. In each case, 1 specimen was immediately embedded in optimum cutting temperature compound, quick frozen to -30 C, processed, stained, and examined. The other sample was placed in Michel's transport medium, held for 7 days, and then processed, stained, and examined. The location, quality, and intensity of immunofluorescent staining (when positive) were then compared in the 2 specimens. There were no differences in the staining patterns between the specimens processed immediately and those held in Michel's medium, thus demonstrating the validity of preserving canine and feline skin biopsy specimens in Michel's medium for immunofluorescent examination.     

Ectoparasites are the major causes of various types of skin lesions in small ruminants in Ethiopia

Ectoparasites are the major causes of skin lesions in animals. Clinical, skin scraping examination, and histopathological studies were conducted to identify and characterize skin lesions in small ruminants caused by ectoparasites. Mange mites, lice, sheep keds, and ticks were collected from the skin of affected animals for species identification. Skin biopsies were collected from affected part of the skin and fixed in 10% neutral buffered formalin for histopathology. Of 1,000 sheep and 600 goats examined, 815 (81.50%) sheep and 327 (54.5%) goats were infested with one or more types of ectoparasites. Sarcoptes scabiei var ovis, Demodex ovis, Psoroptes ovis, Bovicola ovis, Melophagus ovinus, and Amblyomma variegatum and other tick species were identified from sheep. S. scabiei var caprae, Demodex caprae, Linognathus stenopsis, and A. variegatum and other tick species were identified from goats. Gross skin lesions or defects observed on the skin include stained and ragged wool, loss of wool/hair, nodules, crusts, lichenification, and fissuring. Microscopic evaluation of H and E stained skin sections revealed lesions in the epidermal layer such as hyperkeratosis, acanthosis, and melanin inconsistency on the basal cells of the epidermis. Follicular keratosis, perifolliculitis, frunculosis, perivasculitis, and aggregates of inflammatory cells (of acute and chronic type) with fibrosis were experiential in the dermal layer of the skin. Most of the skin lesions caused by ectoparasites are overlapping. Thus, ectoparasites control program should be executed to reduce skin lesions as skins are the major export commodity of the country.     

Evaluation of canine antimicrobial peptides in infected and noninfected chronic atopic skin

Background – Antimicrobial peptides (AMPs) are small immunomodulatory peptides produced by epithelial and immune cells. β‐Defensins (BDs) and cathelicidins (Caths) are the most studied AMPs. Recently, increased cutaneous expression of AMPs was reported in atopic humans and in beagles with experimentally induced atopy. Hypothesis/Objectives – Our goal was to analyse mRNA expression and protein levels of canine (c)BD1‐like, cBD2‐like/122, cBD3‐like, cBD103 and cCath in healthy and naturally affected atopic dogs, with and without active skin infection, along with their distribution in the epidermis using indirect immunofluorescence. Animals – Skin biopsies were taken from 14 healthy and 11 atopic privately owned dogs. Methods – The mRNA levels of cBD1‐like, cBD2‐like/122, cBD3‐like, cBD103 and cCath were quantified using quantitative real‐time PCR. The protein levels of cBD3‐like and cCath were analysed by relative competitive inhibition enzyme‐linked immunosorbent assay, while the distributions of cBD2‐like/122, cBD3‐like and cCath were detected by indirect immunofluorescence. Results – Dogs with atopic dermatitis had significantly greater mRNA expression of cBD103 (P = 0.04) than control dogs. Furthermore, atopic skin with active infection had a higher cBD103 mRNA expression (P = 0.01) and a lower cBD1‐like mRNA expression (P = 0.04) than atopic skin without infection. No significant differences in protein levels (cBD3‐like and cCath) or epidermal distribution of AMPs (cBD2‐like/122, cBD3‐like and cCath) were seen between healthy and atopic dogs. Conclusions and clinical importance – Expression of cBD103 mRNA was greater, while expression of cBD1‐like mRNA was lower in dogs with atopic dermatitis that had active infections. Work is needed to clarify the biological mechanisms and possible therapeutic options to maintain a healthy canine skin.     

