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1.  Characterization of canine monocyte-derived dendritic cells with phenotypic and functional differentiation 
For therapeutic purposes, large numbers of dendritic cells (DCs) are essential. In this study, we used 2% autologous canine plasma, granulocyte/macrophage colony-stimulating factor (GM-CSF), fms-like tyrosine kinase 3 ligand (Flt3L), and interleukin 4 (IL-4) in generating monocyte-derived DCs from peripheral blood mononuclear cells of dogs. The plasma enriched the population of CD14-positive monocytes by greatly enhancing the efficiency of monocyte adherence, the proportion of adherent cells increasing from 6.6% with 10% fetal bovine serum to 15.3% with 2% autologous canine plasma. Culturing the adherent monocytes for 6 d with human GM-CSF, canine IL-4, and human Flt3L significantly increased the yield of DCs, more than 90% of which were CD14-negative. Because, in the presence of lipopolysaccharide (LPS), monocytes that were CD14-positive expressed tumor necrosis factor α much more than DCs with low levels of CD14, it is important to decrease the numbers of CD14-positive cells in generating monocyte-derived DCs. With flow cytometry and real-time reverse-transcriptase-mediated polymerase chain reaction assays, we found that in canine immature DCs (iDCs) the expression of DLA class II molecules, CD1a, CD11c, CD40, and CD86 was high and the expression of CD80, CD83, and CD14 either low or negative. During maturation (stimulated by LPS), the expression of CD1a, CD40, CD83, and CD80 was upregulated. However, the expression of DLA class II molecules, CD11c, and CD86 was not increased in mature DCs. Incubating the iDCs with LPS decreased antigen uptake and increased the cells’ immunostimulatory capacity (assessed by the allogeneic mixed-lymphocyte reaction), indicating that LPS accelerates the functional maturation of DCs. This protocol may facilitate the use of DCs in cellular immunotherapy.
PMCID: PMC1899861  PMID: 17695590
2.  Overexpression of chemokine ligand 7 is associated with the progression of canine transmissible venereal tumor 
Chemokines play multiple roles in the development and progression in a variety of tumors. Chemokine (C-X-C motif) ligand 7 (CXCL7) has been found associated with pro-inflammatory responses, but its role in cancer growth remains unclear. Our previous study showed that R phase tumor infiltrating lymphocytes (TILs) produced large amounts of interleukin (IL)-6 which antagonized transforming growth factor (TGF)-β derived from CTVT to diminish the immune-suppressive microenvironment. Now we intend to determine the expression pattern of CXCL7 and the role of IL-6/TGF-β in CXCL7 induction during spontaneous progressive (P) and regressive (R) phases in canine transmissible venereal tumor (CTVT).
We have demonstrated that CXCL7 expressed at high level in P phase and down-regulated in R phase by western blot and real-time PCR. This suggested that CXCL7 expression was negatively correlated with the tumor growth. Co-culturing TILs with CTVT cells was found to reduce CXCL7 expression, while adding IL-6 blocking antibody reversed it. Moreover, in P phase CTVT, while IL-1β and TGF-β had no obvious effect on CXCL7 expression, IL-6 was found significantly to reduce CXCL7 expression in a dose-dependent manner. The mRNA expression results of CXCL7 receptor, CXCR2, further confirmed the effects of IL-6 concentration on the CXCL7 expression.
CXCL7 overexpression might be associated with the progressive growth of CTVT. The results shown here also suggest the role of CXCL7 in cancer development and the potential as the anti-cancer therapeutic target.
PMCID: PMC3538668  PMID: 23136963
3.  Utilization of IκB–EGFP Chimeric Gene as an Indicator to Identify Microbial Metabolites with NF-κB Inhibitor Activity 
Biological Procedures Online  2010;12:131-138.
NF-κB regulates several important expressions, such as cytokine release, anti-apoptosis, adhesion molecule expression, and cell cycle processing. Several NF-κB inhibitors have been discovered as an anti-tumor or anti-inflammatory drug. The activity of NF-κB transcription factor is negatively regulated by IκB binding. In this study, IκB assay system was established and IκB–EGFP fusion protein was used as an indicator to monitor the effects of substances on the IκB degradation. The results indicated that the chosen hydroquinone could inhibit the IκB degradation and cause the cell de-attachment from the bottom of culture plate. In addition, this system could also monitor the IκB degradation of microbial metabolite of natural mixtures of propolis. Thus, the IκB assay system may be a good system for drug discovery related to microbial metabolite.
