Photodynamic therapy (PDT) is a recently approved treatment modality that involves the sequential administration of a photosensitizer or its precursor and light to generate singlet oxygen for treating diseased tissue. The use of topical aminolevulinic acid (ALA) and blue light for nonhypertrophic actinic keratoses currently represents the only approved dermatologic application for PDT in the U.S.A. ALA is a photosensitizer precursor that is metabolized by cells into protoporphyrin IX (PpIX), which can be subsequently activated by visible light. PDT with topical ALA has been shown to improve psoriasis, but post-treatment hyperpigmentation as well as inconsistent clinical responses despite repeated PDT sessions have limited the development of this treatment approach for psoriasis. Furthermore the use of topical PDT photosensitizers becomes somewhat impractical for treating larger body surface areas in patients with extensive psoriasis. We have recently shown that oral administration of ALA induces preferential accumulation of PpIX in psoriatic plaques. The objectives of this study were to evaluate the effects of PDT with blue light on psoriatic plaques after systemic ALA administration as well as to determine whether systemic ALA-PDT induces apoptosis in lesional T lymphocytes. It has been suggested that induction of apoptosis in lesional T lymphocytes may be indicative of longer remission time following treatment of psoriasis.

Toluidine blue (TBO) is a cationic thiazine dye with an affinity for neoplastic tissues in vivo. The objective of this study was to explore the in vitro photosensitizing potential of TBO and its capacity to induce apoptosis in human leukaemic T cells. Jurkat cells were incubated with TBO for one hour followed by exposure to 11 J cm(-2) of visible light from a slide projector. Cytotoxicity was assessed at 24 hours using a MTT assay. DNA fragmentation was examined at different intervals after photodynamic treatment using a DNA elution-filtration assay with [14C]-thymidine labelled cells. Caspase-3 like activation induced by photodynamic treatment was studied by measuring AC-DEVD-AMC peptide hydrolysis. The MTT assay showed a 97% decrease in optical density 24 hours following photodynamic therapy with 0.15 microg ml(-1) of TBO. Dark toxicity was absent under these conditions. DNA fragmentation was detected as early as 2 hours after photodynamic therapy and reached 68% at 6 hours. At higher TBO concentrations less DNA fragmentation and more dark toxicity was observed. An increase in caspase-3 like activity was also induced by photodynamic therapy with TBO. At the time of light exposure TBO was present in the endoplasmic reticulum and Golgi regions. In conclusion, TBO-based photodynamic therapy has a potent phototoxic effect and induces apoptosis in Jurkat cells.

A sensitive enzyme immunoassay (EIA) for galactomannan antigenemia that avoids the use of radioisotopes was devised. Three carbohydrate-rich antigenic fractions were purified from Aspergillusfumigatus 2085: a cold alkali extract (CA) from mycelium, an acetone-precipitated pyridine extract (APSK-66) from mycelium, and a methanol precipitate from culture filtrate. CA and APSK-66 were further purified by gel filtration and ion-exchange chromatography, respectively. An acid hydrolysate of CA contained only mannose and galactose, as determined by gas-liquid chromatography. Rabbit antisera were raised against conidia, mycelia, and cell walls ofA. fumigatus. By indirect EIA, the best immunoglobulin G response (1/8,000) was obtained against CA in rabbits immunized intravenously with cell walls. Antigenemia was detected by indirect EIA inhibition in heat-dissociated sera of four immunosuppressed rabbits that were infected intravenously but was absent in two uninfected controls. The circulating antigen was resistant to pronase, was adsorbed onto concanavalin A, and had a molecular size of 50 to 100 kilodaltons.

A number of studies have substantiated the pivotal role of innate defense mechanisms in protection against invasive aspergillosis. However, experiments demonstrating increased resistance to lethal intravenous (i.v.) infection with Aspergillusfumigatus conidia in cortisone-treated or untreated mice preinfected with a sublethal dose of conidia and protection of turkeys inoculated subcutaneously with a killed A.fiumigatus germling vaccine against subsequent aerosol challenge led us to speculate that acquired immunity may also contribute to host defense against Aspergilus infection. Five-week-old male BALB/c mice were inoculated i.v. with 1.0 x 104 viable conidia or saline and challenged i.v. with 1.0 x 106 conidia after 7, 15, or 21 days. No protection against challenge was found after 7 days. However, significant and reproducible protection was observed after 15 and 21 days. Mortality was reduced from 90% in control mice to 53% in preinfected mice 40 days after challenge (P = 0.0002). Increased survival was correlated with decreased content of chitin in lungs, liver, and kidneys 4 and 7 days after challenge (P < 0.05). Mice were again inoculated with 1.0 x 104 conidia or saline, and after 21 days, 1.0 x 10' or 2.0 x 108 splenocytes were transferred to naive syngeneic recipients; 2.0 x 10' immune splenocytes conferred significant protection (P = 0.0001) against i.v. challenge with 1.0 x 106 conidia, and mortality decreased from 83 to 48% 40 days after challenge. Transfer of immune serum offered no protection despite the presence of antibody against a hyphal homogenate ofA.fmigatus, which was absent in the sera of control mice. Protection by immune splenocytes was maintained after selective depletion of T cells but was abolished after removal of plastic-adherent splenocytes. Adherent cells were characterized as macrophages by using morphological criteria, nonspecific esterase, and MAC-1 monoclonal antibody. Production of hydrogen peroxide by peritoneal and splenic macrophages from preinfected mice was the same as and lower than, respectively, that from uninfected controls. However, phagocytosis of conidia by peritoneal or splenic macrophages from mice preinfected i.v. or intratracheally was significantly increased after 2 and 3 h of coculture compared with that from uninfected animals, whereas in vitro killing of conidia by splenic macrophages was unaltered. Peritoneal or splenic macrophages from control or preinfected mice failed to kill hyphae in vitro. Killing of hyphae by polymorphonuclear leukocytes was not significantly different between mice preinfected i.v. and uninfected controls. Taken together, the results indicate that acquired immunity mediated by activated macrophages can be demonstrated in experimental murine aspergillosis. Although the mechanism is present biologically, its relevance against the invasive hyphal form ofA. fimigatus is doubtful.

ALA-PDT induces apoptosis and caspase-3-like activation in Jurkat cells.