The role of basophils in the immunopathogenesis of hypersensitivity to fleas (Ctenocephalides felis) in dogs

Biopsies taken of skin test sites from 10 dogs allergic to fleas were fixed in Karnovsky's fixative and embedded in methacrylate. One micron sections were stained with acid Giemsa for identification of basophils. This cell was identified in most biopsies taken at intervals from 1 hour to 48 hours post-injection. The proportion was highest between 4 hours and 18 hours and had substantially fallen by 48 hours. The highest number of basophils recorded as a percentage of the inflammatory infiltrate was 22.1%, with biopsies from 7 of the 10 dogs showing in excess of 10% basophils at some point. The results suggest that cutaneous basophil hypersensitivity may play an immunopathogenic role in flea-bite hypersensitivity in dogs.     

Avidin-biotin-peroxidase complex immunohistochemistry to detect immunoglobulin in formalin fixed skin biopsies in canine autoimmune skin disease.

Avidin-biotin-peroxidase complex immunohistochemistry was used on formalin fixed, paraffin embedded, trypsin digested, skin biopsies to detect immunoglobulin deposition in dogs with autoimmune skin disease. Immunostaining by the avidin-biotin-peroxidase complex technique revealed intercellular and/or basement membrane immunoglobulin deposits in 27 of 28 dogs considered to have autoimmune skin disease by clinical and histological evaluation and in six of 19 dogs considered to have autoimmune skin disease by clinical evaluation but without histological confirmation. Similar immunostaining was not evident in five biopsies of normal skin or in biopsies from four dogs with noninflammatory dermatoses, but was present in biopsies from one of ten dogs considered by clinical and histological criteria to have an inflammatory dermatosis other than autoimmune skin disease. Detection of immunoglobulin deposits in skin biopsies by avidin-biotin-peroxidase complex immunohistochemistry offers numerous advantages over conventional immunofluorescence methods including the opportunity to precisely compare histological and immunological findings.     

Immunodiagnosis of autoimmune skin disease in the dog, cat and horse

Skin biopsies from 47 dogs, 6 cats and 5 horses with suspected autoimmune skin disease were submitted for immunofluorescence from 1978 to 1985. These cases were predominantly Western Australian in origin, although a number were also referred from Queensland and Victoria. In 5 dogs, 2 cats and 2 horses immunoglobulin binding to intercellular cement substance and/or basement membrane was demonstrated by direct immunofluorescence. Antinuclear antibody was also demonstrated in several of these cases. Immunofluorescence was used in combination with histopathological examination to confirm the clinical diagnosis of autoimmune disease in 19/47 dogs, 4/6 cats and 2/5 horses. There was no age, breed or sex predisposition amongst the 19 positive dogs, however there was a higher incidence of antinuclear antibody (54%) than the normal canine population (10%) and other autoantibodies (rheumatoid factor) were sometimes present. Abnormalities in serum protein electrophoresis and serum complement C4 levels were also recorded in this group.     

In Vitro Infectioion of Bovine Hoof Cells and Skin Explants with Treponema Brennaborense and Treponema Denticola

The aim of this study was to establish an in vitro model that permits in vitro infection of bovine skin with Treponema spp . and enables to study the role of treponemes in the pathogenesis of digital dermatitis (DD). In all experiments, incubation with T. denticola or T. brennaborense was carried out simultaneously. Keratinocytes obtained from the claw were cultivated on cover glasses without antibiotics until they reached sub-confluence. Then they were incubated with OMIZ Pat medium containing treponemes for up to 96 h. Every 24 h two cover glasses were fixed and stained with the DAPI method. Skin explants were obtained from typical sites of DD lesions. First the explants were maintained in medium with antibiotics to eliminate bacterial contamination. Subsequently, they were rinsed thoroughly with medium without antibiotics and incubated with Treponema suspensions for 48 h. The treponemes stayed vital under culture conditions for even up to 96 h. They were still showing their typical spiral shaped morphology and adhered to the cultured keratinocytes at all time points. With prolonged incubation time cultured cells began to show morphologic damage and some cells detached from the cover glasses. Light and electron microscopical investigations of the explants revealed that treponemes were adhering to the surface of the epidermis. They were visible in often-enlarged intercellular spaces. In addition treponemes could be detected in deep epidermal layers and in very high concentrations in the dermis. In periodontal disease, spirochetes were observed in enlarged intercellular spaces. Our results support these findings suggesting that treponemes invade the deeper claw tissue via the intercellular spaces of the epidermis. We suggest that the enlargement of the intercellular spaces can improve an increased infection of deeper tissue layers and facilitates the way for infection with other anaerobic bacteria.     

