Enzyme-linked immunosorbent assays of canine apolipoproteins B-100 and A-I

An enzyme-linked immunosorbent assay (ELISA) for canine blood apolipoprotein (apo) B-100 and A-I was developed. The working range for the assay was 1.8 to 28.7 ng/well for apoB-100 and it was 50 to 410 ng/well for apoA-I. The intra- and inter-assay coefficients of variation for the assay for apoB-100 were 5.4 and 6.9%, respectively, and for apoA-I they were 5.8 and 10.6%, respectively. The average concentrations of apoB-100 and A-I in 25 beagles (males, aged 3-4 years) were 0.084 +/- 0.028 (mean +/- SD) mg/ ml and 6.29 +/- 1.55 mg/ml, respectively. The ratios of canine (C) apoB-100 to CapoA-I were 1.41 +/- 0.58%. The respective concentrations in one case of hyperlipidemia with systemic atherosclerosis were 0.454 mg/ml and 11.28 mg/ml (a ratio of 4.03%). These values were larger than those of the controls. These results suggest that the measurements of CapoB-100 and A-I concentrations by this newly developed ELISA are helpful for diagnosis of lipidosis.     

Decreases in serum apolipoprotein B-100 and A-I concentrations in cows with milk fever and downer cows

Milk fever occurring during the peripartum period has been suggested to be caused by fatty liver developed during the nonlactating stage because diseased cows have increased serum concentrations of non-esterified fatty acids (NEFA) and show hepatic lipidosis. In cows with fatty liver and related diseases such as ketosis, serum concentrations of apolipoprotein (apo) B-100 and apoA-I are decreased. The purpose of the present study was to examine whether apoB-100 and apoA-I concentrations are similarly decreased in cows with milk fever. Apolipoprotein concentrations were also measured in cows with downer syndrome, which has been suggested to be related, at least in part, to milk fever. Compared with healthy cows during early lactation, apoB-100 and apoA-I concentrations were decreased in cows with milk fever and also in downer cows. In cows with milk fever, the decreases in apoB-100 and apoA-I concentrations were associated with increased NEFA and decreased cholesterol and phospholipid concentrations. However, in downer cows, serum lipid concentration changes were not as distinct as in cows with milk fever. These results, coupled with previous findings on the decreases in apoB-100 and apoA-I concentrations of cows with fatty liver-related diseases, suggest that fatty liver is involved in the development of milk fever and partly in that of downer cow syndrome.     

Decreases of apolipoprotein B-100 and A-I concentrations and induction of haptoglobin and serum amyloid A in nonfed calves

Reduced feed intake near parturition is suggested to be one of the major causal factors for the development of fatty liver in cows, and nonfeeding has been used as an experimental model for fatty liver. In cows with fatty liver, concentrations of lipoprotein lipids and proteins are decreased. In addition, the acute-phase protein haptoglobin is induced. The purpose of the present study was to examine whether the decrease of lipoprotein concentrations and the induction of acute-phase proteins were similarly reproduced by non-feeding. Holstein female calves (n=5) were nonfed for 3 days and thereafter refed. Serum concentrations of nonesterified fatty acids and beta-hydroxybutyric acid were initially increased by the nonfeeding, and followed by decreases in concentrations of cholesteryl esters, phospholipids, apolipoprotein (apo) B-100 and apoA-I. The apoC-III concentration was not distinctly decreased. Haptoglobin and serum amyloid A were induced during the nonfeeding and refeeding process. Haptoglobin was distributed in different proportions in the high-density lipoprotein, very high-density lipoprotein and the lipoprotein-deficient fractions, whereas almost all serum amyloid A was associated with the high-density lipoprotein fraction. These results suggest that the decreases in lipoprotein concentrations and induction of acute-phase proteins found in cows with fatty liver and those with fatty liver-related diseases such as ketosis are primarily due to the reduced feed intake near parturition.     

