Alopecia areata with lymphocytic mural folliculitis affecting the isthmus in a thoroughbred mare

A 13-year-old, thoroughbred mare was presented with an 8-year history of multifocal, generalized, noninflammatory alopecia and a 3-month history of alopecia, erythema and scaling of the white star on the forehead and muzzle. Histopathological examination of biopsy samples from multiple sites on the body (mane, neck, shoulder, flank and gluteal region) showed a subtle lymphocytic inflammatory infiltrate affecting and surrounding the anagen hair bulbs, consistent with a diagnosis of alopecia areata. The biopsy sample from the star on the forehead showed atrophic hair follicles with perifollicular and mural mononuclear folliculitis affecting the isthmus. Immunohistochemical staining with a CD3 marker confirmed the T-lymphocytic origin of the inflammatory infiltrate in all the samples. The concurrent presence of lymphocytic infiltration at the bulbar and isthmic level of the hair follicles in the same horse is unusual. This finding may represent a variation of the histological appearance of alopecia areata.     

Anti‐isthmus autoimmunity in a novel feline acquired alopecia resembling pseudopelade of humans*

Pseudopelade is a primary scarring (cicatricial) alopecia of humans characterized by lymphocyte‐rich inflammation centred around the hair follicle isthmus. Lymphocyte folliculotropism is associated with isthmus apoptosis and, ultimately, follicular destruction and dermal fibrosis. In a cat, an acquired alopecia was diagnosed as pseudopelade based on the following criteria: (i) an adult‐onset, patchy to diffuse nonpruritic hair loss; (ii) an early folliculo‐destructive phase in which lymphocytes and dendritic cells accumulated in and around the follicular isthmus; and (iii) a late stage in which the lower segments of hair follicles underwent atrophy and were replaced by fibrosing tracts. Additionally, immunological investigations characterized the cytotoxic phenotype of isthmotropic lymphocytes and demonstrated the presence of circulating IgG autoantibodies specific for multiple follicular antigens. Altogether, the results of the present study suggest an immune‐mediated pathogenesis for this case of feline pseudopelade, similarly to that causing alopecia areata in humans and other mammalian species.     

Degenerative mucinotic mural folliculitis in cats

A novel form of mural folliculitis is described in seven cats. Clinically, all cats exhibited generalized alopecia with scaling or crusting that was more pronounced over the head, neck, and shoulders. The face and muzzle of all cats was unusually thickened. Six of seven cats were progressively lethargic but did not demonstrate any other consistent systemic abnormalities. Histologically, there was severe mixed inflammation of the wall of the follicular isthmus in all cats, accompanied by some follicular destruction in five cats. Sebaceous glands were not affected. All cats had variable, but often striking, follicular mucin deposition, as well as epidermal hyperkeratosis and crusting. The cause of the severe mural folliculitis was not identified, and all cats responded poorly to immunomodulating therapy. Follicular mucinosis may be a nonspecific finding, likely reflective of the follicular lymphocytic milieu, and does not always herald follicular lymphoma.     

A case of diet-related lymphocytic mural folliculitis in a cat

A 5-year-old cat developed a raised hair coat and adherent crusting lesions involving the skin of the head, dorsal neck and abdomen. Erosions were present on the lips and eyelids. The footpads were dry and scaly. Histopathology revealed infiltrative lymphocytic folliculitis, moderate lymphocytic infiltration into the epidermis and apoptotic epidermal cells. A restricted diet as the only therapy resulted in gradual resolution of the skin lesions. Despite an improvement in the dermatological condition, the cat increasingly lost all appetite and marked weight loss occurred. The cat died 4 months after presentation. Post-mortem revealed a perforated gastric ulcer and a mild to moderate inflammatory bowel disease. The clinical course of lesion resolution in this cat suggested a diet-related pathogenesis. The late finding of intestinal disease in a patient with diet-related skin disease is still interesting and needs to be evaluated by further case studies.     

