Reovirus type-2-triggered autoimmune cholangitis in extrahepatic bile ducts of weanling DBA/1J mice.

BACKGROUND: Reovirus is a proposed cause of infantile biliary atresia. However, mechanistic insight regarding Reo-2 as a potential cholangiotropic virus is lacking. Furthermore, it is unknown whether Reo-2 infection can induce autoimmune-mediated bile duct injury. METHODS: Lesions of bile ducts in newborn DBA/1J mice infected with Reo-2 were analyzed immunopathologically. RESULTS: Damage to biliary epithelia occurs after Reo-2 infection. In addition, nonsuppurative cholangitis with fibrosis in extrahepatic (especially septal) bile ducts developed following complete viral clearance from the liver. At the inflamed ducts, major histocompatibility complex class I expressing((+)) and FAS(+) cholangiocytes were associated with FAS ligand(+) lymphocytes and tumor necrosis factor-α(+) mononuclear cells (macrophages and lymphocytes). These cholangiocytes were apoptotic and necrotic. Moreover, affected ducts were infiltrated by CD3(+), CD4(+), CD8(+), IFN-γ(+), and FAS(+) lymphocytes. Analysis of blood from Reo-2-infected mice revealed that they developed anticholangiocyte cytoplasm antibodies and had high serum IFN-γ concentration. Notably, there was no increase in Foxp3(+) lymphocytes at inflamed ducts, lymph nodes, and thymi. CONCLUSION: Reo-2 infection induced T-helper cell type 1-dependent injury to bile ducts in weanling mice. The lesions observed in mice may be analogous to those associated with human infantile biliary atresia, which are caused by an autoimmune-mediated process.

Feline infectious peritonitis virus with a large deletion in the 5'-terminal region of the spike gene retains its virulence for cats.

In this study, the Japanese strain of type I feline infectious peritonitis virus (FIPV), C3663, was found to have a large deletion of 735 bp within the gene encoding the spike (S) protein, with a deduced loss of 245 aa of the N-terminal region of the S protein. This deletion is similar to that observed in porcine respiratory coronavirus (PRCoV) when compared to transmissible gastroenteritis virus, which correlates with reduced virulence. By analogy to PRCoV, we expected that the pathogenicity of C3663 may be attenuated in cats. However, two of four cats inoculated with C3663 died of FIP, and a third C3663-inoculated cat showed FIP lesions at 91 days after challenge. These results indicate that the 5'-terminal region of the S gene is not essential for the development of FIP.

Reovirus type-2 infection in newborn DBA/1J mice reduces the development of late allergic asthma.

The aim of the present study was to determine whether or not the development of a helper T (Th) 1 response induced by Reovirus type-2 (Reo-2) infection would protect against the development of Th2-mediated late allergic asthma. This hypothesis was examined by infecting one day old neonatal DB A/1J mice with Reo-2 in an ovalbumin (OVA)-induced late asthma model. Compared with the controls (either infected or uninfected mice with or without OVA sensitization and/or OVA challenge), Reo-2 infection lessened the magnitude of the subsequent allergic Th2-mediated late asthma. In infected mice with allergic late asthma, there was decreased infiltration of interleukin (IL)-4(+), IL-5(+), IL-13(+) and very late antigen (VLA)-4(+) lymphocytes, and eotaxin-2(+) and VLA-4(+) eosinophils, in both bronchial and bronchiolar lesions. Also the expression of vascular cell adhesion molecule (VCAM)-1 and eotaxin-2 on vascular endothelial cells was reduced. Moreover, the systemic production of IL-4, IL-5, tumour necrosis factor-α and OVA-specific IgE was reduced, whereas systemic IFN-γ production was increased. In addition, there was no increase in IFN-α production. Thus the present study suggests that systemic Reo-2 infection at birth may reduce the development of subsequent late allergic asthma by the induction of a Th1 response. Therefore the potential suppressive mechanism(s) that might be induced by Reo-2 infection in newborn mice and their effects on the development of late allergic asthma are discussed.

Mediating Effect of Upper Limb Use on the Relationship Between Upper Limb Performance and Activities of Daily Living: A Longitudinal Mediation Analysis.

