百田 豊

A 12-year-old female Shih-Tzu with hyperadrenocorticism and hypothyroidism developed concurrent refractory generalized demodicosis that did not respond to doramectin treatment. Although amitraz treatment was effective, the dog developed severe diabetes, which resulted in the cessation of amitraz and trilostane. Attempts to control the diabetes were unsuccessful, and its hyperadrenocorticism was left untreated, leading to the recurrence of demodicosis. However, demodicosis went into complete remission with a single dose of fluralaner. Transient erythematous papules appeared on the trunk three days after the administration of fluralaner, but no other adverse reactions were noted. We demonstrated that fluralaner is a potent treatment for demodicosis, and skin eruptions are possible after the first dose of the drug.

Background - Hydration is one parameter of skin barrier function. The Skicon-200EX (R) and Corneometer CM825 (R) are hygrometers used to measure skin hydration in humans based on different measurement methods. The ASA-MX3 (R) is a hygrometer used to obtain measurements at haired skin sites in humans. Hypothesis/Objectives - To validate three hygrometers to measure skin dryness in dogs. Animals - Six clinically normal research dogs. Methods - In vivo evaluation of three hygrometers for three different skin types was performed. Measurement of hydration was performed at five different regional sites. Dry and moist skin were induced by treatment with a sorbent and petrolatum, respectively, and measurements were collected for 120 min. Skin sites with three different hair lengths were evaluated to determine whether hair would interfere with hydration measurements. Results - All three hygrometers obtained measurements at the nonhaired skin sites, except the ASA-MX3 (R) hygrometer at the ear site. At the dry skin sites the Skicon-200EX (R) hygrometer detected a significant decrease of water content for longer than the other devices. At the moist skin sites the Corneometer CM825 (R) and ASA-MX3 (R) hygrometers showed a significant increase in water content. The ASA-MX3 (R) hygrometer was the only device that could obtain measurements at sites with hair. Conclusions and clinical importance - The Skicon-200EX (R) hygrometer was the most sensitive for detecting skin dryness, whereas the Corneometer CM825 (R) and ASA-MX3 (R) hygrometers detected an emollient effect. Only the ASA-MX3 (R) could provide measurements at the haired sites. This study may assist in the selection of a hygrometer based on the purpose of use.

Background - Application of herbal paste and oil to a dog's coat and body before rinsing (often combining with shampooing) is a cosmetic therapy available in Japan. It is highly appreciated by users, who claim that the treatment makes the coat shinier, improves volume and eliminates tangles. However, there has been no scientific evaluation of such treatments. Hypothesis/Objectives - Improvement of hair condition is derived from oils such as sebum and conditioning oils because chemicals are not used. Therefore, we examined nonpolar lipids (the primary lipids in dog hair) and the botanical oils used in this therapy. Animals - Hair samples were obtained from six beagle dogs. Methods - Groups were based on different combinations of the following processes: rinsing, shampooing, herbal therapy and herbal therapy with oil extract. Analysis of lipids was performed by high performance thin layer chromatography. Results - The processes of shampooing and herbal therapy were associated with an equivalent reduction in cholesterol ester and triglyceride (TG). However, hair treated by herbal therapy combined with oil extract had an almost three-fold higher TG content, even after shampooing. Conclusions and clinical importance - This study demonstrated that the herbal therapy was able to coat hair samples with TG that was not removed with rinsing. Further investigation is required to evaluate the possible benefits of the application of botanical products containing lipids, such as TG, on hair coat quality in dogs.

Proliferative and necrotising otitis externa (PNOE) is a very rare disease affecting the ear canals and concave pinnae of kittens. This report describes a 5-month-old cat with PNOE. Histopathological examination confirmed the diagnosis. Treatment was initiated with local injection of methylprednisolone acetate into the lesions. The cat was subsequently treated with clobetasol propionate cream, a potent topical glucocorticoid ointment. The cat showed marked improvement. While topical treatment with tacrolimus, an immunosuppressive agent, is reported to be an effective therapy, to the best of our knowledge, this is the first report to treat PNOE with local corticosteroid therapy.

