Increased expression of fractalkine and its receptor CX(3)CR1 in canine inflammatory bowel disease and their possible role in recruitment of intraepithelial lymphocytes

The interaction between fractalkine/CX(3)CL1 and its receptor CX(3)CR1 has been reported to play an important role in various human inflammatory diseases, including inflammatory bowel disease (IBD) mediated by lymphocyte chemoattraction. The objective of this study was to investigate the role of fractalkine and CX(3)CR1 in lymphocyte migration in canine IBD. IBD was diagnosed in 34 dogs, and 19 healthy beagles were used as normal controls. We quantified intestinal mRNA and protein expression of fractalkine and CX(3)CR1 by real-time RT-PCR and ELISA, respectively, and examined the localization of fractalkine in canine intestine by immunohistochemistry. The expression of CX(3)CR1 and surface antigens on peripheral blood mononuclear cells (PBMCs) and intraepithelial lymphocytes (IELs) was analyzed by flow cytometry. Intestinal fractalkine and CX(3)CR1 mRNA was significantly up-regulated in IBD dogs compared with the healthy control dogs. In addition, fractalkine expression on intestinal epithelial cells was significantly increased in the intestinal mucosa of IBD dogs compared with the healthy dogs. CX(3)CR1(+) PBMCs were significantly elevated in IBD dogs and positively correlated with the histopathological severity of IELs infiltration. These CX(3)CR1(+) PBMCs predominantly expressed markers for cytotoxic T cells. Almost all IELs expressed CD3, and the majority of cells expressed CD8 rather than CD4, which was analogous to the CX(3)CR1(+) PBMCs. These results suggest that the fractalkine-CX(3)CR1 interaction may contribute to the pathogenesis of canine IBD through migration of IELs.     

A case of canine multiple inflammatory colorectal polyps treated by endoscopic polypectomy and argon plasma coagulation

Endoscopic polypectomy and argon plasma coagulation (APC) were performed in a refractory case of inflammatory colorectal polyps in a 7-year-old male Miniature Dachshund. Colonoscopic examination revealed a large sessile polyp and multiple diffuse small polyps, localized to the descending colon and rectum. The case showed a poor therapeutic response to prednisolone and cyclosporine. Under anesthesia, piecemeal resections were performed by polypectomy. APC was carried out to cauterize the polyp remnants. After treatment, reduction of the lesions and the improvement in clinical signs were observed, without recurrence of lesions for at least 10 months. Endoscopic treatment by polypectomy and APC is suggested to be a therapeutic option for refractory cases of inflammatory colorectal polyps in dogs.     

Decreased gallbladder emptying in dogs with biliary sludge or gallbladder mucocele

Biliary sludge in dogs is dismissed commonly as an incidental finding. On the other hand, gallbladder mucocele is reported increasingly in dogs and can lead to biliary obstruction or gallbladder rupture. Cholestasis is suspected to play a role in development of sludge and mucoceles, though there are no data in dogs to support this. We investigated gallbladder emptying, a key factor in biliary flow, in dogs with mobile sludge, immobile sludge, or gallbladder mucocele and in healthy controls. Gallbladder ejection fraction estimated by ultrasonography was used as the index of gallbladder emptying. The ejection fraction at 60 min after eating was significantly decreased in all three abnormal groups. Moreover, all dogs with sludge or a mucocele had gallbladder distension. These changes were the greatest in the mucocele group. Thus, biliary stasis occurs not only in dogs with gallbladder mucocele but also in dogs with biliary sludge. Cholestasis may play a role in the pathogenesis or progression of these diseases in dogs.     

Physicochemical Properties and Molecular Structures of Starches from Millet (Pennisetum typhoides) and Sorghum (Sorghum bicolor L. Moench) Cultivars in Nigeria

Some physicochemical properties and molecular structures of starches from millet (Pennisetum typhoides, Doro and Gero) and sorghum (Sorghum bicolor, red and white) in Nigeria were examined. Starch granules of millet and sorghum were 3–14 μm and 4–26 μm in diameter, respectively. Millet cultivars had similar peak viscosities (204–205 RVU) on pasting, while sorghum showed similar minimum viscosities (155–156 RVU). The actual amylose content (%) calculated from iodine affinity (IA, g/100 g) was 20.1 and 21.4 for sorghum and 21.3 for millet. The IA of amylopectin was high (1.27–1.42) and its average chain lengths were 20–21 with β‐amylolysis limit of 56%. Amylopectins showed a polymodal molecular weight distribution on a molar basis. The distributions differed among the samples with a higher amount of larger molecules in Doro and red sorghum. Weight‐ and molar‐based distributions of debranched amylopectins on HPSEC were polymodal with weight‐based distribution showing presence of long chains. Peak DP values for A+B₁ and B₂+B₃ chain fractions were 13–16 and 42–43, respectively. The (A+B₁)/(B₂+B₃) ratio on molar basis (9.0–11.5) was similar to maize and rice amylopectins. Peak DP on molar‐based distribution for white sorghum and millet amyloses were similar (490–540) and the DP_n range was narrow (1,060–1,300), but weight‐based distribution profiles differed. The average chain lengths were 260–270 with 3.9–4.8 chains per molecule.     