INTRODUCTION: Upper limb performance, frequency of upper limb use, and psychological factors are associated with activities of daily living (ADLs) after stroke. We performed a mediation analysis to investigate how the frequency of upper limb use and some psychological factors mediate the relationship between upper limb performance and ADLs. METHODS: Twenty-two patients with stroke were included in this longitudinal study. We utilized the frequency of upper limb use outcome measures (amount of use and quality of motion of the motor activity log), psychological factors outcome measures (General Self-Efficacy Scale), upper limb performance outcome measures (Fugl-Meyer Assessment (FMA)), and ADLs outcome measure (Functional Independence Measure (FIM) motor subscale (M)). Mediation analysis with a bootstrap sampling procedure was used to assess the indirect effects. RESULTS: Mediation analysis showed that the FMA, as measured by the FIM (M), had significant indirect effects on the amount of use (95% bootstrapped confidence interval (CI): 0.36-2.42) and quality of motion (95% bootstrapped CI: 0.06-1.88). The relationship between upper limb performance and ADLs was mediated by the frequency of upper limb use. CONCLUSION: Our findings suggest that improving the frequency of upper limb use may accelerate post-stroke recovery.

Dysfunction of lacrimal and salivary glands in Sjögren's syndrome: nonimmunologic injury in preinflammatory phase and mouse model.

Sjögren's syndrome (SjS) is a chronic autoimmune disorder characterized by dry eyes and dry mouth due to dacryoadenitis and sialoadenitis with SS-A/Ro and/or SS-B/La autoantibodies in genetically predisposed individuals. Destruction of lacrimal and salivary glands by autoimmune reactions may lead to clinical manifestation. However, the mechanisms behind the decreased volume of secretions in tears and saliva are complex and are not fully understood. Exocrine gland dysfunction may precede autoimmunity (acquired immunity) or represent a process independent from inflammation in the pathogenesis of SjS. The preceded functional and morphologic changes of those tissues by nonimmunologic injury before the development of inflammation at the sites of target organs have been implicated. This paper focuses on the several factors and components relating to glandular dysfunction and morphologic changes by nonimmunologic injury during the preinflammatory phase in mouse model, including the factors which link between innate immunity and adaptive immunity.

An atherogenic lipid profile with low serum paraoxonase-1 activity during nematode infection in rats.

BACKGROUND: Inflammation and oxidative stress are associated with cardiovascular diseases and underlying atherosclerosis. The high density lipoprotein (HDL)-associated paraoxonase-1 (PON1) enzyme is known to be involved in the protection of serum lipids from such oxidation. Nonetheless, the disturbances of lipid profile during nematode-infected model have not yet been studied. Therefore, we aimed to explore the effects of Nippostrongylus brasiliensis infection in male Wistar rats, a model of human gastrointestinal nematode infections, on hepatic PON1 synthesis and the levels of lipid parameters. MATERIALS AND METHODS: Nippostrongylus brasiliensis-infected rats fed standard and high-fat diets. Serum paraoxonase and arylesterase activities were measured on day 0, 2, 4, 7, and 14 post-infection (PI). Hepatic PONs and pro-inflammatory cytokines mRNA expression levels were evaluated in a standard diet-fed groups, and the disturbances in lipid profile as well as the levels of thiobarbituric acid reactive species (TBARS) and oxidized-LDL (Ox-LDL) were measured in high-fat diet-fed groups. RESULTS: We found that N. brasiliensis-infected rats fed the standard diet show a significant reduction in serum PON1 activity and down-regulation of hepatic PON1 mRNA expression as well as up-regulation of hepatic IL-1ß, IL-ß receptor (R), TNF-α, and TNFR1 mRNA expressions in association with hepatic recruitments of Kupffer cells and neutrohils. In the presence of the high-fat diet, N. brasiliensis infection increases serum triglycerides, total cholesterol, LDL/VLDL, TBARS and Ox-LDL as well as decreases serum HDL coinciding with a maximum serum PON1 reduction. CONCLUSIONS: Nippostrongylus brasiliensis infection can induce atherogenic lipid profile and reduce serum PON1 activity.

Therapeutic strategies for SLE involving cytokines: mechanism-oriented therapies especially IFN-gamma targeting gene therapy.

