5-aminolaevulinic acid-based photodynamic therapy induces both necrosis and apoptosis of keratinocytes in plantar warts.

BACKGROUND: 5-aminolaevulinic acid (ALA)-based photodynamic therapy (ALA-PDT) is an effective treatment for proliferative skin diseases. Many studies revealed that ALA-PDT induces apoptosis of cancer cells. However, the mechanism of PDT in warts is not fully elucidated. OBJECTIVE: This study was designed to explore the efficacy of ALA-PDT for plantar warts and its possible mechanism. METHODS: Twenty-five patients with plantar warts underwent four rounds of ALA-PDT at weekly intervals. Therapeutic effects were observed 1 month since last treatment. The adverse reactions were also recorded during and after the procedure. To observe histological changes, 5 patients accepted tissue biopsies before and 24 h after first PDT treatment. RESULTS: Twenty-two patients (88%) showed a complete response. Twenty patients complained of a mild burning sensation during treatment, and four patients developed local edema immediately after red light exposure. Five patients had burns after treatment, which disappeared within 24 h. Histologically, epidermal keratinocytes demonstrated remarkable necrocytosis and apoptosis after PDT. CONCLUSIONS: ALA-PDT is highly effective for treatment of plantar warts. The most common adverse effects observed in patients are pain and edema. ALA-PDT might eradicate plantar warts by activating both necrosis and apoptosis to trigger the death of proliferating keratinocytes.

The lupus-derived anti-double-stranded DNA IgG contributes to myofibroblast-like phenotype in mesangial cells.

Anti-double-stranded DNA (dsDNA) antibodies have been indicated to play a major role in the pathogenesis of lupus nephritis (LN), which is characterized by mesangial alterations, including phenotypic changes. To explore the effects of anti-dsDNA antibodies on the phenotype of mesangial cells (MCs), the anti-dsDNA IgG in sera and histological features of glomeruli were analyzed in the mice models of immune-complex glomerulonephritis. The MCs were cultured in vitro with the addition of anti-dsDNA or non-anti-dsDNA IgG. Compared to the anti-dsDNA-negative controls, the serum positive mice had increased extracellular matrix accumulation and higher alpha-smooth muscle actin expression in the mesangial region. The anti-dsDNA IgG enhanced the synthesis of transforming growth factor beta, alpha-smooth muscle actin, and fibronectin, and even induced the myofibroblast-like morphological features in cultured MCs. Our results indicated that anti-dsDNA antibodies contribute to the phenotypic changes in MCs, which suggests another mechanism of renal injuries in LN induced by anti-dsDNA antibodies.

The influence of temperature on 5-aminolevulinic acid-based photodynamic reaction in keratinocytes in vitro.

BACKGROUND/PURPOSE: Based on the observation that increasing skin temperature could improve 5-aminolevulinic acid (ALA) penetration and accumulation of protoporphyrin IX (PpIX) in the ALA-based photodynamic therapy (PDT), this study was designed to investigate how temperature change varied the therapeutic effect of ALA-based PDT in vitro. METHODS: HaCat cells were cultured with or without ALA at various temperatures. ALA uptake and PpIX accumulation were analyzed before laser irradiation as the baseline. After irradiation, cell death and cytokine secretions in the media, including interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha and basic fibroblast growth factor (bFGF) were assayed, and the morphological changes were recorded. RESULTS: With increasing temperature, the amount of ALA uptake, intracellular PpIX concentration and cell death increased in both the PDT and the non-PDT groups. Secretions of IL-1alpha, TNF-alpha and bFGF also increased and reached a peak around 44-47 degrees C and then declined at a higher temperature. This biphasic response might be due to protein thermolysis that occurs when cells reach beyond thermal tolerance. CONCLUSIONS: Elevating temperature could augment photodynamic reactions to a certain extent, but adverse effects occurred when cells were overheated.

Desferrioxamine shows different potentials for enhancing 5-aminolaevulinic acid-based photodynamic therapy in several cutaneous cell lines.