Prevalence of infiltrative lymphocytic mural folliculitis in equine inflammatory skin diseases

Infiltrative lymphocytic mural folliculitis (ILMF) is a histopathological reaction pattern reported to occur in a small number of equine inflammatory dermatoses. However, the prevalence of ILMF in a variety of equine dermatoses has not been reported. Skin biopsy specimens from 250 horses with inflammatory dermatoses and from 27 horses with physically healthy skin were therefore evaluated. ILMF was present in 82% of the diseased skin specimens examined. ILMF was not seen in physically healthy skin. It appears that ILMF is frequently seen in a wide variety of equine inflammatory dermatoses and therefore is of little diagnostic significance. However, ILMF is not seen in physically healthy equine skin and the presence of lymphocytes in equine hair follicle epithelium should therefore be considered abnormal.     

Treatment of canine Alopecia X with trilostane

Sixteen Pomeranians and eight miniature poodles presenting with clinical signs of alopecia X, elevated blood concentrations of 17-hydroxyprogesterone post stimulation with adrenocorticotropic hormone and increased urinary cortisol/creatinine ratios were treated with trilostane, a competitive inhibitor of 3beta-hydroxysteroid dehydrogenase. Trilostane was given once or twice daily at a mean dose of 10.85 mg kg(-1) day(-1). Adrenal function was evaluated with a follow-up of 28 months in the Pomeranians and 33 months in the miniature poodles. Treatment with trilostane led to complete hair re-growth in 85% of the Pomeranians and in all of the miniature poodles within 4 to 8 weeks. No adverse events attributed to treatment with trilostane were recognized. The hair re-growth might have been the result of a down-regulation of adrenal steroids and/or of the noncompetitive inhibition of the oestrogen receptors at the hair follicle level.     

Idiopathic linear pustular acantholytic dermatosis in a young Brittany Spaniel dog

A unilateral, linear, papular-pustular dermatosis is described in a young Brittany Spaniel dog. The dermatosis appeared to follow Blaschko's lines and extended from the left inguinal region to the medial aspect of the left metatarsal area. The predominant histological finding was an eosinophilic and neutrophilic pustular mural folliculitis with prominent acantholysis of infundibular epithelium. There was a rapid and long-lasting (> 15 months) resolution after oral administration of methylprednisolone (1.6 mg kg(-1)).     

Morphologic characterization of specific granules in Greyhound eosinophils

BACKGROUND: "Vacuolated" eosinophils (ie, eosinophils with empty, nonstaining granules) have been described previously in normal Greyhounds. However, to our knowledge, detailed studies of granules in vacuolated and normal eosinophils in this breed have not been performed. OBJECTIVE: The objective of this prospective study was to characterize some of the morphologic, ultrastructural, and cytochemical staining features of specific (primary) granules in both normal and vacuolated eosinophils in Greyhound blood. METHODS: Morphologic features of eosinophils in Wright's- and Diff-Quik-stained peripheral blood smears from 49 Greyhounds were compared with 200 blood smears from non-Greyhound dogs. Transmission electron microscopy was done on blood from 3 Greyhounds with vacuolated eosinophils and 3 with normal eosinophil granules. Blood smears from 4 of these dogs also were stained cytochemically with alkaline phosphatase (AP), chloracetate esterase (CAE), and alpha naphthyl butyrate esterase (ANBE). The morphologic features and tinctorial properties of vacuolated and normal eosinophils were compared. RESULTS: Twenty-six Greyhounds (53%) had vacuolated eosinophils and 23 (47%) had normal granulated eosinophils in smears stained with Wright's stain. Only 1% of eosinophils were vacuolated in non-Greyhound dogs. Twenty of the 23 (85%) Greyhounds with normal granulated eosinophils on Wright's-stained smears had vacuolated eosinophils in smears stained with Diff-Quik. Ultrastructurally, no morphologic differences were observed between granules of vacuolated and normal eosinophils. Both vacuolated and normal eosinophils in Greyhounds were positive for AP and negative for CAE and ANBE, as expected for normal dogs. CONCLUSION: Vacuolated eosinophils in Greyhounds likely reflect, at least in part, differential staining properties of the specific granules with different hematologic stains. Ultrastuctural and cytochemical features of eosinophil granules were similar in normal and vacuolated eosinophils from Greyhounds.     

