Long-term follow-up of photodynamic therapy with a self-adhesive 5-aminolaevulinic acid patch: 12 months data.

BACKGROUND: Photodynamic therapy with a self-adhesive 5-aminolaevulinic acid (5-ALA) patch shows high efficacy rates in the treatment of mild to moderate actinic keratosis (AK) in short term trials. OBJECTIVES: The purpose of the trial was to follow up patients after successful 5-ALA patch-PDT at 3 month intervals over a total period of 12 months. Patients who had received placebo-PDT or cryosurgery served for comparison. PATIENTS/METHODS: Three months after therapy, 360 patients from two separate randomized parallel group phase III studies (one superiority trial vs. placebo-PDT, one noninferiority trial vs. cryosurgery) were suitable for the follow-up study. Patients had to show at least one successfully treated AK lesion after initial therapy. A total of 316 patients completed the follow-up. RESULTS: Twelve months after a single treatment, 5-ALA patch-PDT still proved superior to placebo-PDT and cryosurgery (P < 0.001 for all tests). On a lesion basis, efficacy rates were 63% and 79% for PDT, 63% for cryosurgery and 9% and 25% for placebo-PDT. Recurrence rates of patch-PDT proved superior to those of cryosurgery (per protocol set: P = 0.011, full analysis set: P = 0.049). While 31% of cryosurgery lesions were still hypopigmented after 1 year, the 5-ALA patch-PDT groups showed hypopigmentation in 0% (superiority trial) and 3% (noninferiority trial) of the treated lesions. CONCLUSION: Twelve months after a single 5-ALA patch-PDT the majority of lesions were still cleared with an excellent cosmetic outcome. 5-ALA patch-PDT proved to be superior to cryosurgery in the noninferiority study setting.

Optimization of photodynamic therapy with a novel self-adhesive 5-aminolaevulinic acid patch: results of two randomized controlled phase III studies.

BACKGROUND: Photodynamic therapy (PDT) is increasingly used for treatment of actinic keratoses (AKs) but is a cumbersome procedure. A thin self-adhesive patch (PD P 506 A) containing 5-aminolaevulinic acid (5-ALA) was developed to facilitate PDT. OBJECTIVES: To investigate efficacy and safety of the patch in comparison with placebo-PDT (superiority design, observer-blinded; study AK 03) and standard therapy, cryosurgery (noninferiority design, open; study AK 04). METHODS: Two separate confirmatory randomized parallel-group phase III studies were set up. In total, 449 patients with up to eight mild to moderate AK study lesions located on the head were treated in 29 German study centres (study AK 03: 103 patients; study AK 04: 346 patients). RESULTS: Twelve weeks after treatment, 5-ALA patch-PDT proved to be superior to placebo-PDT (P < 0.001) and cryosurgery (P = 0.007). Efficacy rates on a lesion basis were 82% (AK 03) and 89% (AK 04) for PDT, 77% for cryosurgery and 19% (AK 03) and 29% (AK 04) for placebo-PDT. Local reactions at the treatment site occurred in almost all patients treated with 5-ALA patch-PDT or cryosurgery. Headache was the only side-effect not related to the treatment site which occurred in more than one patient. CONCLUSIONS: PD P 506 A is an innovative, easy-to-handle 5-ALA patch for PDT of mild to moderate AK lesions. Compared with current PDT procedures, pretreatment (e.g. curettage) is not needed and handling is considerably facilitated. A single PDT treatment results in efficacy rates being statistically significantly superior to placebo and cryosurgery.

PUVA downregulates whn expression in primary mouse keratinocytes.

Psoralen photochemotherapy (PUVA) is one of the most efficient treatment regimens for psoriasis and other skin diseases. In order to evaluate keratinocyte-specific PUVA effects, we investigated the impact of clinically relevant PUVA doses on whn, the 'nude' gene. This transcription factor plays an important role in epidermal homeostasis, and epidermal whn over-expression results in a psoriasis-like phenotype. We demonstrated a persistent down-regulation of whn mRNA 48-72 h after PUVA treatment but not after UVA alone. Using transgenic animals, we also demonstrated dose-dependent down-regulation of whn promoter activity. Finally, whn-null ('nude') keratinocytes were more resistant to PUVA-induced suppression of DNA synthesis than wild-type cells. Our results suggest that whn suppression may be involved in mediating the anti-proliferative effect of PUVA on keratinocytes.

