Immobilization of hybridoma cells with alginate and urethane polymer and improved monoclonal antibody production

Summary Hybridoma cells producing anti--amylase monoclonal antibody were entrapped in calcium alginate and the gels were then coated with urethane polymer. The urethane coating improved gel strength and prevented cell leakage. This immobilization method enabled direct air bubbling in the serum-free medium and a very high cell concentration (3×10⁷ cells/ml gel) was obtained. By using a fluidized-bed reactor, effective removal of the medium in addition to sufficient oxygen supply could be achieved without any special devices and a very high concentration of the monoclonal antibody was continuously obtained.     

Hepatoma-associated alterations of serum alpha 1-antitrypsin in hereditary hepatitis LEC rats as a new animal model of liver disease

1. The LEC rats, a novel animal model of hepatitis and liver tumor, were found to have charge variants of serum alpha 1-antitrypsin (alpha 1AT) at the stage of liver tumor as judged by isoelectric focusing. 2. Treatment of the sera with neuraminidase showed that acidic variants of alpha 1AT in LEC rats seemed to be highly sialylated forms. 3. The concentration and enzymatic activity of serum alpha 1AT in LEC rats were not affected before the onset of hepatitis and even during the development of fulminant hepatitis and hepatocarcinogenesis. This indicates that hereditary hepatitis and subsequent liver tumor in LEC rats do not appear to be associated with alpha 1AT deficiency.     

High expression of an N-acetylglucosaminyltransferase III in 3'-methyl DAB-induced hepatoma and ascites hepatoma

Ascites hepatoma AH-66 and 3'-Me-DAB-induced hepatoma of rats contain highly active N-acetylglucosaminyltransferase III (GnT-III) which catalyzes the addition of N-acetylglucosamine through a beta 1-4 linkage (bisecting N-acetylglucosamine) to the beta-linked mannose of the trimannosyl core of asparagine-linked sugar chains, whereas normal rat liver contains very little. The high activity was also detected in fetal rat liver, newborn rat liver, hyperplastic nodules and various transplantable hepatomas.     

Phosphorylation of rabbit liver cytochrome P-450 LM2 and its effect on monooxygenase activity

The phosphorylation of rabbit liver microsomal cytochrome P-450 LM2 by catalytic subunit of cyclic AMP-dependent protein kinase (W. Pyerin et al. (1983) Carcinogenesis 4, 573) has now been studied in detail with purified soluble form of cytochrome P-450 as well as with the purified protein incorporated into model membranes. The apparent Km values for P-450 of the phosphorylation reaction in all experimental systems were in a range of 2-8 microM, while the Vmax values were dependent on the state of P-450. Upon phosphorylation, the reconstituted enzyme activities with benzphetamine (N-demethylation) and 7-ethoxycoumarin (O-deethylation) as substrates were reduced to 30-40% of control.     

Spin-label studies of erythrocyte deformability. IV. Relation of electron spin resonance spectral change with deformation and orientation of erythrocytes in shear flow.

Electron spin resonance (ESR) spectra of spin-labeled human erythrocytes in shear flow are simulated to derive semi-empirical relations of the ESR spectral change with deformation and orientation of the cells by using a modified theoretical model developed for deformation and orientation of liquid drops. The six observed spectra at different shear stress values were simultaneously simulated by adjusting only two parameters. One parameter can be related to the ratio of the internal to the external viscosity, and the other to the elastic property of the cell membrane. From these results we have derived a semi-empirical relationship between the average deformation index or the orientation angle with a spectral measure, which characterizes the spectral shape change induced by shear stress. Thus, it becomes possible to obtain improved quantitative information on the rheological behavior of red blood cells by using the spin-label ESR method.     

Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

Background

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas.

Methodology/Principal Findings

We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID₅₀/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>10⁵ copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<10⁵ copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV.

Conclusions

The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia.     

Overexpression of Peroxiredoxin 4 Affects Intestinal Function in a Dietary Mouse Model of Nonalcoholic Fatty Liver Disease

BackgroundAccumulating evidence has shown that methionine- and choline-deficient high fat (MCD+HF) diet induces the development of nonalcoholic fatty liver disease (NAFLD), in which elevated reactive oxygen species play a crucial role. We have reported that peroxiredoxin 4 (PRDX4), a unique secretory member of the PRDX antioxidant family, protects against NAFLD progression. However, the detailed mechanism and potential effects on the intestinal function still remain unclear.ConclusionOur present data provide the first evidence of the beneficial effects of PRDX4 on intestinal function in the reduction of the severity of NAFLD, by ameliorating oxidative stress-induced local and systemic injury. We can suggest that both liver and intestine are spared, to some degree, by the antioxidant properties of PRDX4.     

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 10³ TCID₅₀ and 10⁵ TCID₅₀. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.     

Ubiquitin-Specific Peptidase 5, a Target Molecule of Vialinin A,Is a Key Molecule of TNF-α Production in RBL-2H3 Cells

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.     

Protective Effect of Sclerin on a Lead-acetate Injury Affecting the Growth and Porphyrin Metabolism in Rat

While a continuous ingestion of lead acetate added in drinking water suppressed the rat growth, depressing in some degree the level of hepatic δ-aminolevulinate (ALA) dehydratase, a very small amount of sclerin (SCL) added simultaneously in the water restored the growth and dehydratase level. Moreover, subcutaneous injection of SCL to the rat not only maintained the ALA dehydratase level, but prevented a marked depression of the level of mitochondrial ALA synthetase in liver caused by intraperitoneal injection of lead acetate. Injection of SCL alone increased tolerably (about 1.8 times) the mitochondrial ALA synthetase, but little the extramitochondrial synthetase. The treatment by SCL was attended by a initial decrease, then a gradual increase in the activity of microsomal drug metabolizing enzyme.