We developed a simple new endotoxin-specific assay method that uses Limulus amebocyte lysate (LAL) containing a sufficient amount of a water-soluble (1→3)-β-D-glucan derivative as a blocker of the (1→3)-β-D-glucan-mediated coagulation pathway. The addition of 0.1mg/ml or more of carboxymethylated (1→3)-β-D-glucan completely blocked the activation of LAL by (1→3)-β-D-glucan itself. The assay of endotoxin was unaffected by the presence of 1mg/ml carboxymethylated (1→3)-β-D-glucan. Spiked endotoxin was recovered well from β-glucans by the turbidimetric kinetic method with LAL containing 1mg/ml of carboxymethylated (1→3)-β-D-glucan. Besides, this new LAL formulation was applied for an endotoxin-specific assay by the conventional gel-clot method or the chromogenic method. Gram-negative bacteria were specifically detected by the turbidimetric kinetic method with the LAL formulation. This LAL formulation may be used for an endotoxin-specific assay not only in pharmacology but also in clinical microbiology.
We examined the corresponding isomers of catechins and theaflavins for anti-hemolysin activities against Staphylococcus aureus α-toxin and Vibrio cholerae O1 hemolysin. Catechins and theaflavins showed anti-hemolysin activities in a dose-dependent manner. Among the catechins tested, (-)catechin gallate, (-)epicatechin gallate and (-)epigallocatechin gallate having galloyl groups in their molecules showed more potent anti-hemolysin activities against both toxins. On the other hand, free catechins, i.e. (-)catechin, (-)gallocatechin, (-)epicatechin and (-)epigallocatechin had low anti-hemolysin activities against α-toxin. Although (-)catechin or (-)gallocatechin had no effect on cholera hemolysin, (-)epicatechin and (-)epigallocatechin were slightly inhibitory. Among dextrocatechins, (+)epicatechin and (+)epigallocatechin proved to be more effective than (+)catechin and (+)gallocatechin. The anti-hemolysin activities of theaflavins against α-toxin and cholera hemolysin were dependent on the number of the galloyl group in their structure. These results suggest that the tertiary structure of the catechin or theaflavin and the active site of hemolysin, that affects the interaction between them, plays an important role in the antihemolysin activity.
We have developed a simple method for assessing the oxidative metabolic burst of peripheral blood leukocytes with a minute amount of whole peripheral blood by flow cytometry according to the method of Bass et al. with some modification. By this method, we can measure the H2O2 production by both granulocytes and monocytes in the same blood sample. The oxidative product formation by peripheral blood neutrophils can be monitored sequentially in the same mouse infected with E. coli. The mice infected intravenously with 0.1 LD50 of the bacteria showed increased basal activities from an early stage of infection; those infected intraperitoneally with the same dose of the bacteria showed a delayed enhancement. In case of infection with 0.01 LD50, the enhanced basal activities lasted for only a short period of time. The H2O2 production was correlated well with the clearance of the infected bacteria. These results demonstrated that the oxidative-product formation by peripheral blood neutrophils is affected by both the route and the dose of infection.
Improvement of the fermentation-inhibition (FI) test for Mycoplasma pneumoniae was attempted. The sensitivity of detecting the FI antibody to M. pneumoniae in the homologous immune rabbit serum was notably elevated, when such FI medium containing gamma globuline-free horse serum and guinea-pig complement in substitution for unheated horse serum was used. The M. pneumoniae suspension was filtered to remove aggregates of the organisms and used as the antigen for this new FI test. In detection of the serum antibody of patients with M. pneumoniae infections, the new FI test showed much higher sensitivity than conventional FI test and strongly correlated (r=0.84) with high density particle agglutination test known to detect both IgG and IgM antibodies.