Immunoglobulin-E-bearing cells in skin biopsies of horses with insect bite hypersensitivity.

The aim of the present study was to investigate, with immunohistochemistry and in situ hybridisation, if immunoglobulin-E (IgE) and mast cells are involved in the pathogenesis of insect bite hypersensitivity (IBH), an allergic dermatitis of horses. In tissue sections fixed in paraformaldehyde (PFA) for <24 h, significantly more IgE protein-bearing cells were found in the dermis and epidermis of acute and chronic IBH lesions than in skin biopsies from healthy horses (medians = 466, 236 and 110 cells/mm2, respectively; P < or = 0.01). More IgE-mRNA positive (+) cells were observed in the dermis of acute IBH lesions than in the dermis of healthy skin (median = 2.8 vs. 0.0 cells/mm2; P < or = 0.01). Significantly, more mast cells were detected with metachromatic (median = 160 vs. 62 cells/mm2; P < or = 0.001) and tryptase-specific stainings (median = 120 vs. 69 cells/mm2; P < or = 0.001) in the dermis of acute IBH biopsies compared to healthy skin. No chymase+ mast cells were found in any skin biopsy. IBH lesions fixed in PFA for >24 h were compared to dermatomycosis (DM) lesions; IBH biopsies contained a similar number of IgE-protein+ cells to DM biopsies (median = 249 vs. 192 cells/mm2; P = 0.08) but had significantly more IgE-mRNA+, metachromatic and tryptase+ mast cells than DM biopsies. This study suggests an involvement of IgE-mediated immune reactions in the pathogenesis of IBH as well as, sometimes, in dermatomycosis. Using double labelling, cells which expressed IgE protein and contained mast cell enzymes were detected.     

Tissue Expansion in Pig Skin – A Histochemical Approach

In the present study, a porcine model for controlled skin expansion was investigated to improve our understanding of epidermal and vascular responses following stretching. The model is of outstanding importance not only for the clinical use of tissue expansion but provides interesting data for skin physiology and oncology, too. Thirteen out of 15 animals, who underwent silicone tissue expander implantation showed good clinical results. In all of them, skin biopsies were taken at the end of a controlled tissue expansion procedure (final expander volumes 350 or 500 ccm): one tissue specimen was obtained from the centre of the expanded skin area and a second from the neighbouring but nonexpanded skin. The tissue specimens were immediately frozen in liquid nitrogen and processed to 4 microns thick acetone-fixed frozen sections. Lectin histochemistry and immunohistology were performed using the following techniques: direct and indirect immunofluorescence technique (DIFT, IIFT), immunoperoxidase technique (POX) with either 3,3'-diamino-benzidine (DAB) or 3-amino-9-ethyl-carbazole (AEC). The histochemical findings were supplemented by measurements of the number of vital epidermal cell layers, the epidermal thickness (microns), and the papillary vascular count per visual field. There was a significant diminuation of the vascular count (mean +/- S.D. = 55.0% +/- 12.5%; U-test: p less than 5%). By immunohistochemistry, a loss of the basal cell reactivity for the following antibodies was noted: ACAM (against calmodulin), K 8.12 (against keratins 13 +/- 16) and A51-B/H4 (against keratins 8, 14, 18). There was a remarkable increase of filaggrin expression in the uppermost spinal cell layers in expanded skin, which was most pronounced in those specimens with the shortest interval to the last fluid injection into the expander. We gained no evidence for alterations of the expression of suprabasal epidermal keratins, lectin binding sites (UEA I, PNA, ConA, WGA), and vascular lectin- and immunoreactivity due to tissue expansion. The subdermal capsule, which had formed around the silicone expander, was strongly vimentin-reactive. In conclusion, controlled tissue expansion is capable to change the basal cell phenotype--a feature which is shared with a number of conditions with increased proliferative activity and with the epidermis covering different skin tumours. The regular expression of suprabasal keratins and epidermal lectin binding sites provides evidence for a normal epidermal cell differentiation. Furthermore, the porcine skin is a reliable model for studying physiology and pathophysiology of human skin.     

Pemphigus foliaceus: a report of two cases in the dog

The clinical and diagnostic features of two cases of pemphigus foliaceus in the dog are described. Dermatohistopathology and direct immunofluorescence examination of skin biopsies were diagnostic for pemphigus foliaceus. Both dogs required immunosuppressive dosages of prednisolone to achieve control of the condition.     