Background - A closed chamber evaporimeter is suitable for measuring transepidermal water loss (TEWL) in cats because of the compact device size, tolerance to sudden movement and short measuring time. TEWL is a representative parameter for skin barrier dysfunction, which is one of the clinical signs of atopic dermatitis in humans and dogs. Measurement of feline TEWL has been reported, but applicability of this parameter has not been validated. Hypothesis/Objectives - The aims of this study were to determine if tape stripping is a valid experimental model in cats for studying TEWL and to determine if a closed chambered system is a suitable measurement tool for cats. Animals - Ten clinically normal cats. Methods - In order to evaluate variation of the measured values, TEWL was measured at the right and left side of the three clipped regions (axillae, lateral thigh and groin). Subsequently, TEWL was measured using sequential tape stripping of the stratum corneum as a model of acute barrier disruption. Results - The variations between both sides of the three regions showed no significant difference. Sequential tape stripping was associated with increasing values for TEWL. Conclusions and clinical importance - Feline TEWL was shown to reflect changes in the skin barrier in an experimental model using a closed chamber system and has the potential for evaluating skin barrier function in cats with skin diseases.

A 12-year-old female American shorthair cat presented with a one-month history of hematuria and general lethargy. Abdominal ultrasonography revealed complete thickening of the left uterine wall. At a diagnostic laparotomy, a large mass arising from the left uterine horn was discovered, and ovariohysterectomy was performed. Histological diagnosis revealed a T-cell high-grade lymphoma of the uterus. After the ovariohysterectomy, the patient achieved complete remission and was maintained by combination chemotherapy from 14 days after surgery. However, relapse occurred in the urinary bladder wall on day 287, and the patient died of postrenal acute renal failure on day 310. This is the first report of a feline case of primary uterine lymphoma that was treated with ovariohysterectomy followed by systemic chemotherapy.