Antifollicular cell-mediated and humoral immunity in canine alopecia areata

Abstract A combination of cellular and humoral immunological assaults directed against follicular matrical cells is suspected to cause alopecia areata (AA) in humans. The specific aims of this study were to determine whether cytotoxic T lymphocytes (CTL) were increased in the blood and skin lesions of canine AA. Additionally, we wished to determine if circulating antifollicular antibodies were present in the dog. Finally, we aimed to investigate whether bulbar inflammation was associated with decreased matrical cell proliferation and keratinocyte apoptosis. Canine AA lesions were infiltrated by intrabulbar CTL, perifollicular helper T cells and dendritic cells. A higher percentage of CTL was present in canine AA peripheral blood compared with that of normal dogs. Tissue-fixed and circulating antifollicular IgG antibodies were detected. Reduced matrical proliferation and increased keratinocyte apoptosis was observed in inflamed hair bulbs. This study demonstrates the existence of antifollicular cell-mediated and humoral immunological responses in canine AA. Résumé— L'association d'agressions immunologiques cellulaires et humorales dirigées contre les cellules matricielles folliculaires est suspectée d'être à l'origine de l'Alopécie Areata chez l'homme. Le but de cette étude était de montrer si le nombre de lymphocytes T cytotoxiques est augmenté dans le sang et dans les lésions cutanées lors d'Alopécie Areata chez le chien. En plus, nous souhaitions montrer la présence d'anticorps circulants antifolliculaire chez le chien. Enfin, nous voulions voir si l'inflammation du bulbe pileux était associée à une diminution de la prolifération des cellules matricielles et une apoptose des kératinocytes. Lors d'Alopécie Areata chez le chien, existe une infiltration lésionnelle par des lymphocytes T cytotoxiques intrabulbaires, des lymphocytes T auxiliaires périfolliculaires et des cellules dentritiques. Le pourcentage de lymphocytes T cytotoxiques était plus important dans le sang périphérique provenant de sujets à Alopécie Areata que dans le sang d'animaux sains. Par ailleurs, des anticorps antifolliculaire (IgG) circulants et fixés dans les tissus sont détectés. Une réduction de la prolifération des cellules matricielles et une augmentation de l'apoptose des kératinocytes sont observées dans les bulbes pileux atteints. Cette étude démontre l'existence de réponses immunologiques cellulaires et humorales contre le follicule pileux dans l'Alopécie Areata du chien. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunité contre le follicule pileux dans l'Alopécie Areata du chien). Veterinary Dermatology 1996; 7 : 67–79.] Resumen Se cree que la alopecia areata (AA) en la especie humana resulta de la combinación de la acción inmunológica celular y humoral contra células de la matriz folicular. Los objetivos especificos de este estudio fueron determinar si los linfocitos T citotóxicos (CTL) se encontraban aumentados en sangre y en las lesiones cutáneas de animales con AA. Además, descamos investigar la posible presencia de anticuerpos antifoliculares circulantes en el perro. Finalmente, nos propusimos investigar si la inflamación bulbar se encontraba asociada a una disminución en la proliferación de células de la matriz y a la apoptosis de queratinocitos. Las lesiones de la AA canina mostraban infiltración por CTL intrabulbares, células T colaboradoras y células dendríticas. Se encontró un porcentaje mayor de CTL en la sangre periférica de perros con AA respecto a perros normales. Se detectaron anticuerpos IgG antifoliculares circulantes y en tejido fijado. Se observó una disminución en la proliferación matrical y un aumento en queratinocitos apoptóticos en los bulbos foliculares inflamados. Este estudio demuestra la existencia de respuestas inmunitarias antifoliculares de tipo celular y humoral en la AA canina. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunidad antifolicular en la alopecia areata canina). Veterinary Dermatology 1996; 7 : 67–79.] Zusammenfassung— Eine Kombinaton von zellulären und humoralen immunologischen Angriffen, die gegen die follikulären Matrixzellen gerichtet sind, wird als Ursache bei der Alopecia areata (AA) des Menschen vermutet. Die besondere Absicht dieser Studie war, festzustellen, ob die zytotoxischen T-Lymphozyten (CTL) im Blut und in den Hautveränderungen bei kaniner AA erhöht sind. Zusätzlich wollten wir feststellen, ob beim Hund zirkulierende antifollikuläre Antikörper vorkommen. Schließlich beabsichtigten wir, zu untersuchen, ob die bulbäre Entzündung mit verminderter Matrixzellproliferation und Keratinozytenapoptose einhergeht. Die Veränderungen bei kaniner AA wurden von intrabulbären CTL, perifollikulären T-Helferzellen und dendritischen Zellen infiltriert. Verglichen mit dem peripheren Blut gesunder Hunde kam bei kaniner AA eine höherer Prozentsatz an CTL vor. Es wurden gewebsfixierte und zirkulierende antifollikuläre IgG-Antikörper festgestellt. In entzündeten Haarbulbi wurde eine reduzierte Matrixproliferation und eine erhöhte Keratinozytenapoptose beobachtet. Diese Studie zeigt die Existenz von antifollikulären zellvermittelten und humoralen immunologischen Reaktionen bei kaniner AA. [Olivry, T., Moore, P. F., Naydan, D. K., Puget, B. J., Affolter, V. K., Kline, A. E. Antifollicular immunity in canine alopecia areata (Antifollikuläre Immunität bei der Alopecia areata des Hundes). Veterinary Dermatology 1996; 7 : 67–79.]     