In vivo measurement of central corneal thickness in normal chicks (Gallus gallus domesticus) with the optical low-coherence reflectometer

Objective To determine the practicability and accuracy of central corneal thickness (CCT) measurements in living chicks utilizing a noncontact, high‐speed optical low‐coherence reflectometer (OLCR) mounted on a slit lamp. Animals studied Twelve male chicks (Gallus gallus domesticus). Procedures Measurements of CCT were obtained in triplicate in 24 eyes of twelve 1‐day‐old anaesthetized chicks using OLCR. Every single measurement taken by OLCR consisted of the average result of 20 scans obtained within seconds. Additionally, corneal thickness was determined histologically after immersion fixation in Karnovsky’s solution alone (20 eyes) or with a previous injection of the fixative into the anterior chamber before enucleation (4 eyes). Results Central corneal thickness measurements using OLCR in 1‐day‐old living chicks provide a rapid and feasible examination technique. Mean CCT measured with OLCR (189.7 ± 3.34 μm) was significantly lower than histological measurements (242.1 ± 47.27 μm) in eyes with fixation in Karnovsky’s solution (P = 0.0005). In eyes with additional injection of Karnovsky’s fixative into the anterior chamber, mean histologically determined CCT was 195.2 ± 8.25 μm vs. 191.9 ± 8.90 μm with OLCR. A trend for a lower variance was found compared to the eyes that had only been immersion fixed. Conclusion Optical low‐coherence reflectometry is an accurate examination technique to measure in vivo CCT in the eye of newborn chicks. The knowledge of the thickness of the chick cornea and the ability to obtain noninvasive, noncontact measurements of CCT in the living animal may be of interest for research and development of eye diseases in chick models.     

Lipid ingestion from sheep epidermis by Psoroptes ovis (Acari: Psoroptidae)

Skin biopsies from three groups of sheep infested with Psoroptes ovis were cryofixed in liquid nitrogen to preserve outer epidermis with its lipid. From one group of five sheep (Group A), biopsies were taken from relatively healthy skin near the edge of scab lesions where mites had congregated. Frozen vertical sections from these biopsies were stained with Oil Red O and haematoxylin before mounting. Red lipid globules were plentiful within the body cavity of sectioned mites, but ingested lipid could not be distinguished from endogenous mite body stores by this technique. In a second group of five sheep (Group B), enclosed lumbar skin areas were coloured red with the lipophilic stains Oil Red O or Sudan IV. These enclosed coloured skin areas were inoculated with P. ovis and sampled by skin biopsy 1 or 4 days later. Cryofixed biopsies were cut into vertical frozen sections and mounted without staining for examination. Red-coloured lipid within mites, matching red-coloured lipid on outer epidermis, was evidence for the epidermal origin of P. ovis ingesta in the early stages of an infestation. From two other sheep (Group C), cryofixed biopsies were examined by scanning electron microscope and mites were seen with mouthparts embedded in abraded outer epidermis, but the precise depth of epidermal penetration was not determined. A light microscope survey of 3198 frozen vertical skin sections from the first 10 sheep (Groups A and B) showed that inner stratum corneum of the epidermis was the deepest penetration recorded for gnathosomes of P. ovis cryofixed in situ by liquid nitrogen. No structure of P. ovis was identified in dermal tissues.     

In vitro and in vivo Effects of Lufenuron on Dermatophytes Isolated from Cases of Canine and Feline Dermatophytoses*

Lufenuron is a benzyl‐urea phenol compound that inhibits chitin synthesis and is used as an insecticide. Its efficacy in the therapy of dermatophytosis in dogs and cats was evaluated in several clinical studies, with contradictory results. We assessed the in vitro susceptibility of dermatophytes isolated from dogs and cats to lufenuron, and the clinical response of skin lesions to the drug. Dermatophyte cultures isolated from clinical cases were exposed to lufenuron by three different methods: direct application and application of whole blood or subcutaneous tissue samples obtained from a lufenuron‐treated healthy dog. No inhibition of dermatophyte growth was observed in any of the samples after 1 week of incubation. Eight dogs and six cats with skin lesions were included in the in vivo survey. Results indicated that six of seven skin lesions that were diagnosed as being caused by dermatophytes did not respond to lufenuron whereas six of seven skin lesions that were not caused by dermatophytes improved. We concluded that lufenuron, in the way it was administered in this study, had no inhibitory activity on dermatophytes in vitro or in vivo and its clinical use as an anti‐fungal agent is questionable. An immunomodulatory effect of the drug is, however, possible.     