BackgroundCorneocyte surface area (CSA) is as established parameter for skin barrier function in humans. Measurement of canine CSA has been previously reported but has not been validated. Hypothesis/ObjectivesThe aim of this study was to evaluate the validity of CSA as a barrier function parameter in dogs. AnimalsSix clinically normal beagle dogs. MethodsCSA was measured and compared with transepidermal water loss (TEWL) using sequential tape stripping of the stratum corneum as a model of acute barrier disruption. Then, CSA and TEWL were measured at four anatomical sites (groin, lower back, nasal bridge and pinna). The correlation between the two indices was also evaluated. ResultsFrom the results of sequential tape stripping, CSA values gradually decreased with increasing number of tape strippings. The CSA values were inversely correlated with the TEWL ones. The two indices at different sites were variable and were strongly correlated. Conclusions and clinical importanceCanine CSA was demonstrated to be a useful parameter for the canine skin barrier function. The results from the anatomical sites imply that the cephalic sites (nasal bridge and pinna) were lower than others in skin barrier function. Resume ContexteLa CSA (corneocyte surface area) est un parametre etabli pour la fonction de barriere cutanee chez l'homme. La mesure de CSA canine a ete precedemment rapportee mais n'a pas ete validee. Hypotheses/ObjectifsLa but de cette etude etait d'evaluer la validation du CSA en tant que parametre de la fonction barriere chez le chien. SujetsSix chiens beagles cliniquement sains. MethodesLa CSA a ete mesuree et comparee avec la perte hydrique transepidermique (TEWL) utilisant des TS (tape stripping) sequentiels sur le stratum corneum comme modele de rupture de barriere epidermique. Ainsi, la CSA et le TEWL ont ete mesure sur quatre sites anatomiques differents (truffe, chanfrein, bas du dos et pavillons auriculaires). La correlation entre les deux indices a aussi ete evaluee. ResultatsA partir des resultats des TS sequentiels, les valeurs de CSA diminuaient graduellement avec l'augmentation du nombre de TS. Les valeurs de CSA etaient inversement correlees avec celles de TEWL. Les deux indices aux differents sites etaient variables et etaient fortement correles. Conclusions et importance cliniqueLa CSA canine est un parametre important de la fonction barriere cutanee. Les resultats des differents sites anatomiques montrent que la face (chanfrein et pavillons) a une fonction barriere cutanee plus faible que les autres sites. Resumen Introduccionel area superficial de corneocitos (CSA) es un parametro establecido de la funcion de barrera de la piel en humanos. La medicion de la CSA canina ha sido previamente publicada, pero no ha sido validada. Hipotesis/Objetivosel proposito de este estudio fue evaluar la validez de la CSA como un parametro de la funcion de barrera de la piel en perros. Animalesseis perros de raza Beagle clinicamente normales. Metodosse evaluo la CSA y se comparo con la perdida transite de agua (TEWL) utilizando exfoliacion secuencial con cinta adhesiva del estrato corneo como modelo de disrupcion aguda de barrera de la piel. Despues, se midieron CSA y TWEL en cuatro localizaciones atomicas (zona inguinal, zona dorsolumbar, zona dorsal del hocico, y pabellon auricular). La correlacion entre los dos indices fue evaluada. Resultadosde los resultados de la exfoliacion secuencial con cinta adhesiva, los valores de CSA disminuyeron gradualmente tras el incremento del numero de colecciones con cinta adhesiva. Los valores de CSA estuvieron inversamente correlacionados con los valores de TEWL. Los dos indices en las diferentes locaciones fueron variables pero estuvieron altamente relacionados. Conclusion e importancia clinicala CSA es un parametro util para el estudio de la funcion de barrera de la piel canina. Los resultados en las diferentes locaciones anatomicas implican que las zonas cefalicas (zona del hocico y de los pabellones auriculares) fueron mas debiles que las otras localizaciones en cuanto a la funcion de barrera. Zusammenfassung HintergrundBeim Menschen stellt die Korneozyten Oberflache (CSA) einen etablierten Parameter fur die Funktion der Hautbarriere dar. Messungen des caninen CSA sind bereits publiziert, aber nicht validiert, worden. Hypothese/ZieleDas Ziel dieser Studie war eine Evaluierung der Validitat von CSA als Hautbarriereparameter bei Hunden. TiereSechs klinisch normale Beagles. MethodenCSA wurde gemessen und mittels sequentiellem Tape Stripping des Stratum corneum als Modell fur akute Barrierestorung mit transepidermalem Wasserverlust (TEWL) verglichen. Dann wurden CSA und TEWL an vier anatomischen Lokalisationen (Inguinalgegend, kaudaler Rucken, Nasenrucken und Pinnae) gemessen. Die Korrelation zwischen den beiden Messwerten wurde ebenfalls evaluiert. ErgebnisseDie Ergebnisse des sequentiellen Tape Strippings ergaben, dass die CSA Werte bei zunehmender Anzahl der Tape Strippings graduell weniger wurden. Die CSA Werte waren mit den TEWL Werten invers korreliert. Die beiden Messwerte an den verschiedenen Korperstellen waren stark korreliert. Schlussfolgerungen und klinische BedeutungEs konnte gezeigt werden, dass Canines CSA ein wertvoller Parameter zur Evaluierung der Hautbarrierefunktion des Hundes darstellen kann. Die Ergebnisse der unterschiedlichen anatomischen Lokalisationen zeigen, dass die Schadellokalisationen (Nasenrucken und Pinnae) in Bezug auf die Hautbarrierefunktion niedriger lagen als die anderen Werte.