An analysis of factors underlying hypotrichosis and alopecia in Irish Water Spaniels in the United Kingdom

A survey on the occurrence of dermatoses in the Irish Water spaniel (IWS) was carried out in the United Kingdom. A group of 20 dogs was selected and examined clinically. All dogs had a nonpruritic, noninflammatory, regionalized hair loss affecting the same areas of the body in males and females, although an initial cyclical pattern associated with the oestrus cycle was identified. Hormonal investigations showed features suggestive of an abnormality of steroidogenesis. Histopathology revealed features similar to canine recurrent flank alopecia (CRFA) and follicular dysplasia associated with abnormal melanization, as in colour dilution alopecia, although the clinical features did not correlate with those conditions. Dietary changes improved coat and skin quality in most of the cases in this series but the role of the diet was not investigated further. This study suggests that hair loss in IWS is influenced by dietary factors and sexual hormones. Abnormalities of the steroidogenic pathways may contribute to the severity of the condition.     

Comparative immunohistochemical characterization of canine seminomas and Sertoli cell tumors

Primary testicular tumors are the most common causes of cancer in male dogs. Overall, the majority of canine patients should be cured by testicular surgery. However, tumor markers are not well-known in veterinary medicine. We sought to determine using immunohistochemistry whether the combined human testicular tumor markers (placental alkaline phosphatase, OCT3/4, CD30, alpha-fetoprotein, inhibin-alpha, vimentin, c-KIT, and desmin) are expressed in canine seminomas and Sertoli cell tumors (SCTs). We examined 35 canine testicular tumors, 20 seminomas and 15 SCTs. c-KIT was expressed markedly in canine seminomas. Both inhibin-alpha and vimentin were expressed significantly in canine SCTs. The results of this study demonstrate differences and similarities between tumor marker expression of testicular tumors in dogs and humans. All the main markers in current routine use are discussed as well as potential useful markers for benign and malignant tumors, and tumor progression.     