Morphologic and immunohistochemical features of experimentally induced allergic contact dermatitis in Göttingen minipigs

Many preclinical studies in investigative dermatology are performed preferably in pigs because pig skin is more similar to human skin than is rodent skin. A frequently used model is allergic contact dermatitis (ACD); however, this T-cell-mediated skin condition so far is not well characterized in pigs. The present study is aimed at the evaluation of morphologic and immunohistochemical features of experimentally induced acute ACD in G?ttingen minipigs using 2,4-dinitrofluorobenzene (DNFB) as a hapten. Eight minipigs were sensitized with 10% DNFB and challenged 2 weeks later at different sites with 1% DNFB. In addition to clinical examinations, cutaneous blood flow was quantified by laser Doppler velocimetry (Periflux PF3). These examinations were performed before challenge and 8, 24, 48, and 72 hours after challenge. Skin biopsies were taken at the same time points, fixed, sectioned, and stained with Giemsa for histologic evaluation, or with mouse anti-swine monoclonal antibodies (CD1, CD2, CD4, CD5, CD8, CD25, CD45, MHCII) and with one mouse anti-human monoclonal antibody (CD62E) cross-reacting with swine for immunohistochemical evaluation. Positively stained cells were counted per square millimeter of epidermis and dermis by using a video image analyzing system (Videoplan Kontron). Erythema and cutaneous blood flow peaked at 24 hours. The major epidermal changes most pronounced at 48 hours were acanthosis, spongiosis, intracellular edema, exocytosis, and abscesses mainly containing neutrophils and mononuclear cells (MNC). Perivascular infiltrates of MNC as well as neutrophils and eosinophils were the most significant dermal changes, with peak levels at 24-48 hours. In biopsies taken before challenge, CD1+ dendritic cells were found in similar numbers and locations as MHCII+ cells in the epidermis. In the epidermis the maximum CD1+ cell decrease occurred at 24 hours whereas in the dermis the maximum increase in CD1+ stained cells was seen at 72 hours. The dermal infiltrate (CD2+, CD5+, CD25+, and CD45+) was most dense at 48 hours. Between 8 and 48 hours more CD4+ were present than CD8+, cells, whereas at 72 hours CD4+ and CD8+ cells were similar in numbers. These findings closely resemble changes in human ACD. Therefore, DNFB-induced ACD in G?ttingen minipigs is considered to be an appropriate animal model to study immunopathologic mechanisms and pharmacologic intervention.     

Effect of embryo age on the viability of equine embryos after cooled storage using two transport systems

The aim of this study was to compare the viability of 7- and 8-day-old equine embryos cooled and stored for 6 or 24 hours in two different transport systems. Embryos (n = 97) were recovered on day 7 or 8 and assigned to 10 groups (n = 10/group). Embryos within the same age group (D7 or D8) were evaluated immediately after collection (Group-0h) or after storage in an Equitainer at 5°C for 24 hours in 5 ml Emcare Holding Solution (EHS) (Group-E-24h) or 5 ml Ham's F10 (Group-H-24h) or in a refrigerator at 5°C in 500 ml Emcare Flushing Solution (EFS) for 6 hours (Group-B-6h) or 24 hours (Group-B-24h). After collection or storage, embryos were incubated in 1 μg/ml DAPI to determine the percentage of dead cells per embryo (DAPI positive, fluorescent cells). Subsequently, embryos were fixed in 4% paraformaldehyde and re-stained with DAPI to determine the total number of cells. The percentage of dead cells in group-0h and B-6h was similar and significantly lower than for embryos stored for 24 hours in groups B-24h, E-24h, and H-24h. The percentage of dead cells was similar for embryos stored in an Equitainer (groups E-24h and H-24h) and was significantly higher for embryos stored 24 hours in EFS (Group B-24h). Within each storage system (0h, B-6h, B-24h, E-24h, and H-24h) no significant difference in the percentage of dead cells was observed between 7- and 8-day-old embryos. Storage in 500 ml EFS at 5°C for 6 hours resulted in embryos of better quality than after the traditional 24-hour storage in an Equitainer, suggesting that this simplified system offers a good alternative for short-term storage and transport.     