Immunohistochemical detection of apolipoprotein B-100 and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs

Accumulation of lipids and hyalinosis in the splenic arteries of aged dogs are frequently detected by routine histopathologic examinations. The purpose of this study was to pinpoint the localization of canine apolipoprotein B-100 (CApoB-100) and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs (n = 80) through the use of immunohistochemical techniques. CApoB-100 deposits were found in the subendothelial space, extracellular matrix, and atheromatous lesions in the tunica media of the arteries in dogs > or = 6 years of age. Foamy cytoplasm of the infiltrated macrophages was also CApoB-100 immunopositive. In dogs > or = 10 years of age, almost all central arteries were CApoB-100 immunopositive. Hyaline deposits within the wall were characterized by immunopositivity against canine IgA, IgM, IgG, and albumin. Lipid accumulation in splenic arteries may be an age-related lesion and a precursor of the atheromatous plaques associated with splenic hemorrhage and infarcts later in life. In addition, deposition of immunoglobulins, probably mediated by immune complexes, may play an important role in the development of canine vascular diseases similar to human disease.     

Reduced concentrations of apolipoproteins B-100 and A-I in serum from cows with retained placenta.

The purpose of the present study was to evaluate apolipoprotein B-100 and A-I concentrations in cows with retained placenta. Animals used were cows with retained placenta alone (n = 10), those with both retained placenta and ketosis (n = 7), and controls (n = 10). Apolipoprotein B-100 concentrations at 2 to 4 d after parturition were significantly (P < 0.01) decreased in cows with retained placenta alone (mean +/- SD, 0.084 +/- 0.029 mg/ML of serum) when compared with those in control cows (0.154 +/- 0.022 mg/mL). Apolipoprotein A-I concentrations (0.713 +/- 0.177 mg/ML) were also significantly (P < 0.05) lower than those of controls (0.895 +/- 0.159 mg/mL). These decreases were more distinct for apolipoproteins B-100 (55% of controls) than A-I concentrations (80% of controls). Concentrations of apolipoprotein B-100 (0.071 +/- 0.032 mg/mL; P < 0.01) and A-I (0.708 +/- 0.189 mg/mL; P < 0.05) in the cows with both retained placenta and ketosis were also reduced, when compared with values in controls. Other than apolipoproteins, cows with retained placenta alone had significantly (P < 0.01) higher serum nonesterified fatty acids, and lower triglyceride concentrations. Significantly (P < 0.01) higher nonesterified fatty acids and lower triglyceride concentrations were similarly observed in cows with both retained placenta and ketosis.     

Immunohistochemical characterisation of the lesions of canine idiopathic pericarditis

Pericardial tissue was obtained from 14 dogs with idiopathic pericarditis, and from three dogs with pericardial effusion associated with neoplastic disease, for histopathological assessment and characterisation of infiltrating leucocytes by immunohistochemistry. The major pathological change was extensive pericardial fibrosis which was generally accompanied by a mixed inflammatory response that was of greatest intensity at the cardiac surface of the tissue. Perivascular lymphoplasmacytic aggregates were present at the pleural surface and within the fibrosed pericardium. There were no features that clearly distinguished the samples from dogs with neoplastic disease from dogs with idiopathic pericarditis. The pericardial infiltrates were dominated by MAC 387+ monocyte-macrophages and plasma cells expressing immunoglobulin (Ig)A or IgG. CD3+ T lymphocytes and major histocompatibility complex (MHC) class II+ macrophages were less common, although the perivascular aggregates were mixtures of T and B lymphocytes and a proportion of fibroblasts expressed MHC class II. There was no vascular pathology or deposition of immunoglobulin or complement within vessel walls. These findings are consistent with an immune response dominated by humoral effector mechanisms (Th2 immunity) but do not clearly support a primary immune-mediated pathogenesis for idiopathic pericarditis.     

Immunohistochemical detection of canine adenovirus in paraffin sections of liver

An avidin-biotin immunoperoxidase procedure was optimized for detection of canine adenoviral antigens in formalin-fixed, paraffin-embedded liver. Long-term stability of viral antigen was shown by successful demonstration of virus in liver tissue preserved up to six years from dogs with infectious canine hepatitis. This immunohistochemical stain was applied to sections from livers with a wide range of inflammatory lesions. Examination of sections from 53 dogs yielded five livers with small amounts of adenovirus. An additional virus-positive liver was identified from a dog with no hepatic inflammation. Although a cause and effect relationship remains to be determined, these findings suggest a possible connection between canine adenovirus and spontaneous chronic hepatitis.     

