Expression and localization of BCRP,MRP1 and MRP2 in intestines,liver and kidney in horse

Tydén, E., Bj?rnstr?m, H., Tjälve, H., Larsson, P. Expression and localization of BCRP, MRP1 and MRP2 in intestines, liver and kidney in horse. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01140.x. The gene and protein expression and the cellular localization of the ABC transport proteins breast cancer resistance protein (BCRP), multidrug resistance‐associated protein 1 (MRP1) and multidrug resistance‐associated protein 2 (MRP2) have been examined in the intestines, liver and kidney in horse. High gene and protein expression of BCRP and MRP2 were found in the small intestines, with cellular localization in the apical membranes of the enterocytes. In the liver, MRP2 was present in the bile canalicular membranes of the hepatocytes, whereas BCRP was localized in the cytoplasm of hepatocytes in the peripheral parts of the liver lobuli. In the kidney both BCRP and MRP2 were predominantly present in the distal tubuli and in the loops of Henle. In most tissues, the gene and protein expression of MRP1 were much lower than for BCRP and MRP2. Immunostaining of MRP1 was detectable only in the intestines and with localization in the cytoplasm of enterocytes in the caecum and colon and in the cells of serous acini of Brunner’s glands in the duodenum and the upper jejunum. The latter cells were also stained for BCRP, but not for MRP2. Many drugs used in horse are substrates for one or more of the ABC transport proteins. These transporters may therefore have important functions for oral bioavailability, distribution and excretion of substrate compounds in horse.     

Primary liver disease in the horse

The clinical signs of liver disease are highly variable and non-specific. Irrespective of the cause or the duration of liver disease, more specific clinical signs, e.g. hepatic encephalopathy, become apparent in the advanced stages of the disease. Due to the non-specific clinical signs, the possible diagnosis of liver disease is frequently not taken into consideration. However, measurement of the plasma or serum concentrations of total bile acids and gamma glutamyl transferase (gamma GT) may provide valuable diagnostic information. The specific diagnosis can be confirmed by ultrasound examination of the liver and histological examination of a liver biopsy specimen. The most frequently documented liver diseases are acute hepatic necrosis, chronic hepatitis caused by pyrrolizidine alkaloids, cholelithiasis, and haemochromatosis. Immediate treatment with antibiotics and polyionic fluids, in association with supportive nutritional care, is usually necessary to maintain the horse until sufficient liver regeneration occurs to provide adequate function. The use of corticosteroids is contraindicated, except for patients with chronic active hepatitis. In some cases, e.g. portosystemic shunts or cholelithiasis, surgical intervention is indicated. Liver disease may be more common than is currently appreciated since little accurate information on the prevalence or incidence is available.     

Multiple forms of alkaline phosphatase in horse bone,liver, kidney,duodenum, caecum and serum separated by agarose gel electrophoresis with and without neuraminidase pretreatment

Horse bone, liver, duodenum, caecum and kidney alkaline phosphatases were separated by a commercial agarose gel electrophoresis method with and without neuraminidase pretreatment, following the manufacturer's directions. Tissue extracts were obtained in saline solution and ALP extracted from cell membranes by the butanol method. Electrophoresis was performed using a TRIS/barbital/sodium barbital buffer with detergent, pH 8.6 to 9.0, at 250 V for 30 minutes. Bone, liver and kidney untreated extracts showed two ALP bands each, but with different relative migration (compared to albumin migration). When they were preincubated with neuraminidase, the two bone bands showed a marked decrease in their migration, followed by the kidney ALP bands and the most anodic band of liver Both intestinal untreated extracts showed three bands but with different mobilities. After preincubation with neuraminidase, the three bands of caecum mucosa decreased in their migration, and the most anodic duodenum band disappeared, overlapping the second one. When tissue extracts were incubated with wheat germ-lectin (WGL), 74.5% of bone extract ALP and 67.2% of caecum extract ALP precipitated, which demonstrated that the ALP band of both tissues have similar groups in the carbohydrate side chains. Horse serum showed two electrophoretic bands, which increased to three bands when treated with neuraminidase. ALP from hepatocytes was the dominant isoform, followed by a caecum band. Because the electrophoretic mobilities of some of the tissue bands studied were identical, the neuraminidase agarose electrophoretic method appeared to be a satisfactory alternative to separate them.     

Stimulation by phytohaemagglutinin of peripheral blood lymphocytes from horse,pig, sheep and man

Optimal conditions for stimulation by phytohaemagglutinin (PHA) were established for equine, porcine, ovine and human lymphocytes in MEMS medium. Optimal thymidine concentration was determined for assay of cell transformation. With all species tested horse serum gave highest thymidine incorporation. Homologous serum was not more appropriate for lymphocytes of man, pig and sheep. Optimal stimulation was achieved at 20, 0.5–5, 5, and 10–40 μg PHA per 10⁶ cells for human, equine, porcine and ovine lymphocytes, respectively.     