Systemic lupus erythematosus (SLE: lupus) is a chronic complicated autoimmune disease and pathogenesis is still unclear. However, key cytokines have been recognized. Interferon (IFN)-γ and also IFNalpha/beta are of particular importance. Depending on the concept that lupus is a helper T(Th)1 disease and that dendritic cells (DCs) determine the direction of lupus, balance shift of Th1/Th2 and immunogenic/tolerogenic DCs is reviewed for therapy. (IFN)-gamma- and IFN-alpha/beta-targeted (gene) therapies are introduced. These consist of Th1/Th2 balance shift and elimination of IFN-gamma and IFN-gamma-related cytokines such as (interleukin)IL-12 and IL-18. Other approaches include suppression of immunocompetent cells, normalization of abnormal T-cell function, costimulation blockade, B lymphocyte stimulator (Blys) blockade, and suppression of nephritic kidney inflammation. Moreover, balance shift of IFN-alpha/beta and tumor necrosis factor (TNF)-alpha together with regulatory T(Treg) cells are briefly introduced. Clinical application will be discussed.

Systemic treatment of anti-CD4CD25 T cell monoclonal antibody exacerbates sialoadenitis in submandibular glands during the early life in lupus-prone female NZB x NZWF mice.

BACKGROUND: The maintenance mechanisms of peripheral tolerance by CD4(+)CD25(+) T cells before the development of sialoadenitis in secondary Sjögren's syndrome (sSS) are not well understood. The aim of the present study is to examine the effect of reduction of CD4(+)CD25(+) T cells on the development of sialoadenitis during the early life in female NZB x NZWF(1) (B/WF(1)) mice, a model for human sSS. METHODS: Female B/WF(1) mice at 3 days after birth were treated with either anti-mouse CD4(+)CD25(+) T cells rat IgG(1) monoclonal antibody (mAb) or Rat IgG(1)(control). At 25 weeks of age, autoantibodies against nucleus and cytoplasm of ductal epithelial and myoepithelial cells, and histpathology of submandibular glands were examined in the mAb-treated and control groups. Also the development of anti-Ro/SS-A antibodies was examined until 25 weeks of age in both groups. RESULTS: The mAb-treated group showed severe lesions with the development of autoantibodies compared to the control group. CONCLUSIONS: The present results suggest that peripheral CD4(+)CD25(+) T cells may, at least in part, contribute to down-regulate the development of sialoadenitis in submandibular glands of lupus-prone female B/WF(1) mice during their early life.

Enhanced contact hypersensitivity by delayed T-helper 2 response in BALB/c mice.

T-helper (Th) 1/Th2 balance determines the direction of contact hypersensitivity (CHS). To clarify the immunopathogenesis of contact dermatitis, 2,4-dinitrofluorobenzene (DNFB)-induced CHS reaction was compared between the BALB/c and C57BL/6 mice. The two strains were sensitized with DNFB systemically and challenged with DNFB locally. The CHS reaction in BALB/c mice was intense compared with that in C57BL/6 mice at 24 and 48 hours post-DNFB challenge. The dermal lesions were characterized by infiltration of lymphocytes, eosinophils, neutrophils, and macrophages including CD4+ and CD8+ T cells, and interleukin (IL)-4-producing(+) and interferon (IFN)-gamma+ cells in BALB/c mice. In C57BL/6 mice, the composition of inflammatory cells was same as those in BALB/c mice except for eosinophils, CD4+ T cells, and IL-4+ cells. There was no increase in the number of mast cells in the two strains. Local and systemic productions of IL-4 and IFN-gamma in BALB/c mice were higher than those in C57BL/6 mice. Although blood IgE values increased in BALB/c mice, but not in C57BL/6 mice, at 48 hours postchallenge, its value was low. The delayed Th2-like response together with Th1-like response in BALB/c mice may induce strong CHS reaction compared with C57BL/6 mice, which may dominantly develop Th1-like reaction. Moreover, mast cell and IgE do not appear to be involved in delayed CHS.

Use of formalin-fixed paraffin-embedded tissue and single-strand conformation polymorphism analysis for polymerase chain reaction of antigen receptor rearrangements in dogs.

