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Object. The goal of this study was to develop a method of reducing neural xenograft rejection by pretreating the graft with photodynamic therapy (PDT).Methods. Xenograft cell suspensions were prepared from fetal mouse mesencephalon, after which they were incubated for 30 minutes with various concentrations of a photosensitizer, verteporfin for injection, and light exposure. The xenograft cell suspensions were injected into the dopamine-depleted striata of 40 hemiparkinsonian rats assigned to different treatment groups. Four weeks after transplantation, xenograft function (determined by methamphetamine-induced rotation) and survival (determined by immunohistochemical staining for murine neurons) were compared. Group 1 animals (xenografts pretreated with 25 ng/ml verteporfin) and Group 3 animals (no verteporfin pretreatment, but daily administration of cyclosporin A) had significantly better xenograft survival and function compared with control animals (no pretreatment with verteporfin). Group 2 animals (xenografts pretreated with 250 ng/ml verteporfin) had no significant improvement.Conclusions. This work demonstrates improved neural xenograft survival and function when using pretransplant PDT of the graft in a rodent model. The potential benefits of this new therapy are its convenience (one pretransplant treatment) and its compatibility with host immunosuppression.

Background The ability to prolong graft survival, in some cases by depleting donor antigen-presenting cells (APCs), and the subsequent demonstration that lymphocytes stimulated by non-APCs become anergic, suggested that graft survival and tolerance induction might be achieved by manipulating donor APCs to render them incompetent. This possibility was tested in histoincompatible murine skin allograft with photodynamic therapy (PDT). Methods Skin sections (C57BL/6) were exposed in vitro to low doses of benzoporphyrin derivative monoacid ring A (BPD) (verteporfin) and light (A=690+/-10 nm; low-dose PDT) before implantation on recipients (BALB/c). Furthermore, the effect of the treatment on the surface molecules of donor-derived Langerhans cells (LC) was evaluated by fluorescence-activated cell sorter analysis; the effect of treatment on the LC alloreactivity in the mixed epidermal cell lymphocyte reaction was also evaluated. Results Pretreating skin to be grafted with low-dose PDT can significantly prolong the survival of allografts from 9.3+/-2.2 (n=42) days (control group) to 16.9+/-1.7 days (n=20; treated group). Moreover, low-dose PDT significantly down-regulated the major histocompatibility complex and costimulatory (B7) molecules (60-90% reduction) on LC, but not LC-specific endocytic receptor (DEC-205), CD45, intercellulr adhesion molecule 1, LC viabilities, and ectophosphatase activity on LC. Additionally, this treatment significantly suppressed the ability of LC to stimulate alloreactive T cells to proliferate. Conclusions Since engaging T cell receptors in the absence of costimulation results in suboptimal activation of T cells and ultimately anergy, it appears that the immunomodulatory effects of low-dose PDT associated with extended engraftment may depend upon decreased LC expression of major histocompatibility complex and costimulatory molecules.

Benzoporphyrin derivative monoacid ring A (BPD), a hydrophobic chlorin-like porphyrin derivative, which fluoresces strongly at 690 nm, may have potential for both oncologic and nononcologic applications in photodynamic therapy (PDT). To study the influence of cellular characteristics on the uptake of BPD, the murine tumor cell line (P815), and in vitro and in vivo concanavalin A (Con A) -stimulated and unstimulated murine splenic lymphocytes were incubated with 2 micrograms/mL BPD at 37 degrees C for 0-60 min. At various times, cells were lysed and the amount of BPD taken up by cells was quantified by fluorescence measurements. The subsets of cells taking up BPD were analyzed using a panel of monoclonal antibodies and the Coulter XL fluorescence-activated cell sorter. Furthermore, Con A-stimulated and unstimulated spleen cells were incubated with 0-50 ng/mliter of BPD for 1 h prior to exposure to red light (7.2 J/cm2). Cell survival 24 h post-PDT was measured by the MTT assay. We found that the rapidly dividing tumor cell line and mitogen-stimulated murine T cells (mainly CD4+/IL-2R+) took up significantly more BPD (5-10-fold) than do unstimulated splenic lymphocytes. Increased BPD uptake correlated with greater photoinactivation when these cells were exposed to light at a wavelength of 690 nm. These findings suggest that activated cells of the immune system may be a target for photoinactivation by BPD.