Differential gene expression of CYP3A isoforms in equine liver and intestines

Tydén, E., Löfgren, M., Pegolo, S., Capolongo, F., Tjälve, H., Larsson, P. Differential gene expression of CYP3A isoforms in equine liver and intestines. J. vet. Pharmacol. Therap.  35 , 588–595. Recently, seven CYP3A isoforms – CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 – have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin‐isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K_m for the LIPA substrate in the liver and the intestine, while the maximum velocity (V_max) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses.     

Agammaglobulinemia in a horse with evidence of functional T lymphocytes.

Agammaglobulinemia was diagnosed in a 1-year-old Thoroughbred horse on the basis of the following observations: (1) absence of serum immunoglobulins M, A, and G(T); (2) small amounts of serum immunoglobulin G (16 mg/100 ml); (3) absence of specific antibody in the serum of the horse following immunization and challenge exposure to 2 antigens; (4) absence of plasma cells, primary follicles, and germinal centers in a lymph node removed after antigenic stimulation; (5) absence of "natural" serum antibodies to rabbit-erythrocytes which were easily detectable in age-matched control horse serums; and (6) increased susceptibility to infections. There was evidence of functional cell-mediated immunity which included a skin response to injected phytolectins, skin response to antigen challenge following sensitization, and in vitro proliferative response of lymph node cells to phytohemagglutinin. An intact cell-mediated immune response was also supported by the observation that the horse lived to 17 months of age without antibody production, whereas horses with an absence of both antibody production and cell-mediated immunity (combined immunodeficiency) die by 4 months of age without immunologic intervention. The known features of agammaglobulinemia in this horse are similar to those in sex-linked agammaglobulinemia in persons and are unique among the immunodeficiences described in other animals.     

Copper, zinc and manganese concentrations in equine liver, kidney and plasma

Five groups of horses were fed different diets of known trace mineral concentration for a minimum of six months. Copper (Cu), zinc (Zn) and manganese (Mn) concentrations were measured in livers of 125 yearling horses and kidneys of 81 yearling horses as an assessment of trace mineral status. Plasma Cu and Zn determinations were made for all horses.

Mean hepatic Cu concentrations of horses fed diets containing 6.9 to 15.2 mg Cu/kg dry matter (DM) feed were 0.27 to 0.33 μmol/g DM tissue. Plasma Cu concentrations ranged between 22.8 to 28.3 μmol/L. There was no simple mathematical relationship between plasma and hepatic Cu concentrations. Mean hepatic Zn concentrations in horses fed diets containing 25.6 to 52.2 mg Zn/kg DM feed were determined to be between 2.75 to 2.91 μmol/g DM tissue. Mean plasma Zn concentrations in groups of horses were between 11.7 to 13.5 μmol/L. Plasma Zn concentrations were not indicative of hepatic Zn concentration. Hepatic Mn concentrations ranged between 0.13 and 0.14 μmol/g DM tissue.

Renal Zn concentrations ranged between 1.55 to 1.63 μmol/g DM tissue and did not differ with diet. Mean renal Mn concentrations were 0.09 μmol/g DM tissue for all groups of horses. Renal Cu concentrations ranged from 0.36 to 0.47 μmol/g DM tissue and differed with diet.

     

Antibiotic and sulfonamide agents in bob veal calf muscle, liver, and kidney

During the fiscal year 1988, USDA-FSIS detected 3,095 antimicrobial violations in bob veal calves, using the calf antibiotic and sulfonamide test. Of the 3,095 carcass submissions involved, 945 were tested further to identify the causative agents. The results of tests on the available kidney, liver, and muscle specimens are reported. Kidney specimens yielded a specific agent most often (71.2%), with neomycin (42.6%) being cited most among agents found in kidneys. Neomycin was found less frequently in liver (4.5%) and muscle (0.2%). Among all tissues, unidentified microbial inhibitors were either the largest or second largest category found (kidney, 10.5%; liver, 27.1%; muscle, 7.8%), and no other agent exceeded 7.0% (streptomycin in kidney). The proportion of liver and muscle specimens that had unidentified microbial inhibitors is particularly important because the next most common classes were streptomycin in liver at 5.5% and sulfamethazine in muscle at 2%. The frequency of unidentified microbial inhibitors may justify the addition of tests to the FSIS battery for identification of agents. Not all tissues were tested for sulfonamides, hence these agents are likely to have been underreported. Less than 10% of the muscle specimens evaluated yielded an agent, suggesting most calf antibiotic and sulfonamide test-positive carcasses may have been safe with regard to residues in meat, although organs might have been adulterated. Specimens for verification were not selected completely randomly from the population of all calf antibiotic and sulfonamide test-positive animals and calves selected for testing were not chosen strictly by random sampling; therefore, extrapolation of the contents of this report to the bob veal calf industry must be done with caution.     

