Evaluation of cleaning methods for change-over after the processing of cell products to avoid cross-contamination risk.

Introduction: Cell-processing facilities face the risk of environmental bacteria contaminating biosafety cabinets during processing, and manual handling of autologous cell products can result in contamination. We propose a risk- and evidence-based cleaning method for cross-contamination, emphasizing proteins and DNA. Methods: The transition and residual risks of the culture medium were assessed by measuring both wet and dried media using fluorescence intensity. Residual proteins and DNA in dried culture medium containing HT-1080 cells were analyzed following ultraviolet (UV) irradiation, wiping, and disinfectant treatment. Results: Wet conditions showed a higher transition to distilled water (DW), whereas dry conditions led to higher residual amounts on SUS304 plates. Various cleaning methods for residual culture medium were examined, including benzalkonium chloride with a corrosion inhibitor (BKC + I) and DW wiping, which demonstrated significantly lower residual protein and DNA compared to other methods. Furthermore, these cleaning methods were tested for residual medium containing cells, with BKC + I and DW wiping resulting in an undetectable number of cells. However, in some instances, proteins and DNA remained. Conclusions: The study compared cleaning methods for proteins and DNA in cell products, revealing their advantages and disadvantages. Peracetic acid (PAA) proved effective for nucleic acids but not proteins, while UV irradiation was ineffective against both proteins and DNA. Wiping emerged as the most effective method, even though traceability remained challenging. However, wiping with ETH was not effective as it caused protein immobilization. Understanding the characteristics of these cleaning methods is crucial for developing effective contamination control strategies.

Inhibitory effects of kaempferol, quercetin and luteolin on the replication of human parainfluenza virus type 2 in vitro.

The eight flavonoids, apigenin, chrysin, hesperidin, kaempferol, myricetin, quercetin, rutin and luteolin were tested for the inhibition of human parainfluenza virus type 2 (hPIV-2) replication. Three flavonoids out of the eight, kaempferol, quercetin and luteolin inhibited hPIV-2 replication. Kaempferol reduced the virus release (below 1/10,000), partly inhibited genome and mRNA syntheses, but protein synthesis was observed. It partly inhibited virus entry into the cells and virus spreading, and also partly disrupted microtubules and actin microfilaments, indicating that the virus release inhibition was partly caused by the disruption of cytoskeleton. Quercetine reduced the virus release (below 1/10,000), partly inhibited genome, mRNA and protein syntheses. It partly inhibited virus entry and spreading, and also partly destroyed microtubules and microfilaments. Luteolin reduced the virus release (below 1/100,000), largely inhibited genome, mRNA and protein syntheses. It inhibited virus entry and spreading. It disrupted microtubules and microfilaments. These results indicated that luteolin has the most inhibitory effect on hPIV-2 relication. In conclusion, the three flavonoids inhibited virus replication by the inhibition of genome, mRNA and protein syntheses, and in addition to those, by the disruption of cytoskeleton in vitro.

Clinical significance of anti-Epstein-Barr virus antibodies in systemic chronic active Epstein-Barr virus disease.

Systemic chronic active Epstein-Barr virus disease (sCAEBV) is a rare and fatal neoplasm, involving clonally proliferating Epstein-Barr virus (EBV)-infected T cells or natural killer cells. Patients with sCAEBV have abnormal titers of anti-EBV antibodies in their peripheral blood, but their significance is unknown. We retrospectively investigated titers and their relationship with the clinical features of sCAEBV using the data collected by the Japanese nationwide survey. Eighty-four patients with sCAEBV were analyzed. The anti-EBV nuclear antigen (EBNA) antibody, targeting EBNA-expressing EBV-positive cells, was found in 87.5% of children (<15 years old), 73.7% of adolescents and young adults (15-39 years old), and 100% of adults (≥40 years old). Anti-EBNA antibody titers were significantly lower and anti-VCA-IgG antibody titers significantly higher in patients with sCAEBV than those in healthy controls (p < 0.0001). Patients with high anti-VCA-IgG and anti-early antigen-IgG antibody (antibodies against the viral particles) levels had significantly better 3-year overall survival rates than those with low titers, suggesting that patients with sCAEBV have a reduced immune response to EBV-infected cells.