Immunomodulatory effects of the endogenously synthesized photosensitizer, protoporphyrin IX

Problem under investigation. Photodynamic therapy with some photosensitizers has been shown experimentally to modulate in vitro and in vivo immune responses. The goal of this research is to study the therapeutic potential of endogenous protoporphyrin IX (PpIX) as an immunomodulator. Understanding immunomodulatory effects has clinical importance in cancer therapy where PpIX is already used.;Objective/hypothesis. Our objective is to analyze the effects of the endogenous photosensitizer, protoporphyrin IX (PpIX), on cells of the immune system (T cells and monocytes) for their clinical potential. Studies are proposed to first test the hypothesis that activated immune cells, as opposed to resting cells, accumulate PpIX and that these proliferating cells can be targeted for destruction by aminolevulinic acid (ALA) based photodynamic therapy (PDT). If this first hypothesis proves true, the second hypothesis is that specific and nonspecific immune suppression can result from ALA based PDT.;Previous findings. Many reports have shown that photosensitizers with PDT modulate immune responses. Immunosuppression has been shown with transplantation rejection studies in mice. Recipient mice treated with a photosensitizer prior to skin grafting have enhanced survival times of the skin grafts. In addition, mice treated with a photosensitizer plus PDT after sensitization with the hapten dinitrofluorobenzene (DNFB) have shown a decreased contact hypersensitivity response when later rechallenged with DNFB. There has also been work done with autoimmune models such as experimental autoimmune encephalomyelitis and MRL-lpr mice, with delayed onsets of disease symptoms in treated mice. In some of the above examples it is known how the photosensitizers affect immune cells, but for the photosensitizer PpIX very little is known.;Research design/materials/methods. Peripheral blood mononuclear cells (PBMC) from healthy human donors will be used for in vitro assays. Cells will be stimulated by PHA (mitogen), allogeneic mixed lymphocytes (MLR), or antigen (mumps and tetanus). Flow cytometry will be used to measure PpIX fluorescence in addition to phenotyping PpIX+ cells. Phenotyping will utilize antibodies that identify T cells (CD3, CD4, and CD8), monocytes (CD14), dendritic cells (CD83), and also activation markers (CD71 and CD38). Assays used to determine selectivity of cell kill will include activation using secondary MLRs and recall assays. Determination of cell death will include assays to measure necrosis. RT-PCR will be used to measure changes in cytokine production in ALA-PDT treated PBMC. Finally, tritiated thymidine uptake assays will be used to measure cellular proliferation following various treatments.;Preliminary results. Preliminary results show that activated T cells accumulate higher levels of PpIX compared to resting cells. However, CD14+ cells (monocytes and macrophages) accumulate high levels of PpIX in both unstimulated and stimulated cultures.;Intended methods for data analyses. The results of all experiments will be analyzed for statistical significance using Student's unpaired T-test. Winlist software will be used to evaluate flow cytometric data.;Potential significance. The information may be of importance to the oncologists use of ALA-PDT for cancer therapy because it may affect therapeutic outcome. If ALA-PDT modulates in vitro responses, further studies may determine if ALA-PDT can be useful clinically for immunomodulation in either transplantation rejection or autoimmune diseases.