Nesfatin-1 is an anorexic peptide derived from a predursor, nucleobindin-2 (NUCB2), which is distributed in various organs, coexists with ghrelin in the gastric X/A-like cells and closely relates to an appetite control in rodents and humans. Nesfatin-1 may be a significant factor addressing the satiety also in veterinary medicine, however, there are few reports about nesfatin-1 in dogs. In the present study, we detected canine NUCB2/nesfatin-1 mRNA in various tissues, especially abundant in pancreas, gastrointestinal tracts, testis and cerebellum. We examined circulating nesfatin-1 concentrations and NUCB2/nesfatin-1 mRNA expressions in upper gastrointestinal tracts (gastric corpus, pyloric antrum and duodenum) in dogs fed on different types of diets. Plasma nesfatin-1 concentrations in the dogs were approximately 4 ng/ml and they did not change after feeding through the study, however, NUCB2/nesfatin-1 mRNA expressions in pyloric antrum were 1.84-fold higher in the dogs fed on a High fiber/High protein diet (P<0.001), 1.48-fold higher in the dogs fed on a High fat/Low protein diet (P<0.05) and 1.02-fold higher in the dogs fed on a Low fat/High carbohydrate diet (not significant) comparing to those on a control diet. It was concluded that High fiber/High protein and High fat/Low protein diets increased NUCB2/nesfatin-1 production in canine gastrointestinal tracts. These results may set the stage for further investigations of canine NUCB2/nesfatin-1, which may relate to satiety effects in dogs.

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4-5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.

Hyperadrenocorticism (HAC) is a common endocrine disorder in dogs, in which excess glucocorticoid causes insulin resistance. Disturbance of insulin action may be caused by multiple factors, including transcriptional modulation of insulin signal molecules which lie downstream of insulin binding to insulin receptors. In this study, gene expressions of insulin signal molecules were examined using neutrophils of the HAC dogs (the untreated dogs and the dogs which had been treated with trilostane). Insulin receptor substrate (IRS)-1, IRS-2, phosphatidylinositol 3-kinase (P13-K), protein kinase B/Akt kinase (Akt)-2 and protein kinase C (PKC)-lambda were analyzed in the HAC dogs and compared with those from normal dogs. The IRS-1 gene expressions decreased by 37% and 35% of the control dogs in the untreated and treated groups, respectively. The IRS-2 gene expressions decreased by 61% and 72%, the P13-K gene expressions decreased by 47% and 55%, and the Akt-2 gene expressions decreased by 45% and 56% of the control dogs, similarly. Collectively, gene expressions of insulin signal molecules are suppressed in the HAC dogs, which may partially contribute to the induction of insulin resistance.

Regenerative therapy has begun to be clinically applied in humans and dogs to treat neurological disorders, such as spinal cord injury (SCI). Here, we show the therapeutic potential of transplantation of cultured canine bone marrow stromal cells (BMSCs) into mice with SCI. Canine BMSC transplantation therapy was performed, immediately after the spinal cord was injured. Canine BMSC therapy enhanced functional recovery of the hind limbs in mice with SCI. Nestin-positive cells were observed only in the lesion of mice with SCI that received BMSCs. These results suggest that canine BMSCs promote functional recovery in mice with SCI and that migration of nestin-positive cells may contribute to the efficacy of the BMSC treatment.