PCR for antigen receptor gene rearrangement analysis (PARR) is a new diagnostic method for lymphoid neoplasia. In PARR using formalin-fixed paraffin-embedded tissues (PARR-FFPE), control DNA amplification was successful in only three of five samples. The formalin fixation times of the three samples were shorter than those of the others. Analysis of the formalin fixation time and DNA amplification controls suggested that a formalin fixation time of less than one week is appropriate. Additionally, application of single strand conformation polymorphism (SSCP) for PARR provided clearer results than conventional PARR in 16 unfixed tissues and three FFPE tissues. These results show that PARR-FFPE is viable for tissues with an appropriate formalin fixation time and that application of FFPE and SSCP for PARR are useful for diagnosis and retrospective study of canine lymphoid neoplasia.

Superficial necrolytic dermatitis associated with extrapancreatic glucagonoma in a dog.

An 11-year-old Shih Tzu presented with crusting and erythema, mainly on the abdomen and the root of the tail. Based on histopathological findings, blood examinations and necropsy findings, the condition was diagnosed as superficial necrolytic dermatitis associated with a glucagon-secreting extrapancreatic neuroendocrine tumour. Gross necropsy revealed tumour invasion into the spleen, liver, adrenal glands and mesenteric lymph nodes. Immunohistochemical analysis of the neoplastic cells revealed that the tumour was a glucagonoma, consistent with earlier findings of persistent glucagonaemia and hypoaminoacidaemia.

Acute urticaria[corrected]-like lesions in allergen-unexposed cutaneous tissues in a mouse model of late allergic rhinitis.

The mechanisms of distant manifestation after a local allergic reaction are largely unknown. This study examined the development of cutaneous lesions in a mouse model of late allergic rhinitis (LAR). BALB/c mice were sensitized by ovalbumin (OVA) intraperitoneally two times (on days 0 and 10) and challenged by OVA intranasally on day 14. Four days after OVA challenge, nasal and cutaneous lesions including helper T (Th) responses, expression of adhesion molecules and presence of OVA and IgE were examined, and compared with unsensitized and unchallenged (control) mice. Compared with the control group, the LAR group developed LAR characterized by infiltration of lymphocytes and eosinophils, increased IgE values and increased productions of IL-4 and IL-5, but not IFN-gamma. A dominant infiltration of eosinophils and increase in mast cells, attachment of eosinophils to endothelium, intense expression of VCAM-1 on endothelium in venules and VLA-4 expression on eosinophils and mast cells were recognized in the cutaneous tissues. There were no differences in the expression of ICAM-1 on vascular endothelium and LFA-1 on infiltrated leucocytes between the two groups. CLA expression on lymphocytes was not detected, and the binding of OVA and IgE on mast cells and eosinophils was found in the cutaneous lesions in the LAR group, but not in the control group. This study suggests that acute urticaria[corrected]-like lesions in OVA-unexposed cutaneous tissues may be induced by immediate allergic reaction due to the systemic development of Th2-type response in a mouse model of LAR.

Ultrastructure of myoepithelial cells as a target cell in sialoadenitis of submandibular glands of lupus-prone female NZBxNZWF1 mice.

The changes of myoepithelial cells of sialoadenitis in submandibular glands in lupus-prone female NZB x NZWF1 (B/WF1) mice, a model for human secondary Sjögren's syndrome (sSS), were examined ultrastructurally. Inflammatory foci consisting of mainly lymphoid cells (lymphocytes and plasma cells) in the interlobular interstitium began to develop from 18 weeks of ages, and those were found within acini from the age of 25 weeks. These were paralleled with the production of anti-double-stranded deoxyribonucleic acid and anti-Ro/SS-A antibodies with age. Infiltrated lymphoid cells consisted of CD4+ T cells and Ig+ (or IgG2a+) cells. Electron microscopy revealed destruction of myoepithelial cells with lysis of basement membranes contacted with either lymphocytes or plasma cells. These led to the destruction (degeneration and necrosis) of the epithelium in striated and intercalated ducts and acinar epithelium. Further destruction of those cells occurred by the invasion of lymphocytes into the epithelial layers. Small numbers of apoptotic myoepithelium and duct epithelium from the age of 25 to 36 weeks and an increase of those cells in survived mice at 44 weeks of age were observed. The present study suggests that the myoepithelium may be one of the target cells and that the destruction of myoepithelial cells by infiltrated lymphoid cells may precede the destruction of acinar ducts and epithelium in sialoadenitis in sSS.

