Miyako Yoshioka

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrPSc, a protease-resistant misfolded isoform of the cellular prion protein PrPC. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrPSc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrPSc in BSE-affected cattle therefore remains unknown. Methodology/Principal Findings We report here that PrPSc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrPSc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrPSc in blood was not substantiated in the BSE-affected cattle examined. Conclusions/Significance The distribution of PrPSc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrPSc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.

Efficient in vitro amplification of a mouse-adapted scrapie prion protein

Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie prion protein (PrP(Sc)), a major protein component of the infectious agents associated with prion diseases. Although exponential in vitro amplification of hamster scrapie PrP(Sc) has been established, the PMCA used was unsuccessful in achieving good amplification of PrP(Sc) from other animals. Here, we have investigated the cause of the insufficient PrP(Sc) amplification in mice and have developed an improved method suitable for amplification of the PrP(Sc) of the mouse-adapted scrapie prion strain Chandler. Mouse PrP(C), the cellular form of the prion protein, tends to become resistant to proteases during incubation independent of sonication. By adding digitonin to the reaction buffer as a lipid detergent, accumulation of the protease-resistant PrP(C) was inhibited; hence, mouse PrP(Sc) could be amplified to infinite levels. The present study is the first report describing effective amplification of PrP(Sc) of the mouse-adapted scrapie prion and this improved PMCA technique will contribute to prion research that uses mice as experimental animals.

Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11.

The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.

The release of tumor necrosis factor-α, interleukin-1, interleukin-6 and prostaglandin E2 in bovine Kupffer cells stimulated with bacterial lipopolysaccharide

Kupffer cells, in response to gut-derived bacteria, viruses and endogenous endotoxins, are thought to be a major source of proinflammatory cytokines and other mediators. In the present study, we investigated the mode of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1), interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by bovine Kupffer cells in response to LPS in vitro. By semi-quantitative reverse transcription-polymerase chain reaction, both TNF-alpha and IL-1beta mRNAs were expressed between 3 and 12 h after stimulation with 1 microg/ml of LPS. The IL-1alpha transcript also increased at 3 h, and then disappeared at 6 h. Although IL-6 mRNA was slightly expressed without stimulation, it increased and reached a peak after 6 h, and then decreased to normal levels by 24 h. The secretion of TNF-alpha and IL-1 was detected after 3 h and increased until 12 and 24 h, respectively, as detected by bioassay. IL-6 was secreted at a low level without stimulation, peaked 6 h after stimulation and remained elevated until 24 h. The secretion of prostaglandin E2 continued to increase for 24 h. These results suggest that several inflammatory cytokines released from bovine Kupffer cells are regulated in different modes.

A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells

We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and alpha-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 10(6) cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills.

Sensitive detection of scrapie prion protein in soil.

Prion diseases are fatal neurodegenerative disorders that are caused by infectious agents known as prions. Prions are composed primarily of the pathogenic prion protein isoform, PrP(Sc). Because significant levels of infectivity have been detected in excrement from animals infected with scrapie and chronic wasting disease, studies on the dynamics of PrP(Sc) levels in contaminated soil are needed to assess the possible horizontal transmission of prion diseases. Using protein misfolding cyclic amplification, we developed a sensitive detection method for scrapie PrP(Sc) that is mixed with soil. Our detection method has the advantage of not requiring extraction of PrP(Sc) from soil and could provide a sensitivity 1000 to 10,000 times higher than that obtained with an extraction-based method. In addition, we found that PrP(Sc) levels in experimentally contaminated agricultural soils declined to different extents over the course of a 6-month incubation period. Our method appears to be a very useful technique for monitoring PrP(Sc) levels in soil.

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1. After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9. In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.

Kupffer cells are sessile tissue macrophages that have a role in liver defense against endogenous endotoxins. Because little information is available on the role of bovine Kupffer cells, we developed a primary culture method to investigate the function of bovine Kupffer cells. Kupffer cells were isolated from the caudate lobe of calf liver by perfusion with collagenase and pronase. Then, the cells were purified by gradient centrifugation followed by counterflow centrifugal elutriation. With the methods, a mean number of 1.5 x 10(6) Kupffer cells with a final viability of over 98% was obtained from 1 g of the liver. Over 95% of the isolated cells were positive for non-specific esterase activity and had surface molecule of CD68. The cultured Kupffer cells expressed mRNAs of tumor necrosis factor-alpha, interleukin (IL)-1 alpha, IL-1 beta and IL-6 by stimulation for 3 h with lipopolysaccharide. The primary culture of bovine Kupffer cells could be useful to investigate the systemic inflammatory response in bovine liver.

Assessment of prion inactivation by fenton reaction using protein misfolding cyclic amplification and bioassay

An abnormal isoform of the prion protein, associated with transmissible spongiform encephalopathies, retains infectivity even after undergoing routine sterilization processes. We found that a formulation of iron ions combined with hydrogen peroxide effectively reduced infectivity and the level of abnormal isoforms of the prion protein in scrapie-infected brain homogenates. Therefore, the Fenton reaction has potential for prion decontamination.

Spatio-temporal distribution, source, and genotoxic potential of polycyclic aromatic hydrocarbons in estuarine and riverine sediments from southern India.

In order to categorize the distribution, source, and effects of polycyclic aromatic hydrocarbons (PAHs) in aquatic systems of southern India, chemical and toxicological analyses were performed on surface and core sediments, collected from Adyar river, Cooum river, Ennore estuary, and Pulicat lake near Chennai city. The total PAH concentration in surface sediment ranged from 13 to 31,425ng/g with a mean value of 4320ng/g; the concentration was markedly higher in Cooum river compared to that at other sites. The historical PAH dissemination in core samples in the Cooum river, Ennore estuary, and Pulicat lake ranged from 30 to 31,425ng/g, from 8.6 to 910ng/g, and from 62 to 546ng/g, respectively. Surface sediments were predominantly contaminated with low molecular weight (LMW) PAHs. Historical profiles suggest that PAH contamination in the area is now greater than it had been in the past. PAH accumulation in Pulicat lake was distinct from that at other locations where high molecular weight (HMW) PAHs were predominant. DNA damage in HepG2 cells treated with sediment extracts from different locations showed a good correlation with their respective total PAH levels. Statistical analysis revealed that 3-ring and 4-ring PAHs may synergistically contribute to the genotoxic potency compared to others in sediments. The study also showed that a majority of PAHs in the study area indicated a petrogenic origin. Based on the enrichment and toxicological assessment of PAHs in sediments, Cooum river was shown to suffer the highest biological impairment among the studied water bodies.