Protoporphyrin IX (PpIX) is the photosensitizer in 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). Its further bioconversion to heme requires iron and can be suppressed by iron chelators such as desferrioxamine (DFO). To investigate the effectiveness of DFO in enhancing PpIX-based PDT in skin tissue, we selected fibroblasts, HaCat cells and Hep-2 cells as targets co-cultured with ALA, that have different biological characteristics for PpIX conversion. Evaluated interventions included: (1) blank control (no ALA, no DFO); (2) DFO alone; (3) ALA alone; and (4) DFO in combination with ALA. Before photodynamic irradiation, cellular PpIX level and fluorescence were measured. After irradiation, cell death ratio was calculated and morphological changes in the cells were observed. The results showed that the content and photodynamic effects of cellular PpIX presented in the order Hep-2 cells > HaCat cells > fibroblasts, either co-cultured with ALA alone or with ALA plus DFO. DFO was found to have increasing effects on both PpIX level and cell death ratio in the same order. It was found that DFO had different potentials for augmenting ALA-PDT in these cutaneous cell lines. The cells proliferating more rapidly might be more susceptible to enhancement of DFO.

A comparative study on the enhancement efficacy of specific and non-specific iron chelators for protoporphyrin IX production and photosensitization in HaCat cells.

The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). The purpose of this study is to compare the effect of two kinds of iron chelators, desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA) on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark, the concentration of cellular protoporphyrin IX (PpIX) was detected by high performance liquid chromatography (HPLC), and the fluorescence of PpIX was observed at 630 nm emission under confocal laser scanning microscope. For PDT, HaCat cells were irradiated using 632.8 nm laser, and the fractions of apoptotic and necrotic cells were flow cytometrically assayed. Related differences in morphology and ultrastructure of Ha-Cat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone, the addition of DFO or EDTA increased the concentration of cellular PpIX and the fluorescent density of PpIX, and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpIX level, greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpIX production and PDT. Compared to the non-specific iron chelator of EDTA, the specific chelator, DFO, showed more potential for the enhancement.

[Effects of elevating temperature on photodynamic reaction in laryngeal squamous carcinoma cells induced by delta-aminolevulinic acid].

OBJECTIVE: To investigate effect of temperature on delta-aminolevulinic acid (ALA) induced photodynamic reaction in laryngeal squamous carcinoma cells. METHODS: Human laryngeal squamous carcinoma cells of the line Hep-2 cells were co-cultured with 2 mmol/L ALA (Group A) or without ALA (Group B) at the culturing temperature 19 - 46 degrees C. Three hours later cellular protoporphyrin IX (PpIX) level was determined by high performance liquid chromatography with fluorescent detection. Laser scanning confocal microscopy was used to observe the fluorescent strength in the Hep-2 cells. The ratio of cell death (including necrosis and apoptosis) was detected with flow cytometer before and after photodynamic reaction. RESULTS: In Group A the PpIX level at the temperature of 19 degrees C was (0.25 +/- 0.06) microg/L, and raised to (1.07 +/- 0.11) microg/L at 46 degrees C. There was no cellular PpIX detected in Group B at any temperature. Before laser radiation the cell death ratios of both groups were the same at the temperature 19 - 37 degrees C, and at the temperature 37 - 46 degrees C. After laser radiation the cell death ratio of Group A raised from 31.11% to 98.92% as the temperature went up steadily, but the results of Group B showed the same curve as before laser radiation. At any same temperatures the cell death ratios of Group A were all significantly higher than those of Group B (all P < 0.05), and as the temperature was elevated the difference between the 2 groups raised from 28.99% to 59.26%. CONCLUSION: Moderate higher temperature enhances the PpIX production and photodynamic reaction in human laryngeal squamous carcinoma induced by ALA in vitro.

A Comparative Study on the Enhancement Efficacy of Specific and Non-specific Iron Chelators for Protoporphyrin IX Production and Photosensitization in HaCat Cells / 华中科技大学学报（医学）（英德文版）

The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). The purpose of this study is to compare the effect of two kinds of iron chelators,desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA) on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark,the concentration of cellular protoporphyrin IX (PpIX) was detected by high performance liquid chromatography (HPLC),and the fluorescence of PpIX was observed at 630 nm emission under confocal laser scanning microscope.For PDT,HaCat cells were irradiated using 632.8 nm laser,and the fractions of apoptotic and necrotic cells were flow cytometricaUy assayed. Related differences in morphology and ultrastructure of HaCat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone,the addition of DFO or EDTA increased the concentration of cellular PpIX and the fluorescent density of PpIX,and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpIX level,greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpIX production and PDT. Compared to the non-specific iron chelator of EDTA,the specific chelator,DFO,showed more potential for the enhancement.