During phacoemulsification cavitation bubbles are formed. These bubbles are believed to be one source of damage to corneal endothelium seen after phacoemulsification. Free radicals are induced whenever cavitation bubbles implode. The aim of this study was to confirm the initiation of free radicals by phacoemulsification and to correlate the power of ultrasound in the phacoemulsification process to the amount of free radicals formed, using both in vitro and in vivo techniques. The formation of free radicals was determined by adding luminol to a buffer and measuring the chemoluminescence in vitro and in rabbit eyes (Lumacounter 2080 or a single-photon-counting apparatus) during phacoemulsification. The data obtained show that free radicals are formed during phacoemulsification and that the amount of free radicals correlates with the power of ultrasound. Furthermore, the radical formation could be inhibited by the radical scavengers SOD, Healon and Healon GV. These results were achieved both in vitro in the test tube and in vivo in rabbit eyes. By showing that the addition of SOD to the irrigation buffer during phacoemulsification decreases the corneal endothelial cell damage, we show that free radicals could have a role in postoperative complications seen clinically.

Five flavonoid compounds were isolated from two Polypodium species (P.decumanum and P.triseriale) with the common name Calaguala. Structure elucidation was carried out using different NMR techniques and revealed the presence of one new glycoside (kaempferol 3-O-beta-D-xylopyranosyl-(1-2)-beta-D-arabinopyranoside) (1), two known flavonoid glycosides, rutin and kaempferol 3-O-alpha-D-arabinopyranoside (2,3), the trimeric proanthocyanidin, selligueain (4), and the coumarinic acid derivative, melilotoside (5). The compounds were tested for their activity in PAF induced exocytosis in human neutrophils but none of the compounds showed PAF specific activity. Instead, they showed more general effects on the neutrophil including inhibition of the spontaneous elastase release (5) and potentiation of the release induced by PAF (1). Selligueain was found to inhibit the proteolytic enzyme, elastase in vitro.

A rapid and convenient protein-gel blot technique for qualitative detection of antigens/allergens in pollen allergen extracts and IgE/IgG antibodies in patient sera has been developed. The antigens were separated by isoelectric focusing in agarose gel and transferred to nitrocellulose by capillary migration. After incubation of the nitrocellulose strips with serum from allergic patients, the binding of the patient’s specific IgE or IgG antibodies was analyzed by using isotope-labelled or enzyme-labelled anti-IgE or anti-IgG. The time needed for detection with isotope-labelled antibody was approximately 20 h and with enzyme-labelled antibody 2 h. The immunoprint technique is easy to use, which renders it suitable for routine use in allergy research and quality control.

The allergen composition of moulds (Alternaria alternata and Aspergillus fumigatus) and mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae) was studied by electroblotting. The allergens were separated by SDS-gPAGE and transferred to a nitrocellulose membrane by electroblotting. Specific IgE antibodies directed against mould and mite allergens were utilized as probes to detect the allergens. The bands were localized on the nitrocellulose membrane by means of enzyme- or isotope-labelled antibody. Circulating specific IgE antibodies in mould and mite sensitive patient sera were also analyzed and characterized with respect to their IgE specificity. Under appropriate experimental conditions this electroblotting technique could also be used for quantitation of individual allergens by scanning the isotope- or enzyme-stained nitrocellulose strips.

Calaguala, an extract from the fern Polypodium decumanum, has been used to treat psoriasis and related immunological disorders. In an effort to explain Calaguala's medicinal effects the inhibitory activity of the extract in two platelet activating factor (PAF) related models has been investigated. In the first model, PAF was used to induce release of the proteolytic enzyme elastase in human neutrophils. Calaguala inhibited this effect with an IC50 of 0.1 mg/ml. The known PAF antagonist ginkgolide BN 52021 was used as a positive control and had an IC50 of 0.034 mg/ml. In the second model the inhibition of biosynthesis of PAF in neutrophils using lyso-PAF and labeled acetyl-CoA was studied. Also in this assay Calaguala showed a dose-dependent activity, the IC50 being 0.2 mg/ml. Since recent findings have indicated that PAF might be involved in the pathogenesis of psoriasis, it is possible that the activity shown by Calaguala in these PAF assays may contribute to the clinical efficacy of the extract. The PAF induced exocytosis assay was further used to guide the fractionation of the crude extract. From the acetone supernatant the nucleoside adenosine was isolated as an active principle. Pure adenosine dose-dependently inhibited the exocytosis induced by PAF (IC50 = 0.024 micrograms/ml) but was inactive in the biosynthesis assay. Adenosine is most probably one of the bioactive compounds of Calaguala responsible for its therapeutic properties.