The generation of leukotrienes (LTC4, LTD4, LTE4, and LTB4; 12-epi-LTB4 isomer) from human granulocytes by thiol-activated toxins (streptolysin 0, alveolysin from Bacillus alvei, and theta toxin from Clostridium perfringens) is described. The release occurs under noncytolytic conditions. Although LTB4 is the major component after calcium ionophore stimulation, more LTC4 as compared with LTB4 is released with the toxins. The 5-lipoxygenase pathway of toxin-mediated activation can effectively be inhibited by caffeic acid, a lipoxygenase inhibitor. The toxins also induce the release of leukotriene-metabolizing enzymes such as y-glutamyltranspeptidase, which transfers LTC4 into LTD4, and dipeptidase, which metabolizes LTD4, into LTE4. Dipeptidase activity is more pronounced than they -glutamyltranspeptidase activity but still does not reach the levels obtained when cells were triggered with opsonized zymosan.
A blocking factor (BF) was obtained from normal and rat reaginic serum which inhibited the passive cutaneous anaphylactic (PCA) reaction of mouse and rat IgE in rat skin. Several chromatographic procedures on DEAE- and CM-cellulose at various pH values were compared in an attempt to obtain BF with high activity. BF-containing fractions after DEAE chromatography at pH 5,4 revealed two components on SDS-polyacrylamide gel: one at 50,000–60,000 and the other at 65,000–70,000 daltons. The latter component was also obtained when normal or rat reaginic serum was adsorbed on Sepharose anti rat IgG. The eluate inhibited the PCA reaction and demonstrated two bands on SDS-polyacrylamide gel, one at 150,000 (IgG) and another at 65,000–70,000 daltons. Purified IgG and rat albumin did not inhibit the PCA reaction. Antisera against BF were obtained with the component at 65,000–70,000 daltons. Anti-BF revealed a specificity for two bands in the cathodal position when assayed against normal rat serum; one of which was IgG. Anti-BF recognized one component in the DEAE fraction when analyzed against the DEAE fraction which contained blocking activity. BF is immunologically distinct from IgG. After immunoadsorption of normal or rat reaginic serum on Sepharose anti-BF columns, an eluate was obtained which showed blocking activity in the PCA reaction and two components on SDS-polyacrylamide gel, one at 150,000 and the other at 65,000–70,000 daltons. Evaluation of blocking factor by isoelectric focussing obtained after various procedures revealed an isoelectric point of 4.7. BF shows dual functions: it inhibits and at high dilutions also enhances the PCA reaction. It is suggested that this factor has a modulatory role in the allergic disease process.
Recently, we described the presence of a blocking factor (BF) in rat serum which inhibited the histamine release from rat mast cells in vivo and in vitro. The blocking activity was demonstrated in human serum as well. Purification of the human-BF was carried out in a similar way as previously described for the rodent molecule. Both protein fractions produced a marked suppression of histamine release from rat mast cells and human basophils in a dose-dependent fashion. Qualitative analysis of the purified preparations demonstrated a major component with a molecular weight of 70,000 daltons. In human serum the blocking factor was identified as transferrin by serological and biochemical methods. It is suggested that this molecule may play an important role in regulating histamine release during allergic and inflammatory reactions.
Higher mg/kg doses led to higher plasma concentrations but did not lead to increased clinical efficacy. Anti-inflammatory dose doxycycline (40-mg controlled-release formulation) conferred peak anti-inflammatory efficacy in the treatment of rosacea.
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