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5 ± 1.5%) and CEC-32 (RIF 7.0 ± 0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R_low. Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2 ± 0.3%) similar to that of R_low (1.1 ± 0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.     

A comparison of the potency of several Brucella allergens used to detect brucellosis in cattle

The potency of Brucella allergens prepared from a smooth Brucella abortus strain S-99, mucoid strain Leewarden, rough strain 45/20, and rough Brucella melitensis strain B-115 was assessed. The potency of these allergens was compared with that of a standard allergen prepared from smooth Brucella abortus S-99 that efficiently detected bovine brucellosis in other studies. Eight cattle experimentally inoculated with Brucella abortus 544 were tested with the allergens 4 and 10 weeks after infection, and again 8 months after infection. All the allergens effectively detected infection but there was a clear distinction in the mean skin reactions 48 and 72 h after injection of the allergens. The skin reactions provoked by the allergens prepared from smooth or mucoid strains of Brucella were most pronounced 48 h after injection. Skin reactions provoked by allergens prepared from rough strains of Brucella were strongest 72 h after injection. Allergens prepared from smooth or mucoid Brucella strains were more potent in detecting brucellosis than those prepared from rough strains of Brucella.Abbreviations Bruc/OCB Brucellergen OCB - cfu colony-forming units - CFT complement fixation test - ID-DLO Institute voor Dierhouderij en Diergezondheid-Dienst Landbouwkundig Onderzoek - ICFTU international complement fixation units - IU international units - LPS lipopolysaccharide - SAT serum agglutination test - SDTH skin delayed-type hypersensitivity     

不同日龄滩羊皮肤组织化学特征比较

为比较2、35日龄滩羊皮肤毛囊的发育特点与血管内皮生长因子（vascular endothelial growth factor，VEGF）和血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）的分布特征，探究出生后滩羊被毛生长发育的变化特点，试验应用常规HE染色及改良Masson胶原纤维染色、Gomori银氨法染色、磷钨酸染色等特殊染色观察2与35日龄滩羊皮肤组织结构特点；应用免疫组织化学法结合免疫荧光染色法观察VEGF及VEGFR2在2与35日龄滩羊皮肤组织中的分布定位，并用IPP图像分析软件进行定量分析。结果显示：与2日龄滩羊皮肤组织比较，35日龄滩羊表皮与真皮间界限更加清晰，毛囊结构发育完整；毛囊密度显著降低（P<0.05）；胶原纤维与弹性纤维含量增加，形成网格状分布。免疫组化及免疫荧光结果显示，VEGF及VEGFR2在滩羊皮肤表皮及毛囊外根鞘、皮脂腺上均有表达。统计表明，VEGF及VEGFR2在2日龄滩羊皮肤组织中的表达量均显著高于35日龄（P<0.05）。综合上述结果，滩羊毛囊发育过程中，胶原纤维和弹性纤维增加明显；VEGF与VEGFR2通路在毛囊角质形成中起直接调节作用。     

Immunoperoxidase staining for the detection of autoantibodies in canine autoimmune skin disease; comparison to immunofluorescence results

Skin sections from 22 dogs with autoimmune skin disease were stained with anti-canine IgG, IgM and IgA using an immunobridge immunoperoxidase method. Eight cases of lupus erythematosus, three cases of pemphigus vulgaris, and 11 cases of pemphigus foliaceus were included. Results of previously performed, direct immunofluorescence tests for the detection of canine immunoglobulin on skin were available on 17/22 cases. The immunoperoxidase method yielded an overall positive result in 59% (5/8 lupus erythematosus, 2/3 pemphigus vulgaris and 6/11 pemphigus foliaceus) versus an overall positive result of 47% for direct immunofluorescence (3/5 lupus erythematosus, 2/2 pemphigus vulgaris and 2/10 pemphigus foliaceus). The immunobridge immunoperoxidase method compared favorably to direct immunofluorescence testing of canine skin for autoantibody in cases of lupus erythematosis and pemphigus vulgaris, and was superior in cases of pemphigus foliaceus. This method should prove useful as an aid in the diagnosis of canine autoimmune skin disease.     

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.