Signaling pathways in cell death and survival after photodynamic therapy.

Photodynamic therapy (PDT) is a cytotoxic treatment, which can induce cells to initiate a rescue response, or to undergo cell death, either apoptosis or necrosis. The many signaling pathways involved in these processes are the topic of this review. The subcellular localization of the photosensitizer has been shown to be a key factor in the outcome of PDT. Mitochondrial localized photosensitizers are able to induce apoptosis very rapidly. Lysosomal localized photosensitizers can elicit either a necrotic or an apoptotic response. In the plasma membrane, a target for various photosensitizers, rescue responses, apoptosis and necrosis is initiated. Several protein phosphorylation cascades are involved in the regulation of the response to PDT. Finally, a number of stress-induced proteins play a role in the rescue response after PDT. Notably, the induction of apoptosis by PDT might not be crucial for an optimal outcome. Recent studies indicate that abrogation of the apoptotic pathway does alter the clonogenic survival of the cells after PDT. Further studies, both in vitro and especially in vivo could lead to more efficient combination therapies in which signaling pathways, involved in cell death or rescue, are either up- or downregulated before PDT.

Photodynamic treatment of pooled coumarin plasma for external quality assessment of the prothrombin time.

AIMS: To determine the conditions of photodynamic inactivation of vesicular stomatitis virus (VSV) added to pooled coumarin plasma and the effects of the photodynamic treatment on the prothrombin times and international normalised ratio (INR) in a Netherlands national external quality assessment scheme. METHODS: Pooled coumarin plasma samples were illuminated with visible light in the presence of 1 microM methylene blue. Inactivation conditions for VSV in pooled coumarin plasma were determined using an end point dilution assay. Plasma illuminated for 20 minutes was mixed with red blood cells and mailed to participants of the Netherlands external quality assessment (EQA) scheme. Prothrombin times and INRs were determined with various thromboplastin reagents. RESULTS: Photodynamic treatment using 1 microM methylene blue and 700 W/m2 caused 4.7 log inactivation of VSV in pooled coumarin plasma. Fibrinogen and coagulation factors II, V, VII, and X were decreased slightly by the treatment. These conditions caused prolongation of the prothrombin time in EQA surveys. The magnitude of the effect was different for various thromboplastin reagents. The increase of the INR was negligible when measured with the Thrombotest reagent. With other reagents, an approximately 5-16% increase of the INR was observed. Interlaboratory variation of the INR was not affected by photodynamic treatment. CONCLUSIONS: Photodynamic treatment of pooled coumarin plasma is very effective for the inactivation of some enveloped viruses such as VSV, but has only a limited effect on the prothrombin time and INR. Photodynamic treatment can be used to improve the viral safety of coumarin plasma for EQA of the prothrombin time and INR.

Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation.

Recombinant adenovirus vectors are popular tools for gene transfer and gene therapy. However biosafety constraints require that all handling of the vectors and vector-containing samples is restricted to dedicated containment laboratories, unless they had undergone a validated virus-inactivation procedure, which decontaminates the samples from any active virus. In this study we evaluated the feasibility of photodynamic treatment (PDT) with visible light to inactivate recombinant adenovirus vectors in biological samples, with minimum associated effects on other biological activities. Several photosensitizers were tested for their capacity to inactivate a model human adenovirus vector, AdCMVLuc, upon illumination. Four photosensitizers (methylene blue (MB), rose bengal (RB), uroporphyrin (UP) and aluminum phthalocynine tetrasulphonate (AIPcS4)) could inactivate the adenovirus, as measured by expression of the luciferase reporter gene and by plaque assay. Of these, MB demonstrated to be the most effective sensitizer in phosphate-buffered saline (PBS), giving > 7 log10 inactivation of the adenovirus. DNA isolated from MB- and light-treated virions was inefficient as a template for transcription. Furthermore, Southern blot analysis revealed fragmentation of the viral DNA. Based on its preference for DNA, MB is suited for adenovirus inactivation in blood plasma. Spiking experiments in which AdCMVLuc was added to plasma samples demonstrated a reduction (> 4 log10-fold) of reporter gene expression to almost background levels. In contrast to MB, photodynamic treatment with RB, UP or AIPcS4 did not lead to DNA damage. Although alterations of the viral capsid could not be detected, the binding pattern of the particles to target cells was significantly changed. Taken together, our data demonstrate that PDT is an efficient, convenient and useful method for the inactivation of adenovirus vectors in biological samples.

