Absorbed EVELISA: a diagnostic test with improved specificity for Johne's disease in cattle.

The use of enzyme-linked immunosorbent assays (ELISAs) is recommended for Johne's disease (JD) control in dairy herds. In 2006, we developed a novel ELISA test for JD, named EVELISA (ELISA using ethanol extract of Mycobacterium avium subsp. paratuberculosis), which showed higher sensitivity than commercial ELISA tests. To further investigate the performance of EVELISA, we obtained 38 serum samples from cattle in a JD-free herd with suspected cases of serological false-positive reactions. When these samples were tested using the EVELISA and a commercial ELISA test, more than 70% of the samples were falsely identified as JD positive. Antibodies in the serum samples reacted strongly with antigens of various environmental mycobacteria, suggesting the presence of cross-reactive antibodies in the samples. The possible cross reactions in the EVELISA were inhibited markedly by the use of Mycobacterium phlei antigens for antibody absorption. When these samples were tested, 8 samples were classified as positive for JD by the EVELISA with the antibody absorption, whereas 27 samples were classified as positive for JD by the commercial ELISA. For an estimation of tentative sensitivity and specificity, the ELISA tests were performed on 38 serum samples from JD-negative herds with no suspected cases of serological false-positive reaction and 68 samples from cattle diagnosed as positive for M. avium subsp. paratuberculosis infection by fecal culture test. Sensitivity and specificity of the EVELISA with preabsorption of serum with M. phlei ("ethanol vortex absorbed-ELISA" or EVA-ELISA) were estimated to be 97.1% and 100%, respectively, whereas those of the commercial ELISA were 48.5% and 97.4%, respectively. Further, in 85 fecal culture-negative cattle in JD-positive herds, higher sensitivity of the EVA-ELISA than the commercial ELISA was demonstrated by a Bayesian analysis. This study indicates that the EVA-ELISA may form a basis for a sensitive diagnostic test with a higher level of specificity than that of the current commercial ELISA test.

Developmental responses of 2-cell embryos to oxygen tension and bovine serum albumin in Wistar rats.

To improve rat embryo culture conditions, responses of Wistar 2-cell embryos from 2 breeders to oxygen tension (5 vs 20%) and bovine serum albumin (BSA) (0 vs 3 mg/ml) were examined using rat 1-cell embryo culture medium (mR1ECM). Supplementation of 3 mg/ml BSA significantly stimulated and accelerated development to the blastocyst and expanded blastocyst stages during 72 and 96 h culture, while reduced oxygen tension stimulated cell division. Fetus development after transfer of blastocysts obtained from 72 h culture under 5% O2 with BSA was significantly higher than those cultured under atmospheric oxygen without BSA. However, the nuclear numbers of in vitro cultured blastocysts and fetus development after embryo transfer were still significantly lower than in vivo developed blastocysts, indicating the current culture condition is still suboptimal.

Modeling the biological response of normal human cells, including repair processes, to fractionated carbon beam irradiation.

To understand the biological response of normal cells to fractionated carbon beam irradiation, the effects of potentially lethal damage repair (PLDR) and sublethal damage repair (SLDR) were both taken into account in a linear-quadratic (LQ) model. The model was verified by the results of a fractionated cell survival experiment with normal human fibroblast cells. Cells were irradiated with 200-kV X-rays and monoenergetic carbon ion beams (290 MeV/u) at two irradiation depths, corresponding to linear energy transfers (LETs) of approximately 13 keV/µm and 75 keV/µm, respectively, at the Heavy Ion Medical Accelerator in Chiba of the National Institute of Radiological Sciences. When we only took into account the repair factor of PLDR, γ, which was derived from the delayed assay, the cell survival response to fractionated carbon ion irradiation was not fully explained in some cases. When both the effects of SLDR and PLDR were taken into account in the LQ model, the cell survival response was well reproduced. The model analysis suggested that PLDR occurs in any type of radiation. The γ factors ranged from 0.36-0.93. In addition, SLD was perfectly repaired during the fraction interval for the lower LET irradiations but remained at about 30% for the high-LET irradiation.

Improvement of iron-mediated oxidative DNA damage in patients with transfusion-dependent myelodysplastic syndrome by treatment with deferasirox.

Myelodysplastic syndrome (MDS) is characterized by dysplastic and ineffective hematopoiesis, peripheral blood cytopenias, and a risk of leukemic transformation. Most MDS patients eventually require red blood cell (RBC) transfusions for anemia and consequently develop iron overload. Excess free iron in cells catalyzes generation of reactive oxygen species that cause oxidative stress, including oxidative DNA damage. However, it is uncertain how iron-mediated oxidative stress affects the pathophysiology of MDS. This study included MDS patients who visited our university hospital and affiliated hospitals (n=43). Among them, 13 patients received iron chelation therapy when their serum ferritin (SF) level was greater than 1000 ng/mL or they required more than 20 RBC transfusions (or 100 mL/kg of RBC). We prospectively analyzed 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in peripheral blood mononuclear cells (PBMC) obtained from MDS patients before and after iron chelator, deferasirox, administration. We showed that the 8-OHdG levels in MDS patients were significantly higher than those in healthy volunteers and were positively correlated with SF and chromosomal abnormalities. Importantly, the 8-OHdG levels in PBMC of MDS patients significantly decreased after deferasirox administration, suggesting that iron chelation reduced oxidative DNA damage. Thus, excess iron could contribute to the pathophysiology of MDS and iron chelation therapy could improve the oxidative DNA damage in MDS patients.