Systemic administration of olygodeoxynucleotides with CpG motifs at priming phase reduces local Th2 response and late allergic rhinitis in BALB/c mice.

Oligodeoxynucleotides (ODN) with CpG motifs (CpG ODN) induce T helper (Th)1-type reaction. We aimed to evaluate the therapeutic effect of CpG ODN in the development of late allergic rhinitis induced by ovalbumin (OVA), which is one of Th2 diseaes, in BALB/c mice. Effects of a single dose of synthetic CpG-ODN (50 microg) intraperitoneally (i.p.) at the priming phase (on day 0) by OVA on the development of late eosinophilic rhinitis at respiratory areas were compared to the control mice treated with its vehicle (ODN without CpG motifs; 50 microg). Animals were again sensitized by OVA (on day 10) i.p., and 4 days after second sensitization animals were challenged by OVA intranasally (on day 14). Four days after challenge, eosinophilic reactions, nasal lesions and local cytokine values were examined. Compared to the control group, the CpG ODN-administration increased production of OVA-specific Th1 cytokine (interferon-gamma) and decreased productions of ovalubmin-specific Th2 cytokines [interleukin (IL)-5 and IL-13] in nasal cavity fluids, supernatants of splenocytes and/or sera. Also, eosinophilia and increased total IgE values were decreased in mice treated with the CpG ODN compared to the control group. Moreover, nasal lesions with infiltration of eosinophils were prominently reduced by the CpG ODN-treatment compared to the control mice. The present study suggests that the systemic administration of CpG ODN at the priming phase may reduce local OVA-specific Th2 responses, resulting in decreased nasal pathology in the late allergic eosinophilic rhinitis.

Detection of proteolytic cleavages of diabetes-associated protein IA-2 beta in the pancreas and the brain using novel anti-IA-2 beta monoclonal antibodies.

Insulinoma-associated protein (IA)-2 beta, an inactive member of the protein-tyrosine phosphatase (PTP) family, is a major autoantigen in type-1 diabetes mellitus. IA-2 beta exists mainly in a 60-kDa form, and is frequently located in the dense-core secretory vesicles of pancreatic beta cells. As IA-2 beta gene-deficient mice exhibit impaired insulin secretions, IA-2 beta is probably involved in insulin secretions. In the present study, we characterized the major forms of IA-2 beta in the brain and pancreas of normal and non-obese diabetic (NOD) mice. Novel monoclonal antibodies (mAbs) against IA-2 beta revealed that this brain protein was of multiple compositions incorporating the 60-, 64-, 67- and 71-kDa forms, which were designated as IA-2 beta 60, IA-2 beta 64, IA-2 beta 67 and IA-2 beta 71, respectively. On the contrary, only the 60-kDa isoform of IA-2 beta was expressed in the mouse pancreas and in the mouse pancreatic beta cell line, MIN6. Sequence analyses revealed that IA-2 beta 60, IA-2 beta 64 and IA-2 beta 71 (brain-derived immunoprecipitated IA-2 beta isoforms) contained alternative NH2- termini starting from Glu489, Ala464, and Ser414, respectively, while IA-2 beta 60 (an MIN6-derived immunoprecipitated IA-2 beta isoform) contained those from Glu489. Consistent with the lack of an NH2-terminal region of IA-2 beta, the isoforms were recognized by their respective mAbs characterized with different epitope regions. Furthermore, Western blotting and immunohistochemistry demonstrated that NOD mice expressed similar isoforms present in the brains and pancreatic islets of C57BL/6J, BALC/CA and ICR mice, accordingly. Taken together, these results suggest that IA-2 beta undergoes at least three distinct proteolytic cleavages.

Maxillofacial rhabdomyosarcoma in the canine maxillofacial area.