Photodynamic sterilization of red cells and its effect on contaminating white cells: viability and mechanism of cell death.

BACKGROUND: Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates. Various lipid-enveloped viruses can be inactivated with only limited red cell damage. Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative. STUDY DESIGN AND METHODS: White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions. Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake. Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V). RESULTS: Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake. Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant. Some of the cells die by apoptosis, which is induced within 30 minutes after treatment. Treatment with AIPCS4 damages only monocytes; other cell populations are not affected. CONCLUSIONS: Physicochemical properties of the photosensitizers partly determine their effect on white cells. Differences in intracellular localization are likely to be responsible for the effects observed.

Inhibition of various steps in the replication cycle of vesicular stomatitis virus contributes to its photoinactivation by AlPcS4 or Pc4 and red light.

Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.

Oxidized low-density lipoprotein as a delivery system for photosensitizers: implications for photodynamic therapy of atherosclerosis.

Photodynamic therapy is a promising new strategy in the treatment of cardiovascular diseases. Photodynamic therapy for vascular diseases may be improved by the specific delivery of photosensitizers to the atherosclerotic lesion. In this study, we studied whether oxidatively modified low-density lipoprotein (OxLDL) could be used as a specific carrier for photosensitizers, thereby using the scavenger receptor expressed on macrophages as a target. The photosensitizer aluminum phthalocyanine chloride (AlPc) was incorporated into OxLDL, and its photodynamic effects were studied. Macrophages (RAW 264.7) were incubated with various concentrations of OxLDL-AlPc for different periods. After illumination of the cells with red light, cytotoxicity was observed that was dependent on the time of illumination and incubation. Macrophages incubated with OxLDL-AlPc that were not illuminated revealed no cytotoxicity. The uptake of the OxLDL-AlPc complexes was mediated by scavenger receptors expressed on macrophages. In the presence of the polyanion polyinosinic acid, a specific ligand for scavenger receptors, no cytotoxicity could be observed. Serum incubations of the OxLDL-AlPc complexes revealed that these complexes stay intact after incubation. No redistribution of AlPc to other plasma (lipo-) proteins could be detected, and 80-90% of the AlPc remained associated with the OxLDL particle. These results indicate that OxLDL may function as a specific delivery system for photosensitizers to the scavenger receptors expressed on the macrophages in the atherosclerotic lesion, increasing the beneficial effects of photodynamic therapy for cardiovascular diseases.

Transfusion-transmitted diseases: risks, prevention and perspectives.

During the past decades major improvements in blood safety have been achieved, both in developed and developing countries. The introduction of donor counseling and screening for different pathogens has made blood a very safe product, especially in developed countries. However, even in these countries, there is still a residual risk for the transmission of several pathogens. For viruses such as the human immunodeficiency virus (HIV), and the hepatitis viruses B and C, this is due mainly to window-period donations. Furthermore, the threat of newly emerging pathogens which can affect blood safety is always present. For example, the implications of the agent causing new variant Creutzfeld-Jakob disease for transfusion practice are not yet clear. Finally, there are several pathogens, e.g. CMV and parvo B19, which are common in the general donor population, and might pose a serious threat in selected groups of immunosuppressed patients. In the future, further improvements in blood safety are expected from the introduction of polymerase chain reaction for testing and from the implementation of photochemical decontamination for cellular blood products. The situation in transfusion medicine in the developing world is much less favorable, due mainly to a higher incidence and prevalence of infectious diseases.

