To overcome the lack of availability of fresh human whole blood for pyrogen detection, we explored the feasibility of utilizing cryopreserved pooled human blood to detect the responses of the pro-inflammatory cytokines IL-6 and IL-1β to LPS. Whole blood was obtained from five donors and incubated with LPS. The quantities of pro-inflammatory cytokines were measured using ELISA, and the results were compared among the samples. After the blood was cryopreserved with Dimethyl sulfoxide (DMSO) (10% v/v) and stored for 4 mo at -196℃, the detection limits of the IL-6/IL-1β responses to LPS were 0.2/0.4 endotoxin units (EU)/ml, respectively, and IL-6/IL-1β release increased in response to LPS in a dose-dependent manner. When these experiments were performed in three separate laboratories, the within-laboratory reproducibility of the IL-6/IL-1β responses was 100%/86.7%, 93.3%/100%, and 86.7%/80%, and the inter-laboratory reproducibility was 92.9%/85.7%, 64.3%/63.6%, and 57.1%/66.7%, respectively. The sensitivity (the probability of correctly classifying positive samples) and specificity (the probability of correctly classifying negative samples) of the IL-6/IL-1β tests were 81.7%/82.5% and 100%/100%, respectively. The results of this study suggest that cryopreserved pooled blood is a convenient and viable alternative for evaluating in vitro pyrogenicity. Additionally, maintaining cryopreserved pooled blood promotes safety for the user because it is released only after pretesting for infection parameters and has lower variation than fresh donations from a variety of donors.

To investigate whether Bombyx mori Linnaeus (Lepidoptera: Bombycidae) intestinal microorganism play a role in the host defence system against viral pathogens, a lipase gene from the silkworm intestinal bacterium Bacillus pumilus SW41 was characterized, and antiviral activity of its protein against B. mori nucleopolyhedrovirus (BmNPV) was tested. The lipase gene has an open-reading frame of 648 bp, which encodes a 215-amino-acid enzyme with a 34-amino-acid signal peptide. The recombinant lipase (without signal peptide) was expressed and purified by using an Escherichia coli BL21 (DE3) expression system. The total enzyme activity of this recombinant lipase reached 277.40 U/mg at the optimum temperature of 25°C and optimum pH value of 8.0. The antiviral test showed that a relative high concentration of the recombinant lipase reduced BmNPV infectivity in vitro, which resulted in decreased viral DNA abundance and viral occlusion bodies. Besides, the preincubation method also suggested that the lipase probably directly acting on the budded virions. The results suggest that the lipase from intestinal bacterium B. pumilus SW41 is a potential antiviral factor for silkworm against BmNPV.

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Two mono-Schiff base manganese(III) and cobalt(II) complexes with benzo-10aza-crown ether pendants (MnL 1 2 Cl, CoL 1 2 ), and the analogues with morpholino pendants (MnL 2 2 Cl, CoL 2 2 ) have been synthesised and employed as models for hydrolase enzymes to promote the hydrolysis of p-nitrophenyl picolinate (PNPP) in buffer solution. A kinetic model of PNPP cleavage catalysed by these complexes is proposed. The effects of the structures of the complexes and reaction temperature on the rate of PNPP hydrolysis have been examined. All four complexes exhibit high catalytic activity and the rate increases with pH. In comparison with the crown-free analogues MnL 2 2 Cl and CoL 2 2 , the crowned Schiff base complexes MnL 1 2 Cl and CoL 1 2 exhibit higher catalytic activity for promoting PNPP hydrolysis.