Immunohistochemical characterization of intraocular metastasis of a canine transmissible venereal tumor

Little has been published on intraocular metastasis of transmissible venereal tumors (TVT) in dogs. This report presents a 4-year-old male Labrador Retriever with a previous history of subcutaneous TVT which underwent total remission after treatment with vincristine. The dog presented with clinical signs of uveitis and increased intraocular pressure (IOP) in both eyes. After enucleation of the left eye, a diagnosis of TVT was made based on morphology, histology and immunohistochemistry (IHC). IHC staining for vimentin, S-100 protein, cytokeratin and HMB45 was performed to differentiate this lesion from TVT, lymphoma, melanoma, carcinomas, neurogenic tumors and fibrosarcoma. The IHC findings supported the diagnosis of TVT for this round cell tumor.     

Detection and molecular characterization of two canine circovirus genotypes co-circulating in Vietnam

BackgroundCanine circovirus is reported in dogs in many countries, including the USA, China and Thailand. It has been detected in healthy dogs and dogs with diarrhea, hemorrhagic gastroenteritis, and vasculitis. It comprises five genotypes and is frequently found as a coinfection with canine parvovirus-2 (CPV-2).AimTo characterize canine circovirus genotypes co-circulating with CPV-2 in Vietnam.MethodPCR assessment of 81 CPV-2-positive fecal samples from Vietnamese diarrheic dogs up to seven months of age for other viral enteric pathogens, including canine bocavirus, canine adenovirus, paramyxovirus, canine coronavirus, porcine circovirus-3 and canine circovirus. In addition, eight selected full genome sequences of Vietnamese canine circovirus were analyzed and used for phylogeny.ResultsIn total 19.8% of samples were found to be positive for canine circovirus. Phylogeny revealed that the Vietnamese canine circovirus strains were clustered in two different genotypes (genotype-1 and -3). The genetic diversity among Vietnamese canine circovirus was 86.0–87.2%. The nucleotide discrepancy among both genotypes altered the deduced amino acid sequence in 14 and ten residues of the replicase and capsid proteins, respectively. Genetic recombination analysis revealed that the Vietnamese canine circovirus-6 strain has the American and Chinese canine circovirus as its major and minor parents, respectively. Only a single dog revealed triple detections of CPV-2c, Canine circovirus and canine adenovirus (1.2%).ConclusionThe co-circulation of two different genotypes of canine circovirus and CPV-2c in dogs in Vietnam has been illustrated.Clinical relevanceThe mortality rate with CPV-2 only (22%) doubled in dogs with canine circovirus and CPV-2 co-infection.     

Establishment and characterization of a canine sebaceous epithelial cell line derived from an eyelid mass

Little is known about the pathological roles of sebaceous glands in canine skin diseases, as most examinations have been conducted with cultured human sebaceous epithelial cell lines. To our knowledge, there is no available canine sebaceous epithelial cell line. The purpose of this study was to establish a canine sebaceous epithelial cell line and characterize it. An eyelid mass in a dog was surgically resected for treatment, and it was histologically diagnosed as sebaceous epithelioma. Collected tissue was conducted for culture, and the growing epithelial-like cells were passaged. The cells showed continuous proliferation for over 6 months. After 40 passages, the cells were named CMG-1. Lipid droplets in the cytoplasm of CMG-1 cells were confirmed by Oil Red O staining. As reported in studies with human sebaceous epithelial cell lines, lipogenesis in CMG-1 cells was promoted by linoleic acid, whereas transforming growth factor-β (TGF-β) suppressed it. Additionally, real-time PCR revealed that the expression levels of chemokines and cytokines, including CC chemokine ligand (CCL)-2, CCL-20, CXCL-10, Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1α, IL-1β, and IL-8, were significantly increased in CMG-1 cells following treatment with lipopolysaccharide. In conclusion, we successfully established a new canine sebaceous epithelial cell line. Our data indicated that lipogenesis and inflammatory responses were quantitatively evaluable in this cell line. CMG-1 cells could be useful for the pathological analysis of sebaceous gland diseases in dogs.     

