Comparative study of in situ hybridization and immunohistochemistry for the detection of porcine circovirus 2 in formalin-fixed, paraffin-embedded tissues.

In situ hybridization and immunohistochemistry with different types of antibody (monoclonal vs. polyclonal, natural vs. synthetic) was compared to detect porcine circovirus 2 (PCV2) in formalin-fixed, paraffin-embedded tissues from pigs with experimentally and naturally occurring postweaning multisystemic wasting syndrome. PCV2 DNA and antigen was detected in tissues from both experimentally and naturally infected pigs by in situ hybridization and immunohistochemistry, respectively. Statistical evaluation revealed that more PCV2 positive signals were significantly detected in both experimentally and naturally infected pigs by in situ hybridization compared with immunohistochemistry (P<0.05). The results of this study demonstrated that in situ hybridization proved more sensitive than immunohistochemistry for the detection of PCV2 in formalin-fixed, paraffin-embedded lymph node tissues.

Radioprotective effects of fucoidan on bone marrow cells: improvement of the cell survival and immunoreactivity.

Fucoidan is a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and has a variety of biological effects including mobilization of hematopoietic progenitor cells. Recently, we demonstrated that fucoidan stimulates the antigen-presenting functions of dendritic cells. In this study, we investigated the radioprotective effects of fucoidan on bone marrow cells (BMCs), which are the main cellular reservoir for the hematopoietic and immune system. To evaluate the effects of fucoidan, we assayed cell viability and immune responses. In a viability assay, fucoidan significantly increased the viability of BMCs. Based on the results of flow cytometric analysis, the increased viability of fucoidan-treated BMCs was attributed to the inhibition of radiation-induced apoptosis. Furthermore, fucoidan altered the production of immune-related cytokines from BMCs and increased the capability of BMCs to induce proliferation of allogeneic splenocytes. Taken together, our study demonstrated that fucoidan has radioprotective effects on BMCs with respect to cell viability and immunoreactivity. These results may provide valuable information, useful in the field of radiotherapy.

Antigenic diversity of Theileria major piroplasm surface protein gene in Jeju black cattle.

Piroplasms are tick-transmitted, intracellular, hemoprotozoan parasites that cause anorexia, fever, anemia, and icterus. Theileriosis is caused by Theileria sergenti and causes major economic losses in grazing cattle in Japan and Korea. In May 2003, we examined the antigenic diversity of the major piroplasm surface protein (MPSP) gene in 35 healthy Jeju black cattle that were born and raised at the National Institute of Subtropical Agriculture. On microscopic examination of Giemsa-stained blood smears, 9 of 35 cattle had intra-erythrocytic piroplasms. Hematological data were within normal range for all 35 cattle. Amplification of DNA from all blood samples using universal MPSP gene primers showed mixed infections with C, I, and B type Theileria spp. Type C was identified in 20 of 35 blood samples, and type B was identified in 17 samples. Allelic variation was seen in type B.

Effects of root canal sealers on lipopolysaccharide-induced expression of cyclooxygenase-2 mrna in murine macrophage cells.

Cyclooxygenase-2 (COX-2) is a useful biomarker of the inflammatory potential of biomaterials in vitro. In this study we investigate the effects of soluble extracts from 3 selected root canal sealers (AH26, Sealapex, and N2 Universal) on COX-2 mRNA expression in cultured murine macrophage cells. Root canal sealers and the addition of lipopolysaccharide (LPS) both produced significant increases in COX-2 mRNA expression in RAW 264.7 macrophages. In addition, both Sealapex and N2 Universal produced a synergistic 6- to 8-fold increase in COX-2 mRNA expression, whereas AH26 did not demonstrate synergy with LPS. These results suggest that LPS and certain root canal sealers have a synergistic effect on the inflammatory responses of macrophages. Under the conditions of this in vitro study, the results suggest that one potential mechanism of periapical inflammatory reactions might be the synergistic effects of certain root canal sealers on LPS-induced COX-2 expression by macrophage cells.

One-step immunochromatography assay kit for detecting antibodies to canine parvovirus.

This study was performed to determine the feasibility of using whole serum to detect antibodies to canine parvovirus (CPV) under nonlaboratory conditions and to evaluate the performance characteristics of an immunochromatography assay kit. Precise detection of levels of antibody against CPV in puppies can be used to determine a vaccination schedule, because maternal antibodies frequently result in the failure of protective vaccination, and can also be used to determine the antibody levels of infected puppies. Several methods for the titration of CPV antibodies have been reported, including the hemagglutination inhibition (HI) assay, which is considered the "gold standard." These methods, however, require intricate and time-consuming procedures. In this study, a total of 386 serum specimens were tested. Compared to the HI assay, the rapid assay had a 97.1% sensitivity and a 76.6% specificity (with a cutoff HI titer of 1:80). This single-step assay could be performed rapidly and easily without special equipment. The kit provides a reliable method for detection of anti-CPV antibody where laboratory support and personnel are limited.

Development of a sandwich ELISA for the detection of Listeria spp. using specific flagella antibodies.

Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x 10(5) cells/0.1 ml at 22 degrees C. and 1 x 10(6) cells/0.1 ml at 30 degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 10(4) cells/0.1 ml at 22 degrees C and 30 degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.

Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation.

Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsalpha) or HP1(Hsbeta), but not HP1(Hsgamma), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsalpha) or HP1(Hsbeta) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-DeltaHP1(Hsalpha), GFP-DeltaHP1(Hsbeta), or GFP-DeltaHP1(Hsgamma) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsalpha) or HP1(Hsbeta). In contrast, cells expressing mutant GFP-DeltaHP1(Hsalpha) or GFP-DeltaHP1(Hsbeta) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsalpha) or HP1(Hsbeta) suppressed tumorigenicity, whereas expression of mutant HP1(Hsalpha) or HP1(Hsbeta) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsalpha) or HP1(Hsbeta) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state.

Gene regulation by human orthologs of Drosophila heterochromatin protein 1.

Heterochromatin protein 1 (HP1) functions in gene silencing, transcriptional regulation, and chromatin remodeling. Drosophila HP1 enhances position effect variegation and has also been shown to repress transcription of some euchromatic genes and activate some heterochromatic ones. The purpose of this study was to determine if human orthologs of Drosophila HP1 differentially regulate transcription by identifying genes that are targets for regulation by HP1 family proteins in human cells. Using a gene expression array, we identify several genes regulated by overexpression of the three human HP1 family proteins HP1(Hsalpha), HP1(Hsbeta), and HP1(Hsgamma). We show further that overexpressed HP1(Hsalpha) and HP1(Hsbeta) repress the transcription of four human genes while overexpressed HP1(Hsgamma) enhances transcription of the same genes. These results show that different human HP1 family proteins can potentially repress or activate the same genes.