Shelf-life of photodynamically sterilized red cell concentrates with various numbers of white cells.

BACKGROUND: Phthalocyanines are useful sensitizers for the photodynamic sterilization of red cell concentrates. The use of the phthalocyanine Pc4 (HOSiPcOSi(CH3)2(CH2)3N(CH3)2) and red light is very efficient in killing various viruses. The addition of scavengers of Type I photodynamic reactions and the use of cremophor to deliver Pc4 give protection to the red cells. STUDY DESIGN AND METHODS: Various red cell components, either white cell-enriched, buffy coat-removed, or white cell-reduced, have been used to study the effect of photodynamic treatment with Pc4 on hemoglobin and potassium leakage and on ATP and glucose levels after prolonged storage. RESULTS: After treatment, storage interval-dependent damage to the red cells could be observed. In components with 26 x 10(9) white cells per L, virus inactivation was less efficient than that in components with no or 2 x 10(9) white cells per L. Similarly, red cells were less affected by the treatment in components with a large number of white cells. Pretreatment storage and use within 1 week after photodynamic treatment induce less damage to the red cells at the moment of transfusion. CONCLUSION: Various improvements in the treatment protocol may ultimately lead to the implementation of photodynamic treatment in transfusion practice. In this respect, the white cell content of the red cell concentrates should be taken into account.

Primary targets for photoinactivation of vesicular stomatitis virus by AIPcS4 or Pc4 and red light.

Phthalocyanines are useful sensitizers for the photodynamic sterilization of red blood cell concentrates. The mechanism of photoinactivation of lipid-enveloped viruses is not completely understood. Vesicular stomatitis virus (VSV) was used as a model virus to study the primary targets of photoinactivation by aluminum phthalocyanine tetrasulfonate (AIPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Inactivation conditions for VSV in buffer were determined using an end point dilution assay, and viral RNA synthesis in host cells was measured to determine the loss of infectivity in a direct way. The very rapid decrease in the viral RNA synthesis after photodynamic treatment was correlated with respect to different potential primary targets that are involved in different steps of the viral replication cycle. Damage to the viral proteins, induced by treatment with AIPcS4 or Pc4 and analyzed by gel electrophoresis, could not account for the observed loss of infectivity. Binding of VSV to host cells was only slightly impaired after photodynamic treatment with both sensitizers and could therefore not be responsible for the rapid decrease in viral RNA synthesis in cells. A very strong inhibition of viral RNA polymerase activity after treatment with AIPcS4 and red light was detectable using an in vitro assay. This decrease correlated well with the loss of infectivity, indicating that either the RNA or the viral RNA polymerase is the primary target for photoinactivation of VSV with AIPcS4. Treatment with Pc4 did not cause inhibition of viral RNA polymerase activity to an extent that could account for the observed very rapid loss of infectivity. It was therefore concluded that neither the viral proteins nor the binding to the host cells nor the RNA or RNA polymerase are the primary targets for photoinactivation of VSV by Pc4.

In vitro fluence rate effects in photodynamic reactions with AIPcS4 as sensitizer.

It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AIPcS4) is more efficient in photosensitization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m2 as compared to 500 W/m2. Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo, does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explantation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni- or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.

The influence of merocyanine 540 and protoporphyrin on physicochemical properties of the erythrocyte membrane.

The interaction of the red cell membrane with merocyanine 540 or protoporphyrin led to four phenomena, most probably interrelated. (i) The morphology changed from the normal discoid to an echinocytic form. This morphological change persisted when followed over a period of 24 h. (ii) Simultaneously, cell deformability was decreased, as revealed by viscosity measurements and a cell-filtration technique. (iii) Both drugs caused swelling of the erythrocytes in isotonic medium, due to a very-short-term increased permeability of the membrane, also for larger molecules such as lactose. The pathway of this temporary leak seems to be unrelated to the Na+/K+ -ATPase, the K+/Cl- and the Na+/K+/Cl- cotransport systems, the Ca2+-activated Gardos pathway, the oxidation/deformation-activated leak pathway and the so-called residual transport route. Despite the morphological changes, K+-leakage induced by mechanical stress was not increased. (iv) During osmotic swelling, the critical hemolytic volume was found to be increased in the presence of either merocyanine 540 or protoporphyrin. The increase critical volume protected erythrocytes against osmotic hemolysis.