Immunohistochemical staining for S100 protein in the diagnosis of canine amelanotic melanoma

Formalin-fixed, paraffin-embedded tissue samples of canine amelanotic melanomas and normal canine tissues were studied immunohistochemically for the presence of S100 protein. Use of the avidin-biotin complex procedure demonstrated variable amounts of S100 protein in the tumor cell cytoplasm and nuclei in 26 of 31 tumors. S100 protein was not observed in some other common canine skin tumors stained by the avidin-biotin complex technique. These were a mast cell tumor, fibrosarcoma, mammary gland adenocarcinoma, histiocytoma, transmissible venereal tumor, and a thyroid gland adenocarcinoma. Among normal tissues the presence of S100 protein was demonstrated in chondrocytes in the trachea, myoepithelial cells in the breast, melanocytes in the skin, some sweat glands and ducts in the skin, stellate cells in the pituitary, and interdigitating reticulum cells in the lymph node and in Peyer's patches. These results indicate that the avidin-biotin complex procedure for demonstrating S100 protein is a useful diagnostic tool in the diagnosis of canine amelanotic melanoma.     

Induction of fatty liver in cows by ethionine administration and concomitant decreases of serum apolipoproteins B-100 and A-I concentrations.

Ethionine, an analogue of methionine, induces fatty liver in rats by inhibiting protein synthesis, including that of apolipoproteins in liver. Ethionine was administered to cows to elucidate the participation in fatty liver development of impaired triglyceride secretion from liver attributable to decreased apolipoprotein synthesis. The administration resulted in a significant increase of liver triglyceride contents. Several apolipoproteins were found to have decreased concentrations. In particular, apolipoprotein B-100 in very low-density (0.95 to 1.006 g/ml) lipoprotein and in low-density (1.006 to 1.063 g/ml) lipoprotein fractions was greatly reduced. The decreases of apolipoprotein B-100 concentrations in the 2 lipoprotein fractions were at least partly correlated to the decreased triglyceride concentrations in the respective fractions. Decreased concentrations of apolipoprotein A-I in high-density (1.063 to 1.210 g/ml) lipoprotein were also observed, although not as distinctly as with apolipoprotein B-100. Total cholesterol and phospholipid concentrations in low- and high-density lipoprotein fractions were decreased. The decrease in cholesterol was attributed to reduced concentrations of cholesteryl esters. It was suggested that the impaired lipid secretion from liver attributable to the decreased apolipoprotein concentrations has a role in ethionine-induced fatty liver of cows.     

高能低蛋白饲粮对蛋鸡肝脏apo B-100基因mRNA表达的影响

以海蓝褐蛋鸡为试验对象,探讨高能低蛋白日粮对蛋鸡肝脏载脂蛋白B-100mRNA表达关系的影响。选300日龄海蓝褐蛋鸡60只,随机分为2组,每组30只,分别饲喂正常饲粮(对照组,代谢能:2.75MJ/kg,粗蛋白:17%)、高能低蛋白饲粮(试验组,代谢能:2.99MJ/kg,粗蛋白:12.9%)。于试验0、30、60d每组选取体质量相近的试验蛋鸡6只,取肝脏样品,应用半定量逆转录聚合酶链式反应技术测定不同时期apoB-100mRNA在肝脏中的表达水平;结果显示:试验组载脂蛋白B-100mRNA表达在试验60d显著低于对照组(0.01P≤0.05)。说明高能低蛋白日粮能导致蛋鸡肝脏载脂蛋白B-100mRNA表达水平下降。     

Immunohistochemical detection of growth hormone (GH) in canine hepatoid gland tumors

The aim of this study was to detect immunohistochemically means growth hormone (GH) in 24 hepatoid gland adenomas and 5 hepatoid gland carcinomas and to compare the difference of immunoreactivity between types of tumors. The tumors were classified according to the WHO standards. Tissue sections which were prepared from formalin-fixed, paraffin wax-embedded tissues from 25 male and 4 female dogs were carried out immunostaining using polyclonal primary anti-hGH and EnVision method. Of 24 hepatoid gland adenomas (perianal gland adenomas) 23 (95.8%) were positive. All 5 hepatoid gland carcinomas (perianal gland carcinomas) were positive. No statistically significant differences in percentage of labelled cells between malignant and benign tumors were seen. The present demonstration of GH in hepatoid gland tumors adds new data on GH in extra-pituitary tissues and hormon-dependent tumors.     

Immunohistochemical detection of intermediate filament proteins in formalin fixed normal and neoplastic canine tissues.