Thymoma in a dog with a part of granular cell proliferation and concurrent lymphoma cells

A 12-year-old male Shiba dog showed anemia and the swelling of systemic lymph nodes. X-ray and post mortal examinations revealed a anterior mediastinal mass. Histologically, the tumor mass consisted of four different elements; cord-like proliferation of cuboidal epithelial cells, tubular or cystic structures lined with ciliated epithelial cells, proliferation of large round-shaped epithelial cells with PAS-slightly positive granular cytoplasm, and diffuse proliferation of neoplastic lymphocytes. Epithelial cells in cord-like or cystic structures were strongly positive for cytokeratin. Granular or foamy cells were negative for all markers examined and had myelin-like bodies in the cytoplasm by electron microscopy. The neoplastic lymphocytes in the tumor mass were considered being derived from concurrent multicentric lymphoma. Based on these findings, the present case was diagnosed as thymoma with a part of granular cell proliferation and concurrent lymphoma cells.     

固沙林庇护区内土壤-植物系统的变化特征

用定位实测法,系统研究了科尔沁沙地24龄人工固沙杨树(Populussimonii)林庇护区内土壤-植物系统的变化特征,同时分析了土壤-植物系统中植物特征与土壤养分之间的相互影响程度。结果表明:(1)与对照点(流动沙丘)相比较,林地迎风区6H(H为平均树高)、3H、林地中央、林地背风区林缘、6H和8H处0～20cm层土壤中的粗沙含量降低26%～69%,粉、粘粒含量增加539%～1731%。土壤中全C、全N、速效P含量分别增加68%～1002%,40%～840%和45%～276%。(3)与对照点相比较,各观测点植物群落的物种优势度有所改变,物种丰富度提高了3～16种,覆盖度增加了43%～54%,地上生物量增加了72%～381g/m2。(3)相关分析结果表明,在林地庇护区内的土壤-植物系统中,植物的物种多样性、枯落物量和根量与土壤C、N含量的相互影响关系更为密切。     

Use of models to evaluate carbon sequestration in agricultural soils

ABSTRACT

The Rothamsted Carbon (RothC) model, which is one of widely used soil carbon (C) models, was validated against long-term experimental datasets in Japan and modified to suit Andosols and paddy soils reflecting unique soil C turnover mechanisms in these soils. Nationwide soil C calculation system was developed by combining these modified models and spatial model input data such as weather, soil type, land use, and agricultural activities. The model was validated in China and Thailand by using long-term field experimental datasets, too. Further studies especially in tropical Asia will be needed. Matching conceptual model C pools with measurable fractions have been big challenges. Using various plant materials, two conceptual pools of plant litter, decomposable plant material (DPM) and resistant plant material (RPM), in the RothC were successfully identified. It was achieved by comparing the default proportions of DPM and RPM pools in the RothC and proportions in plant material fractions determined by two-step acid hydrolysis with H₂SO₄. The trial to match all of five C pools in the model, however, remains unachieved though a study was conducted comparing not only the size of C pools but mean residence time of the pools. A web-based decision support tool called ‘Visualization of CO₂ absorption by soils’ was developed. This allows users to easily calculate changes in soil C, CH₄ and N₂O emissions, and fossil fuel consumption. With this tool, farmers can see how to improve the environmental sustainability of their products and this tool may help spread mitigation options widely. Soil C sequestration can help achieve climate change mitigation and sustainable agricultural production. Importance of long-term field observations should be more highlighted because long-term experiments have supported the development of modeling approaches. I hope models will be more widely used by decision makers. Collaboration between modeling and monitoring studies is important.     

Multiplex PCR and Genescan analysis to detect immunoglobulin heavy chain gene rearrangement in feline B-cell neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms.     

Screening of therapeutic targets for canine mast cell tumors from a variety of kinase molecules

Options of systemic treatment for canine MCT have been still limited and most canine cases with MCTs eventually undergo relapses even after achievement of a remission. Thus additional therapies are required to establish for the tumor. To identify the novel candidate therapeutic targets for canine MCT, the mRNA expression and phosphorylation statuses of several receptor or non-receptor kinases as well as the inhibitory effect of 95 specific inhibitors on the growth were assessed in three canine MCT cell lines (HRMC, VIMC1 and CMMC1). Among the 14 targets, the mRNAs of 11, 7 and 7 kinases were amplified in HRMC, VIMC1 and CMMC1, respectively. The mRNAs of VEGFR3, PDGFRα, SRC, YES, LCK and FYN were detected in all cell lines. The phosphorylation of 12, 8 and 7 kinases was observed by using specific antibody arrays in HRMC, VIMC1 and CMMC1, respectively. DTK, EPHB6, AMPKα1, CREB, STAT5a and STAT5b were phosphorylated in all cell lines. The 10, 9 and 17 inhibitors exhibited the biological activity against the growth of HRMC, VIMC1 and CMMC1, respectively. Only three inhibitors such as SB218078 (for Chk1), PDGF RTK inhibitor IV (for PDGFR) and radicicol (for Hsp90) suppressed the growth of all three cell lines. The present study indicated that several kinases, such as Chk1, PDGFR and Hsp90, could be used as therapeutic targets in the treatment for canine MCT. Further studies and clinical trials are warranted to apply the inhibitors for the treatment of the tumor.     

Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene

OBJECTIVE: To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1). SAMPLE POPULATION: Madin-Darby canine kidney (MDCK) epithelial cell line. PROCEDURES: The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus-based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays. RESULTS: P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1. CONCLUSIONS AND CLINICAL RELEVANCE: Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.