Efficient folding/assembly in Chinese hamster ovary cells is critical for high quality (low aggregate content) of secreted trastuzumab as well as for high production: stepwise multivariate regression analyses.

When developing cell culture processes for therapeutic antibodies, the low content of aggregated proteins is the most critical because administering aggregated antibody molecules might result in adverse effects such as immunogenicity. To characterize cells with high productivity and quality, we determined factors that are closely related to antibody titer, which is a productivity indicator, and the area percentage of high molecular weight species in cultivated media, which is equivalent to aggregate content and is used as a quality indicator. We examined the factors influencing antibody titer and aggregate content using various data from 28 cell lines throughout their culture periods from growth to death phases. Our study using correlation analysis revealed that statistically significant correlations between factors and indicators changes with sampling points, hence we thought that various factors would influence each indicator simultaneously. To understand the relationship between these factors and titer/aggregates contents, we performed stepwise multiple linear regression analyses and deduced a multiple linear model for each indicator. The titer was found to positively associate with specific growth rate and specific production rate and negatively with intracellular heavy chain content. The aggregate content was found to positively associate with protein disulfide isomerase mRNA level and negatively with light chain secreted into culture media, specific production rate, intracellular light chain content, and specific growth rate. Our observations suggest that correct and efficient assembling and/or folding of an antibody molecule in an endoplasmic reticulum are important for high titer and low aggregates contents.

Use of flow cytometry to separate Leucocytozoon caulleryi gametocytes from avian blood.

The highly pathogenic avian protozoan Leucocytozoon caulleryi infects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelled L. caulleryi gametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified as L. caulleryi gametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation of L. caulleryi gametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specific L. caulleryi gene expression.

Cacao procyanidins reduce plasma cholesterol and increase fecal steroid excretion in rats fed a high-cholesterol diet.

Cocoa powder is rich in polyphenols, such as catechins and oligomeric procyanidins, and has a hypocholesterolemic effect in humans. This study evaluated the principal active components and potential mechanism(s) for the hypocholesterolemic effect of polyphenolic substances from cocoa powder in rats. Male Wistar rats were fed a 1% high-cholesterol diet (HC) or a high-cholesterol diet containing 1% polyphenol extract from cocoa powder (PE) or a mixture of 0.024% catechin and 0.058% epicatechin (CE) for 4 weeks. We also examined the effects of these polyphenolic substances on micellar cholesterol solubility in vitro. The PE group had significantly lower plasma cholesterol concentrations, and had significantly greater fecal cholesterol and total bile acids excretion than the HC group. The CE group diet did not influence plasma cholesterol concentrations, or fecal cholesterol or total bile acids excretion. Micellar solubility of cholesterol in vitro was significantly lower for procyanidin B2 (dimer), B5 (dimer), C1 (trimer) and A2 (tetramer), which are the main components of polyphenol extract from cocoa powder, compared to catechin and epicatechin. These results suggest that oligomeric procyanidins from cocoa powder are the principal active components responsible for the hypocholesterolemic effect, and inhibit the intestinal absorption of cholesterol and bile acids through the decrease in micellar cholesterol solubility.

Preventive effects of soyasapogenol B derivatives on liver injury in a concanavalin A-induced hepatitis model.

To shed light on the structure-activity relationship, various soyasapogenol B derivatives were synthesized and evaluated for preventive effects on liver injury in the concanavalin A (Con A)-induced hepatitis model in mice. Con A injection into mice induces some pathophysiology of human liver disease such as autoimmune or viral hepatitis. Two hydroxyl groups on the A ring of soyasapogenol B are required for amelioration of liver damage. Modification of the C-22 hydroxyl moiety with an acyloxy or alkyloxy group, or removal of the hydroxyl group, resulted in a greatly enhanced percentage of alleviation. Among the series of soyasapogenol B derivatives examined, six compounds exhibited preventive effects on liver damage.