Normal and well differentiated neoplastic canine tissues were immunohistochemically stained for keratin, vimentin and desmin intermediate filament proteins using commercially available monoclonal antibodies. Keratin was detected in 56 of 57 carcinomas, vimentin in 59 of 62 sarcomas and desmin in three of four muscle cell tumors. Most normal and neoplastic tissues expressed only one type of intermediate filament; exceptions were one hemangiosarcoma and one pulmonary carcinoma in which there was coexpression of vimentin and keratin proteins. Since immunohistochemical detection of intermediate filaments has tissue-specific distribution in the majority of well differentiated canine neoplasms, these stains may be useful in the differential diagnosis of anaplastic canine tumors. However, the monoclonal antibodies to cytokeratin which were tested in this study failed to detect intermediate filaments in liver, pancreas and salivary glands which suggests that these antibodies may also be unable to detect epithelial tumors derived from these tissues. In addition, in nine neoplasms, the normal tissues adjacent to neoplastic cells failed to stain for the intermediate filament normally expressed. When this occurs, evaluation of intermediate filament expression is invalid for the determination of tissue of origin of the neoplastic cells.     

Immunohistochemical detection of P-glycoprotein (clone C494) in canine mammary gland tumours

Elevated levels of P-glycoprotein have been reported in multidrug-resistant tumours in both humans and dogs. In the present study, we investigated the expression of P-glycoprotein in 57 canine mammary gland tumours, 10 mammary gland hyperplasia and seven normal mammary glands by immunohistochemistry. Tissue sections were incubated with an anti-Pgp monoclonal antibody and visualized with En Vision-DAB polymer. Normal and hyperplastic mammary tissues were negative or showed slight cytoplasmic immunoreactivity. Neoplastic cells in benign mammary tumours showed diffuse cytoplasmic staining, in contrast to malignant tumours that showed mainly a membranous staining pattern for Pgp (C494). We observed statistically significant differences among all the different groups of tissues analysed except for benign tumours versus hyperplasia (P = 0.221). Receiver-operating characteristic analysis showed that the best cut-off point to differentiate the threshold to differentiate negative from positive tissue samples was 18.40% of immunostained cells. These results provide a first indication that routine evaluation of Pgp expression in canine mammary gland tumours, taking into consideration a cut-off point for positivity, may be useful for selecting cases for chemotherapy.     

Immunohistochemical detection of p27 and p21 proteins in canine hair follicle and epidermal neoplasms

Trichoblastomas, trichoepitheliomas, and squamous cell carcinomas in the skin of dogs were analysed by immunohistochemistry for the nuclear expression of p27, p21 and proliferating cell nuclear antigen (PCNA). High levels of p27 were present in trichoepitheliomas and trichoblastomas compared with squamous cell carcinomas. Detectable p21 was found in trichoepitheliomas and squamous cell carcinomas, but trichoblastomas had low level of p21 nuclear reactivity. Low levels of PCNA were detected in trichoepitheliomas and trichoblastomas compared with squamous cell carcinomas. The results suggested that nuclear p27 acts as a cyclin-dependent kinase (CDK) inhibitor in trichoepitheliomas and trichoblastomas. Nuclear p21 expression is involved in the induction of epithelial differentiation and seems to be unrelated to CDK inhibition.     

Immunohistochemical investigation of canine episcleritis

OBJECTIVES: To identify macrophages, B cells and T cells in archived canine episcleral biopsies and to correlate these findings with the clinical presentation and therapeutic outcome. PROCEDURES: Archived formalin-fixed biopsies were immunohistochemically labeled for CD18, CD79a, and CD3 to identify macrophages, B cells and T cells, respectively. Slides were digitally photographed and positive cells were manually counted. Signalment, duration of illness, affected eye(s), treatment, and therapeutic outcome were reviewed for each dog. Dogs were divided into groups based on clinical presentation (unilateral episcleritis, bilateral episcleritis or nodular granulomatous episclerokeratitis (NGE). RESULTS: Twenty-four cases were evaluated. There were 19 episcleritis (13 unilateral, six bilateral) and five NGE cases. The mean age for clinical manifestations of unilateral episcleritis was 6.8 years, bilateral episcleritis was 8.7 years, and NGE was 3.8 years. The Cocker Spaniel was over-represented in the episcleritis groups. All NGE cases were Collies. Approximately 50% of the unilateral episcleritis cases resolved and did not require long-term therapy. Almost all cases of bilateral episcleritis and NGE required continuous medical therapy to maintain remission. There was a significantly higher percentage of B lymphocytes in biopsies from lesions that required ongoing medical therapy to maintain lesion remission than in the lesions that resolved, and for which medications were discontinued (P = 0.0471). CONCLUSIONS: The prognosis for resolution of NGE and bilateral episcleritis without long-term medical therapy is poor. There is a significant difference in the inflammatory cell population in episcleritis that resolved with medical therapy vs. episcleritis that required ongoing medical therapy.     