Calculation of dose intensity and comparison of published methods using a cohort of canine T-cell lymphoma patients undergoing CHOP-based chemotherapy

Methods of calculating and reporting dose intensity (DI) of CHOP-based protocols in the veterinary literature vary. The goal of this retrospective study is to examine the prognostic significance of the average percentage of planned DI received in a cohort of canine T-cell lymphoma patients treated with a modified CHOP protocol with corresponding toxicity and efficacy data. Our data set of 40 dogs was analysed using various previously published methods for calculating DI. Median progression-free survival and overall survival were 91 and 196 days, respectively. Receiving a higher percentage of planned DI was not found to be associated with patient outcome. Outcomes remain poor for dogs with T-cell lymphoma treated with CHOP-based chemotherapy irrespective of received DI. Standard methods of DI calculation and reporting should be adopted in veterinary oncology to enable repeatable and rigorous comparisons of published chemotherapy protocols and to ascertain the potential prognostic relevance of DI in canine lymphoma patients.     

Susceptibility of canine and feline Escherichia coli and canine Staphylococcus intermedius isolates to fluoroquinolones

OBJECTIVES AND DESIGN: 1) A prospective study to determine in vitro concentrations for a range of fluoroquinolones, gentamicin and amoxycillin-clavulanate required to inhibit growth of recently collected, feline and canine Escherichia coli and canine Staphylococcus intermedius isolates. 2) A comparative retrospective study to compare the minimum inhibitory concentrations (MICs) of ciprofloxacin, enrofloxacin and amoxycillin-clavulanate for archived canine E coli and S intermedius isolates collected ten to twenty years earlier, with those for recently collected isolates. PROCEDURE: Susceptibility was assessed using disk diffusion, agar dilution susceptibility testing and Epsilometer tests (E-tests) for both recently collected and archived isolates. RESULTS: All feline E coli isolates and recently collected canine S intermedius isolates were susceptible to all fluoroquinolones. There was a statistically significant increase in the MIC range of ciprofloxacin and enrofloxacin for recently collected E coli, and in the MIC range of amoxycillin-clavulanate for recently collected S intermedius isolates compared to archived isolates. Twelve of 59 recently collected canine E coli isolates were resistant to both ciprofloxacin and enrofloxacin. Resistant canine E coli isolates were associated with complicating host or infection site factors. CONCLUSION: This is the first report comparing the MICs for all veterinary fluoroquinolones currently available in Australia for a representative sample of canine and feline E coli and canine S intermedius isolates. Importantly, this study identified 12 of 59 canine E coli isolates resistant to fluoroquinolones and identified the development of low level resistance in canine E coli to ciprofloxacin and enrofloxacin and canine S intermedius to amoxycillin-clavulanate.     

Lipomatosis of the canine parotid gland: case report with a literature review

In this report, we describe a case of lipomatosis in the left parotid gland of an eight-year-old female Shetland sheepdog and review the relevant literature. Preoperative diagnosis of lipomatosis with Tru-Cut biopsy presented difficulties in this case. The postoperative diagnosis was based on the gross appearance of a non-infiltrative, circumscribed swollen mass in the parotid gland and the histological appearance of normal adipocytes that infiltrated into the parotid gland without involving surrounding structures. Additionally, flotation of the whole parotid gland in formalin solution together with well-maintained residual lobulation and a well-maintained configuration on the cut surface were identified as subsidiary characteristics of lipomatosis of the parotid gland. Lipomatosis of the parotid gland has been rarely reported in dogs and is a poorly understood condition.     

Evaluation of a hematology analyzer with canine and feline blood

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.     

Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.