Background - The measurement of transepidermal water loss (TEWL) is one of the parameters that can be used to assess skin barrier function. The variability and reliability of TEWL measurements in dogs have been controversial, and the hair coat has been considered as one of the factors that may cause variation of TEWL values. Hypothesis/Objectives - The aims of the study were to establish a suitable procedure for measuring feline TEWL, to evaluate the influence of hair coat on TEWL measurements and to assess variations of TEWL at different anatomical sites. Methods - Transepidermal water loss was measured using a closed-chamber evaporimeter, the VapoMeter (R). We compared three adjacent sites in the groin area of 10 clinically normal, domestic short hair cats. One site was unclipped, the second was trimmed with scissors and the third was shaved using electric clippers. Values of TEWL were obtained for 48 h after trimming with scissors and clippers. Five sites were clipped (upper back, lumbar back, lateral thigh, axillae and groin), and the TEWL was measured. Results - The mean and SD of TEWL values of the clipper-trimmed site were the smallest, followed in order by the site trimmed with scissors and the unclipped site. The TEWL values were statistically constant in the clipper-trimmed site, while the values in the unclipped sites were not. There was no statistically significant difference in TEWL values between all of the anatomical sites except for the axillae. Conclusions and clinical importance - Hair clipping of sites with electric clippers is recommended for TEWL measurement in cats.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the C-C family chemokines, which mobilizes monocytes from bone marrow to the site of inflammation. To evaluate the clinical utility of canine MCP-1 as a blood test item, we measured serum MCP-1 concentrations in normal and ill dogs. Reference interval of canine MCP-1 was established as 115.6-176.9 pg/ml. Serum MCP-1 concentrations increased in the dogs affected with neoplastic (518.0 ± 84.8 pg/ml), inflammatory (257.0 ± 42.5 pg/ml) or other diseases (360.3 ± 45.2 pg/ml). The results showed high sensitivity of MCP-1 to detect neoplasia and inflammation. Moreover, MCP-1 increased in some cases in which C-reactive protein didn't increase. MCP-1 might be helpful as a screening blood test marker for detection of neoplasia and inflammation in dogs.

血液生化学検査は，獣医臨床において疾病の診断，治療効果の判定に重要な役割を果たしている。生化学測定値の評価においては基準値の設定が重要であり，ヒトでは年齢や性差など患者の属性によって基準範囲が異なる項目も知られている。一方，獣医領域においてはこれらに加え，品種によって基準値が異なる可能性も考えられるが，充分なデータがないのが現状である。本研究では，健常と考えられる犬から採取しドライケミストリ法で測定した検査データ（n=3,303）を基に，主な20 項目について犬種別に比較検討した。その結果，ほとんどの項目では顕著な犬種差はみられなかったが，クレアチニン（CRE）では小型犬が低く大型犬が高めに分布するなど，いくつかの項目で犬種差が認められた。臨床現場で犬種別の基準範囲は常用する必要は無いが，CRE 等において境界付近の値を示す症例では，診断の一助となる状況もあると思われた。

A novel canine tumor cell line designated as the CMS-C cell line was established from pleomorphic rhabdomyosarcoma (RMS) raised in the prostate gland of a 14-year-old intact male mixed-breed dog. CMS-C cells displayed the same immunohistochemical characteristics (positive for vimentin and desmin and negative for cytokeratin and smooth muscle actin) as the original tumor cells and express myoD1 and UCP3, known as striated muscle-specific molecules, as shown by RT-PCR assay. Therefore, the established CMS-C cell line appears to be of rhabdomyoblast cell origin. The CMS-C cell line established from pleomorphic RMS will be a useful tool for further studies about canine RMS.

Artificial pancreas technology, involving "closed-loop" controls with real-time blood glucose monitoring, has been increasing in reliability as its potential for clinical use and application grows. One such device, based on this technology, is the STG-22 (Nikkiso Co., Ltd., Tokyo, Japan) artificial pancreas apparatus. In order to assess the reliability and accuracy of the device for measuring blood glucose, it is important to compare its readings to those obtained using a 'gold standard' method, such as the hexokinase method. Therefore, in the present study, canine blood [glucose] measurements using the STG-22 were compared to those obtained using a previously established commercial reagent, Quickauto-Neo GLU-HK. Furthermore, two different sample types (whole blood versus plasma constituent) were compared to determine which sample type results in more accurate and optimal readings with the STG-22. Given that the STG-22 was not primarily designed for canine blood samples, results for canine blood samples were not accurate. Measurements performed by the STG-22 with whole blood were significantly lower than reference [glucose] counterparts. Alternatively, an opposite trend was observed with plasma measurements that were significantly higher. A conversion format using the following formula, Hexokinase [glucose] = STG-22 [glucose] x 1.407 + 1.532, was observed with canine samples in our study.