Immunohistochemical detection of uroplakin III,cytokeratin 7, and cytokeratin 20 in canine urothelial tumors

Immunohistochemistry for uroplakin III (UP III), cytokeratin 7 (CK 7), and cytokeratin 20 (CK 20) using commercially available antibodies was done in normal canine urinary bladder and 72 canine urinary bladder tumors that had been fixed in formalin and embedded in paraffin. Prolonged fixation (3-28 days) did not significantly alter the immunostaining for UP III. There was moderate reduction in the intensity for CK 7 and CK 20 after 1 week of fixation. UP III was detected in superficial (umbrella) cells and some intermediate cells of the normal urinary bladder, 7 of 7 transitional cell papillomas (TCPs), 50 of 55 transitional cell carcinomas (TCCs), and 4 of 5 metastatic TCCs. Staining was typically outlined in the plasma membrane, but diffuse or focal cytoplasmic staining was also observed. Intracytoplasmic lumina were usually positive for UP III. One squamous cell carcinoma of the bladder, 4 nonepithelial bladder tumors, and 285 nonurothelial tumors from different nonurinary locations were negative for UP III. CK 7 was detected in 7 of 7 TCPs, 53 of 54 TCCs, and 5 of 5 metastatic TCCs. The staining for CK 7 was diffuse cytoplasmic. CK 20 was detected in 1 of 7 TCPs, 37 of 54 TCCs, and 1 of 5 metastatic TCCs. The staining with CK 20 was cytoplasmic and weaker than with antibodies to UP III or CK 7. There was concurrent expression of UP III, CK 7, and CK 20 in 36 of 54 TCCs. UP III is a specific and sensitive marker for canine transitional epithelial (urothelial) neoplasms, detecting 91% of TCCs. Negative results may be observed with anaplastic tumors.     

Immunohistochemical demonstration of keratin in canine neuroepithelioma

Morphological features and immunoreactivity for cytokeratin (CK), glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) of three canine neuroepitheliomas and three canine ependymomas were investigated. Neuroepitheliomas were in three German shepherds as intradural-extramedullary solitary masses, with spinal cord displacement between T10 and L2. Histologically, they contained tubules and acini, lined by epithelial cells with focal squamous metaplasia, rosette-like structures, and polygonal to spindle-shaped cells between tubules. Acini were empty or filled with a homogeneous, eosinophilic periodic acid-Schiff (PAS)-positive material. Mitotic indices varied from low to moderate. Ependymomas occurred in the third (two cases) and fourth ventricle in adult boxers. Histologically, they were composed of cells with an ill-defined, scant amphophilic cytoplasm, with a central round euchromatic nucleus; cells formed pseudorosettes, with a central fibro-vascular stroma. Neuroepitheliomas stained for CK, but ependymomas did not. Both failed to stain for GFAP, NSE, or phosphotungstic acid hematoxylin (PTAH). Thus, antibodies to cytokeratin are useful to distinguish neuroepitheliomas from ependymomas.     

Immunohistochemical detection of arteriolar hyperplasia in canine liver biopsy samples using the claudin-5 antibody

Claudins are key tight junctional proteins between adjacent epithelial, mesothelial or endothelial cells, which are responsible for the permeability of the paracellular space. This paper describes that the endothelial cells of normal hepatic arterioles, portal venules and portal lymphatics as well as the endothelium of sinusoids from dogs show strong membranous claudin-5 cross-reactivity. In 25 liver biopsy samples taken from dogs with portal vein hypoperfusion, an increased number of arterioles was detected in the portal areas (PAs) by the use of humanised anti-claudin-5 antibody. The increased number of hyperplastic hepatic arterioles per PA was 5-6, 8-12 and 15-20 in the case of small, medium-sized and large PAs, respectively. It is suggested that the claudin-5 marker can improve the detection of hepatic arteriolar proliferation in the PAs of liver samples.