Treatment with 8-MOP and UVA enhances MHC class I synthesis in RMA cells: preliminary results.

The response of psoriasis and cutaneous T-cell lymphoma to treatment with 8-methoxypsoralen (8-MOP) and long wavelength ultraviolet light (UVA) is only partly understood. Psoralens form photoadducts within the DNA after activation by UVA and this damage leads to the inhibition of DNA synthesis. Additionally, it has been shown that different forms of DNA damage can induce a stress response, leading to upregulation of selected products. Among these are the major histocompatibility complex (MHC) class I genes. Thus the aim of the present study was to assess the rate of synthesis of MHC class I proteins in murine T-cell lymphoma cells (RMA) after treatment with 8-MOP and UVA. RMA cells were treated with 8-MOP (50-200 ng ml-1) and UVA (1 J.cm-2) and metabolically labelled with 35S-methionine 4 and 24 h after treatment. MHC class I synthesis was determined by immunoprecipitation of the cell lysates with an anti-Kb monoclonal antibody, Y3. After 4 h, treated and untreated cells demonstrated no differences in the rate of MHC class I synthesis. However, after 24 h a dose-dependent increase in MHC class I synthesis was observed. This increase in MHC class I expression could be responsible, at least partly, for the responses observed in patients treated with photopheresis.

Increased surface expression of class I MHC molecules on immunogenic cells derived from the xenogenization of P815 mastocytoma cells with 8-methoxypsoralen and long-wavelength ultraviolet radiation.

In a previous study we demonstrated that the treatment of the highly tumorigenic cell line, P815, with 8-methoxypsoralen and long-wavelength ultraviolet radiation resulted in the production of several immunogenic clones (tum-). Mice inoculated with the tum- cells survived much longer than mice inoculated with the original tumorigenic cells (tum+). It was suggested that the increased survival of mice treated with the tum- clones arose as a result of an increased antigenicity derived from the phototreatment. In this report we show that the tum- cells have a greater density of class I MHC molecules on their surface (50-157% compared to P815). Class I MHC density on the cell surface is required to elicit targeted cytotoxic responses. These results can be considered in terms of human class I MHC assays which show that many human tumor cells have a reduced expression of class I MHC. Because other DNA damaging agents have also been shown to enhance class I expression, it is suggested that in addition to the cytotoxic effects of these agents, other pleiotropic effects must be considered. Photochemotherapy may phenotypically alter cells so that the enhanced expression of class I MHC molecules on the surface of phototreated cells may be associated with the clinical responses observed in cutaneous T cell lymphoma patients.

Lymphocyte adhesion to brain capillary endothelial cells in vitro.

The presence and upregulation of adhesion molecules on bovine brain endothelial cells (BBEC) were investigated. Monolayers of BBEC were incubated with lipopolysaccharide (LPS), interleukin-1 beta (rhIL-1 beta), and interleukin-6 (rhIL-6) to simulate in vitro an inflammatory site in the cerebral capillaries. Adhesion of lymphocytes to BBEC increased 4.1-fold after stimulation of the endothelial cells for 4 h with 5 or 10 ng/ml LPS. Lymphocyte adhesion increased after incubation of the BBEC for 4 h with IL-1 and was increased 3.7-fold using 100 ng/ml IL-1. BBEC pre-incubated with IL-6 for 4 h also showed an increase in adhesion of lymphocytes, and cells pretreated with 100 ng/ml IL-6 showed a 3-fold increase in lymphocyte adherence. Specific monoclonal antibodies directed against CD11a, CD18, and VLA-4 were able to block adherence of lymphocytes to stimulated BBEC. These results indicate that the in vitro activation of BBEC may serve as a model for the study of inflammation of the blood-brain barrier.