The use of transcutaneous photodynamic therapy (PDT) has been investigated in the prevention of adjuvant enhanced arthritis in MRL/lpr mice. Mice receiving adjuvant were treated with PDT at 10-day intervals starting on the day of adjuvant administration. PDT was carried out by intravenous injection of the photosensitizer, benzoporphyrin derivative-monoacid ring A, followed by its transcutaneous activation with light. Adjuvant-injected animals displayed a delayed onset and reduced incidence and severity of arthritis when compared to untreated animals. Most importantly, inflammatory structural damage to cartilage and bone tissues was prevented by PDT. PDT was found to have no adverse effects on animals as assessed by mitogen responses, hematopoiesis, and serum enzyme levels. As mitogen-activated MRL/lpr splenocytes were shown to be more susceptible to in vitro photodynamic treatment, it is postulated that the observed effects were the result of selective destruction of adjuvant-activated lymphocytes in the circulation and/or joints.

Photodynamic therapy is now recognized as a legitimate therapy for both palliative and potentially curative treatment of solid tumors. This represents a major achievement for investigators who have committed their careers to PDT. Scientific research characteristically runs well ahead of accepted views and technologies. This Symposium-in-Print perhaps provides a snapshot of the potential for this technology during the next two decades.

The capacity of photosensitizers and light to ablate cancerous tissues and unwanted neovasculature constitutes the classical application of photodynamic therapy (PDT). Cell death results from either necrotic or apoptotic processes. The use of photosensitizers and light at doses which do not cause death has been found to affect changes in certain cell populations which profoundly effect their expression of cell surface molecules and secretion of cytokines, thereby altering the functional attributes of the treated cells. Cells of the immune system and the skin may be sensitive to modulation by 'sub-lethal PDT.' Ongoing studies have been conducted to assess, at the molecular level, changes in both lymphocytes and epidermal cells (EC) caused by treatment with low levels of benzoporphyrin derivative monoacid ring A (BPD) (a photosensitizer currently in clinical trials for cancer, psoriasis, endometriosis and age-related macular degeneration) and light. Treatment of skin with BPD and light, at levels which significantly enhanced the length of murine skin allograft acceptance, have been found to down-regulate the expression of Langerhans cell (LC) surface antigen molecules [major histocompatibility complex (MHC) class II and intracellular adhesion molecule (ICAM)-1] and the formation of some cytokines (tumor necrosis factor-alpha (TNF- (alpha) ).

In this study, the effect of transdermal photodynamic therapy (PDT) using benzoporphyrin derivative monoacid ring A (BPD) on the development of the immunologically mediated contact hypersensitivity (CHS) response against the hapten dinitrofluorobenzene (DNFB) and on the duration of skin allograft acceptance has been evaluated. In the CHS model it was found that the treatment of hairless strain mice with whole-body transdermal PDT using BPD (1 mg/kg) and LED light (15 J/cm2) resulted in a profound suppression of the CHS reaction if treatment was applied either 48 or 24 hours prior or up to 72 hours after sensitization of abdominal skin with DNFB. Less inhibition of the CHS response was observed if PDT was given one day before the ear challenge with DNFB which was applied 5 days following the initial DNFB sensitization. However, DNFB-exposed, PDT-treated mice retained the capacity to respond maximally to the unrelated contact sensitizer oxazolone. These results are consistent with other models of experimentally induced immune tolerance. allogeneic skin graft studies demonstrated that pretreatment of skin with BPD and light, at levels that did not cause significant tissue damage, significantly enhanced the length of engraftment. Using a separate protocol, photodynamic treatment of recipient mice at various times after transplant had no significant effect on allograft acceptance. Irradiation of skin in the presence of BPD may significantly inhibit the initiation of certain immunological responses within these tissues.