Three dogs had a diagnosis of maxillofacial rhabdomyosarcoma. These dogs were treated with surgery and/or radiotherapy, and had poor clinical responses. The tumor tissues in all three cases were observed around the upper premolar teeth with ulcerative lesions and CT examinations in each case revealed extensive bony involvement into the maxilla. Two cases were subjected to surgical excision of the tissues, followed by an external radiation therapy. The other case was only treated with palliative radiation. Outcomes of the treatment of all the cases were quite poor because of the invasive and refractory nature of the tumor cells, leading to the local recurrence and lung metastasis early in the clinical course. All dogs died within two months of the first admission.

Co-localization of chondromodulin-I (ChM-I) and bone morphogenetic protein-6 (BMP-6) in myoepithelial cells of canine mammary tumors.

To compare the roles of chondromodulin-I (ChM-I) and bone morphogenetic protein-6 (BMP-6) in ectopic mesenchymal tissue formation in canine mammary gland tumors, 33 tumors and 2 normal mammary glands were examined. Immunohistochemical analysis revealed co-expression of ChM-I and BMP-6 in canine mammary tumors. In mixed tumors, newly formed woven bone with ossified cartilage matrix was observed in 4/9 cases. The osteoblasts lining the woven bone showed moderate immunoreactivity to ChM-I and BMP-6. Almost all chondrocytes and proliferative myoepithelial cells within the basement membrane showed intense immunoreactivity to both, and the myoepithelial cells adjacent to the mature cartilage showed the most intense immunoreactivity. The immunoreactivity to ChM-I and BMP-6 of the interstitial myoepithelial cells in the myxomatous stroma varied in each focus of mixed tumors. Similar findings were found in complex adenomas. In simple adenomas, hyperplasic myoepithelial cells within the basement membrane showed moderate immunoreactivity to both markers. Western blot analysis detected a 25 kDa band of ChM-I in fresh tissue samples from three mixed tumors. Our results support the hypothesis that proliferating myoepithelial cells with ChM-I and BMP-6 expression play important roles in mesenchymal metaplasia in canine mammary tumors.

Ultrastructure of goblet-cell metaplasia from Clara cell in the allergic asthmatic airway inflammation in a mouse model of asthma in vivo.

Mucus overproduction from goblet cells, a characteristic feature of the allergic asthmatic inflammation induced by ovalbumin (OVA) in mice, was examined morphologically. In OVA-untreated (normal) mice, there were no goblet cells in intrapulmonary bronchus and bronchiole. However, goblet cells with or without hyperplasia in the mucosa of inflamed bronchus-bronchiole were recognized in the allergic asthmatic mice. The non-ciliated epithelium containing electron lucent granules (mucus) showed many similarities to Clara cells, which have characteristic secretory granules and many mitochondria, except for the less-developed smooth endoplasmic reticulum seen in normal mice. Ciliated Clara cells with or without mucus were rarely recognized. In addition, mucus was found in neither ciliated nor basal epithelium. The present study suggests that goblet-cell metaplasia in the bronchus and bronchiole of inflamed mucosa may be derived, at least in part, from Clara cells.

Evaluation of cytokine messenger RNA expression in peripheral blood mononuclear cells from dogs with canine demodicosis.

Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-gamma as well as TNF-alpha in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-beta mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-beta mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-beta mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-beta mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.

Detection of the anti-P53 antibodies in dogs with tumors.

To detect the anti-P53 antibodies of dogs with tumors, a GST-recombinant canine (rc) P53 fusion protein was expressed and purified. Immunoblot analysis was performed using this GST-rcP53 fusion protein as an antigen and serum samples from dogs suffering from tumors as primary antibodies. Out of 16 serum samples obtained from various tumor cases, four samples showed reaction with GST-rcP53. In contrast, serum from other 12 dogs with tumors, four dogs with non-neoplastic diseases and two control healthy dogs (as controls) did not show any reaction with GST-rcP53 in immunoblotting. The p53 gene mutation and the P53 protein expression were examined, using the tumor tissues to explore the relationship between the existence of the GST-rcP53 bands, gene mutations of p53 and the accumulation of P53 protein. One case, which showed a clear GST-rcP53 band, had a point mutation of the p53 cDNA and showed nuclear accumulation of P53 protein. These results suggest that the anti-P53 antibodies are also produced in tumor dogs with p53 gene mutations.