PMCID: PMC3055915  PMID: 21406073
Microbial metabolite; Antioxidant; IκB; EGFP; Hydroquinone; Propolis
4.  Serum acute phase proteins and swine health status  
The purpose of this study was to investigate the relationship between swine health status and the concentration of the serum acute phase proteins, haptoglobin (HP), and C-reactive protein (CRP). A total of 378 clinically healthy pigs from farms A and B, plus 20 pigs culled from farm A due to poor growth, were used in this experiment. Each pig was examined and blood samples were collected during slaughter. The HP concentration was measured by using an HP-hemoglobin binding assay. The CRP concentration was measured by using a CRP enzyme immunoassay. Gross and histopathological lesions were examined and recorded at slaughter. Representative samples were then collected in order to isolate pathogens. Swine enzootic pneumonia, found in 47.7% of the pigs, was the most common lesion. Other lesions included pleuropneumonia (32.7%), suppurative pneumonia (10.3%), fibrinous pericardititis (4.3%), Ascaris migration in the liver (33.9%), and intestinal serositis (3.0%).
On farm A, the percentage of pigs with 1 or more lesions was 88.2%. For culled pigs from farm A, the mean serum concentrations of HP and CRP were 2.23 ± 0.14 mg/mL and 252.93 ± 11.62 μg/mL, which were significantly higher than concentrations in clinically normal pigs (1.42 ± 0.02 mg/mL and 84.88 ± 2.61 μg/mL, respectively, P < 0.01). Moreover, among clinically normal farm A pigs, the mean HP concentration in pigs with lesions (1.43 ± 0.02 mg/mL) was significantly higher than in pigs without lesions (1.32 ± 0.07 mg/mL) (P < 0.05). However, the mean serum CRP concentrations in these animals were not significantly different.
On farm B, the percentage of pigs with one or more lesions was 50.0%. Interestingly, the mean serum HP concentration in clinically normal pigs with lesions was significantly lower in farm B pigs (1.23 ± 0.07 mg/mL) than in the farm A pigs (1.43 ± 0.02 mg/mL; P < 0.01). However, serum CRP concentrations in farm A and B pigs were not significantly different. Serum HP concentration, which is a better indicator of inflammatory reactions in pig herds than serum CRP concentration, provides an important marker for swine health status.
PMCID: PMC280713  PMID: 14620865
5.  Serum C-reactive protein in dairy herds 
The purpose of this study was to determine the relationship between the serum level of C-reactive protein (CRP) and lactation and health status. Blood samples were collected every 2 wk for 12 mo from 29 randomly selected dairy cattle on 3 farms. At the time the blood samples were collected, the stage of pregnancy, lactation status, breeding records, general health condition, reproductive status, and body condition score were recorded for each cow. Serum CRP was detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis and western immunoblotting. C-reactive protein levels were measured with a densitometer and expressed as an optimal dose value. C-reactive protein levels were correlated with the body condition score, lactation status, and animal health (P < 0.05), but not with ambient temperature, animal age, or parity. C-reactive protein levels increased with milk production, peaking during high lactation (2 to 4 mo of pregnancy), and decreased when lactation ceased. In addition, the CRP level was highest during naturally occurring infections, such as mastitis and other tissue inflammation. Thus, the CRP level can confirm the presence of inflammation. The stress effect of taking blood samples as measured by the CRP level, was also examined. The CRP level became rapidly elevated 12 h after the blood samples were taken but returned to normal 36 h later. In conclusion, the stresses resulting from overall poor health, heavy lactation, and blood sampling caused the elevation of serum CRP. C-reactive protein is a marker or tool for evaluating the health status of a herd. C-reactive protein should also be considered as a useful criteria to assess the stress levels and may be useful in early surveillance of disease conditions in a dairy herd.
PMCID: PMC227036  PMID: 12760474

Results 1-5 (5)