The photosensitizer benzoporphyrin derivative monoacid ring A (VerteporfinR or BPD) has maximum absorption characteristics (690 nm) and biodistribution characteristics which permit activation of the drug in capillaries of the skin without causing skin photosensitivity (transdermal PDT). This permits targeting of cells in the circulation for selective ablation. Since BPD has been shown to accumulate preferentially in activated lymphocytes and monocytes, studies have been undertaken to determine the effect of transdermal PDT on murine models for rheumatoid arthritis (the MRL/lpr adjuvant enhanced model) and multiple sclerosis (the experimental allergic encephalomyelitis (EAE) model in PL mice). Localized transdermal PDT with BPD was found to be completely successful in preventing the development of adjuvant enhanced arthritis in the MRL/lpr mouse as well as improving the underlying arthritic condition of these animals. In the EAE model, in which an adoptive transfer system was used, it was found that transdermal PDT of recipients was effective in preventing EAE if treatments were implemented up to 24 hours after cell transfer but was not effective if given later, indicating the requirement for circulating T cells for effective treatment.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Benzoporphyrin derivative monoacid ring A (BPD) is taken up rapidly (within 30 minutes) by most cells. Rapidly dividing tumor cell lines and mitogen activated murine T lymphocytes were found to take up significantly more (5 - 10 fold) BPD than do normal splenic lymphocytes making them a potential PDT target. Experiments have shown that BPD can be activated in the blood of animals by whole body irradiation with red light, shortly after intravenous administration, in the absence of skin photosensitivity. During the treatment time, plasma levels of BPD were between 0.7 and 1.0 (mu) g/mL. The light treatment resulted in between 70 and 80% photoinactivation of circulating BPD. When L1210 tumor cells were preincubated with BPD and injected i.v. into mice immediately before total body light treatment, significant reductions in circulating clonogenic tumor cells were observed in blood samples taken immediately following treatment. This `transdermal' treatment has been shown to be effective in preventing the development of hind limb arthritis in MRL/1pr mice.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

In vitro experiments with benzoporphyrin derivative monoacid ring A (BPD) confirmed earlier studies that it was taken up rapidly (within 30 min) to maximum concentrations by all cells tested. It was also confirmed that rapidly dividing tumor cell lines and mitogen-activated murine T lymphocytes took up significantly more (5-10-fold) BPD than did normal splenic lymphocytes. Further experiments were undertaken to determine whether BPD could be activated by whole-body irradiation with red light in the blood of animals, shortly after intravenous (i.v.) administration, in the absence of skin photosensitivity. It was found that shaved and depilated mice injected i.v. 60 min earlier with BPD at between 0.5 and 1.0 mg/kg could tolerate 160 J/cm2 of broad-band red light (560-900 nm) delivered, at a relatively low rate, over a 90 min time interval without developing skin photosensitivity or general phototoxicity. During the treatment time, plasma levels of BPD were between 0.7 and 1.0 μg/mL. The light treatment resulted in between 70 and 80% photoinactivation of circulating BPD. When LI 210 tumor cells were preincubated with BPD and injected i.v. into mice immediately before total-body light treatment (160 J/cm2 of 590-900 nm light delivered over 90 min), significant reductions in circulating clonogenic tumor cells were observed in blood samples taken immediately following treatment. This indicated that sufficient light was being delivered to BPD in the blood flowing in the peripheral vasculature to effect cytotoxicity to cells containing the photosensitizer without causing either vascular or skin photosensitivity. Thus, activation of this photosensitizer in the circulation can be achieved by transdermal light exposure without causing skin photosensitivity provided that light exposure is performed at a time when the first phase of plasma clearance is complete and when the drug has not yet accumulated in skin.