The effect of fluconazole (Fez) on the cyclosporine (CsA) dosage was investigated in renal transplanted dogs receiving CsA-based immunosuppressive therapy. Initially, CsA was administered orally twice daily to raise the blood trough level between 400 and 600 ng/ml. After the addition of Fez, the CsA dosage was adjusted to maintain its therapeutic blood concentration. Fcz significantly decreased CsA dosage in both normal and renal transplanted dogs, but a higher dosage of CsA was needed in renal transplanted dogs. In conclusion, Fcz decreases required CsA dosage and thereby reduces the cost of immunosuppressive therapy in canine renal transplantation. (C) 2010 Elsevier Ltd. All rights reserved.

Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin alpha 3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin beta 1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin beta 1 in immunostaining as well as in flow cytometry and augmented integrin beta 1-dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin beta 1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin beta 1, which is associated with integrin beta 1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.

Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.

A member of tetraspanin CD151 is a scaffold protein of laminin-binding integrins and it plays an important role in stable interaction between cells and basement membrane. Although the upregulation of CD151 in tumor cells is thought to accelerate tumor invasion and metastasis, detailed pathological investigation on CD151 and its association with integrins has not been well documented, yet. In the present study, we showed that the expression levels of CD151 and its associated integrin subunits in epidermal carcinoma cell HSC5 were higher than those in immortalized epidermal cell HaCaT. By the stimulation of epidermal growth factor, CD151 was dissociated from cell surface and dispersed in the cytoplasm, and alpha 3 beta 1 integrin was concomitantly internalized. To understand the significance of CD151 in tumor cell dynamics, CD151 in HSC5 was knocked down (HSC5(CD151-)), and the expression of integrin subunits and matrix metalloproteinases (MMPs) were investigated. In HSC5(CD151-), striking morphological alteration on Matrigel and laminin, and cytoskeletal rearrangements were demonstrated. alpha 3 beta 1 integrin was internalized in part, and alpha 6 beta 4 integrin was re-distributed from basal site to cell periphery. Quantitative RT-PCR, Western blot and zymography revealed that the expression levels of MMP2, MMP7 and MMP9 were markedly downregulated in HSC5(CD151-). Immunoprecipitation assay demonstrated that MMP7 was co-immunoprecipitated with CD151. In double stainings, MMP7 was colocalized with CD151 at the leading edge of lamellipodia under migratory status. These results elucidated the importance of CD151 as one of the key molecules for integrin-dependent carcinoma-stroma interaction. It is indicated that CD151 might contribute not only to cell stabilization by associating with adhesion complexes but also to cell migration by inducing integrins re-localization and MMPs production.

Adenosine deaminase (ADA), an enzyme involved in purine metabolism, has been shown to be of clinical importance in several diseases in humans. To investigate whether ADA is of any clinical significance in cats, plasma adenosine deaminase (P-ADA) and T cell adenosine deaminase (T-ADA) activities were measured in feline immunodeficiency virus (FIV) negative and positive cats. The AIDS-related complex (ARC) group showed a significant elevation in P-ADA activity compared to the asymptomatic carrier (AC), and FIV-negative groups (P<0.005). T-ADA activity was significantly elevated in FIV-positive cats compared to the FIV-negative group (P<0.05) and this elevation was attributed to the increase in the ARC group (P<0.01). A correlation was found between P-ADA and TADA activities in the FIV-negative group. T-ADA activity and CD4(+) cell number showed a strong negative correlation in FIV-positive cats (P<0.0005). CD4(+) cell numbers were significantly reduced in the ARC group compared to the healthy controls (P<0.005). Our results showed that T-ADA is increased in FIV-positive cats during the ARC stage. These results also suggest that ADA may be an indicator of T cell activation in the ARC stage of FIV infection.