Intermittent hypoglycemia in a horse with anaplastic carcinoma of the kidney

Clinically apparent hypoglycemia is rare in adult horses. Hypoglycemia is a well-recognized paraneoplastic syndrome in humans and dogs with non-insulin-secreting tumors and may occur in horses as well. Hypoglycemia associated with non-insulin-secreting tumors is believed to result from production of an abnormal form of insulin-like growth factor II. Neoplasia should be considered in the differential diagnosis for adult horses with hypoglycemia.     

铜鱼肠道、肝胰脏对四种蛋白质饲料的离体消化率测定

采用体外消化法研究了铜鱼(Coreiusheterodon)对鱼粉、豆粕、菜粕和棉粕4种饲料蛋白质的离体消化能力。试验结果表明:①铜鱼对鱼粉、豆粕、菜粕和棉粕的干物质离体总消化率为菜粕50.18%>鱼粉50.13%>豆粕49.11%>棉粕32.90%,而对粗蛋白的离体总消化率为:鱼粉41.92%>豆粕40.88%>菜粕23.68%>棉粕21.39%;②铜鱼对4种饲料原料的干物质和粗蛋白体外消化能力为:中肠>肝胰脏>前肠>后肠。     

Cellular characterization of multidrug resistance P-glycoprotein, alpha fetoprotein, and neovascular endothelium-associated antigens in canine hepatocellular carcinoma and cirrhotic liver

P-glycoprotein (P-gp), which is encoded by the multidrug resistance gene (MDR-1); alpha fetoprotein (AFP); and vascular endothelium-associated antigens are well-known markers for human and canine hepatic diseases. We obtained liver tissues from 5 dogs with hepatocellular carcinoma (HCC) and 12 dogs with cirrhosis, and we performed histopathologic and immunohistochemical evaluations using anti-P-gp, anti-AFP, anti-CD31, and anti-CD34 antibodies. P-gp was expressed at higher levels in HCC than in cirrhotic livers ( P < .01), and was most commonly localized in biliary canaliculi and small ductuli. AFP was localized mainly in the cytoplasm in HCC ( P < .01) and in a few cases of cirrhosis. In both HCC and cirrhosis, the AFP-positive cells were morphologically similar to normal hepatocytes and showed an even cytoplasmic distribution of AFP. The endothelial markers CD31 and CD34 were used to investigate vascular distribution. CD31 was expressed strongly in the portal area and parenchyma in HCC, but it was rarely observed in the parenchyma in cirrhosis. CD34 expression could not be detected in both HCC and cirrhosis. This study constitutes the first comprehensive study of P-gp, AFP, and endothelial markers in canine HCC and cirrhosis. The importance of these markers in HCC and cirrhosis in dogs was demonstrated and provides a more accurate basis for a definitive diagnosis of HCC and cirrhosis in dogs.     

Ultrasonographic examination of the omasum, liver, and small and large intestines in cows with right displacement of the abomasum and abomasal volvulus

OBJECTIVE: To describe ultrasonographic appearance of the liver, small and large intestines, and omasum in cows with right displacement of the abomasum (RDA) and with abomasal volvulus (AV) and to determine whether RDA and AV can be differentiated on the basis of ultrasonographic findings. ANIMALS: 17 cows with RDA, 9 cows with AV, and 10 healthy control cows. PROCEDURES: A linear transducer was used to examine the abomasum, liver, omasum, and small and large intestines from the right side. Results-The liver was imaged less frequently in cows with RDA or AV, compared with control cows. In 9 cows with RDA or AV, the liver could not be imaged. The small intestine was imaged less frequently in cows with RDA or AV than in control cows; in cows with AV, the small intestine could not be imaged in the 8th, 9th, or 10th intercostal space. The large intestine was imaged less frequently in the 11th and 12th intercostal spaces and the cranial region of the flank in cows with RDA or AV. The omasum was also imaged less frequently in the 8th and 9th intercostal spaces in cows with RDA or AV. Cows with RDA or AV could not be differentiated on the basis of ultrasonographic findings. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with control cows, cows with RDA and AV had changes in positioning and therefore extent of ultrasonographic imaging of the liver, omasum, and small and large intestines; however, these findings were not useful in differentiating between cows with RDA and AV.     

Biochemical and functional characterization of lymphocytes from a horse with lymphosarcoma and IgM deficiency

Neoplastic lymphocytes from a horse with lymphosarcoma and IgM deficiency were analyzed for ability to grow in culture; surface and cytoplasmic IgM; functional activity in blastogenesis, cytoxicity, and suppressor assays; and activities of six enzymes involved in purine and pyrimidine metabolism. The cells lacked surface and cytoplasmic IgM. They had elevated activity of adenosine deaminase and reduced activity of purine nucleoside phosphorylase. Neoplastic cells were nonresponsive in blastogenesis assay and did not kill allogeneic lymphocyte target cells or YAC-1 targets in a lectin-dependent cytotoxicity assay, however, the cells were active in a suppressor assay. They were grown for 16 weeks in cultures supplemented with interleukin 2, during which time the cells retained suppressive activity. These results are consistent with a T cell lymphoma of suppressor cell origin, and may explain the deficiency of IgM observed in some horses with lymphoreticular neoplasms.