Blepharochalasis is a rare condition characterized by recurrent episodes of eyelid edema lead to an atrophic eyelid skin with fine wrinkles and peculiar bronze discoloration. A 32-year-old female presented with loose and redundant skin of the bilateral eyelids. We diagnosed her disease as blepharochalasis by clinical features and by disappearance of elastic fibers from the dermis in the biopsied specimen. Because elastic fibers diminish in the late phase of blepharochalasis, we performed RT-PCR to analyze the mRNA expression of elastin, a major component of elastic fiber. Elastin mRNA expression in the patient's cultured fibroblasts had not decreased compared with that in the control fibroblasts. This result suggests that environmental factors or other matrix components of elastic fibers may be involved in the loss of elastic fiber.

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

The laminin alpha4 chain, a component of laminin-8/9, is expressed in basement membranes of endothelial cells, the peripheral nerves, and muscle fibers. The localization and functions of laminin alpha4 chain in the skin have not been elucidated. By immunostaining with specific antibodies, we demonstrate here that the alpha4 chain is located in the basement membrane zones of blood vessels and is also associated with fibroblast-like cells in the dermis. Western blot showed that cultured fibroblasts secreted a laminin trimer containing the alpha4 chain. We have also focused on the cell adhesion activities of the human laminin alpha4 LG4 module since the corresponding LG4 module of laminin alpha3 was previously identified as active for cell adhesion. Recombinant human alpha4 LG4 was active for heparin-dependent fibroblast adhesion. Screening assays with 19 synthetic peptides covering the entire alpha4 LG4 module identified three peptides (HA4G82: TLFLAHGRLVYM; HA4G83: LVYMFNVGHKKL; and HA4G90: TEATWKIKGPIYL) as active sites for heparin- and heparan sulfate-dependent cell adhesion. Serine-substituted peptides demonstrated that two basic residues, His and Arg, within HA4G82 were essential for cell adhesion activity. The cell surface heparan sulfate proteoglycans (HSPGs), syndecan-2, -4, and glypican were stably expressed in 293T cells to estimate whether they function as cell adhesion receptors. 293T cells overexpressing syndecan-2 or -4 bound to recombinant alpha4 LG4 and to HA4G82, but parental or glypican-1-overexpressing 293T cells did not. Therefore, syndecan-2 and -4 could mediate cell adhesion to the laminin a4 LG4 module. Our study suggests that the laminin alpha4 LG4 module may play an important role in cell adhesion and/or vessel wall formation in the skin by interacting with syndecan-2 and/or -4.

The LG4 module of the laminin alpha3 chain (alpha3 LG4), a component of epithelial-specific laminin-5, has cell attachment activity and binds syndecan (Utani, A., Nomizu, M., Matsuura, H., Kato, K., Kobayashi, T., Takeda, U., Aota, S., Nielsen, P. K., and Shinkai, H. (2001) J. Biol. Chem. 276, 28779-28788). Here, we show that recombinant alpha3 LG4 and a 19-mer synthetic peptide (A3G756) within alpha3 LG4 active for syndecan binding increased the expression of matrix metalloproteinase-1 (MMP-1) in keratinocytes and fibroblasts. This induction was inhibited by heparin and required de novo synthesis of proteins. In keratinocytes, A3G756 up-regulated interleukin (IL)-1beta and MMP-1 expression and an IL-1 receptor antagonist thoroughly inhibited A3G756-mediated induction of MMP-1. A3G756 also activated p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-related kinase (Erk). Studies with specific inhibitors of MAPKs showed that p38 MAPK activation was necessary for both IL-1beta and MMP-1 induction, but Erk activation was required only for MMP-1 induction. In fibroblasts, IL-1 receptor antagonist did not block A3G756-mediated induction of MMP-1. These results indicated that induction of MMP-1 by alpha3 LG4 is mediated through the IL-1beta autocrine loop in keratinocytes but the mechanism of the induction in fibroblasts is different. Our study suggests that the laminin alpha3 LG4 module may play an important role in tissue remodeling by inducing MMP-1 expression during wound healing.