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Chronic inflammatory skin disorders are frequently associated with impaired skin barrier function. Selective phosphodiesterase-4 (PDE4) inhibition constitutes an effective therapeutic strategy for the treatment of inflammatory skin diseases. We now report the pharmacological anti-inflammatory profile of DRM02, a novel pyrazolylbenzothiazole derivative with selective in vitro inhibitory activity toward PDE4 isoforms A, B and D. DRM02 treatment of cultured primary human and mouse epidermal keratinocytes interfered with pro-inflammatory cytokine production elicited by interleukin-1α and tumor necrosis factor-α. Similarly, DRM02 inhibited the production of pro-inflammatory cytokines by human peripheral blood mononuclear cells ex vivo and cultured THP-1 monocyte-like cells, with IC(50) values of 0.6–14 µM. These anti-inflammatory properties of DRM02 were associated with dose-dependent repression of nuclear factor-κB (NF-κB) transcriptional activity. In skin inflammation in vivo mouse models, topically applied DRM02 inhibited the acute response to phorbol ester and induced Th2-type contact hypersensitivity reactivity. Further, DRM02 also decreased cutaneous clinical changes and expression of Th17 immune pathway cytokines in a mouse model of psoriasis evoked by repeated topical imiquimod application. Thus, the overall pharmacological profiling of the PDE4 inhibitor DRM02 has revealed its potential as a topical therapy for inflammatory skin disorders and restoration of skin homeostasis.

Photodynamic therapy (PDT) is clinically approved for the treatment of several types of cancer as well as age-related macular degeneration, the leading cause of blindness in the elderly. PDT using the photosensitizer verteporfin has been previously shown to induce rapid apoptosis via a mitochondrial-caspase activation pathway. The impact of PDT on other cellular organelles such as the endoplasmic reticulum (ER) is undefined. The effect of PDT on intracellular Ca2+ ([Ca2+]i) in control and Bcl-2-overexpressing HeLa cells was assessed. A greater [Ca2+]i transient was observed for Bcl-2 overexpressing cells in response to PDT. The PDT-induced Ca2+ release was due to the emptying of Ca2+ from ER and possibly mitochondrial stores and was not due to an influx of Ca2+ from the medium. For Bcl-2-transfected cells, the release of Ca2+ was incomplete as determined by a further [Ca2+]i transient produced by the addition of the Ca2+ ionophore ionomycin after PDT. Furthermore, extrusion of Ca2+ was not hindered while ER-mediated sequestration of Ca2+ was impaired after PDT. Impairment of ER-mediated sequestration of Ca2+ may be due to the immediate caspase-independent depletion of sarco/endoplasmic reticulum Ca2+ ATPase-2 (SERCA2) that occurred in response to PDT in birth HeLa/Neo and Bcl-2 overexpressed HeLa cells. In summary, PDT induced the rapid degradation of SERCA2 and release of ER and mitochondrial Ca2+ stores. Although overexpression of Bcl-2 did not protect against SERCA2 degradation, it may influence the release of Ca2+ from ER and mitochondrial stores in PDT-treated cells.

The high turnover of endothelial cells (EC) in atherosclerosis suggests that an increase in the frequency of both cell proliferation and cell death is important in the pathogenesis of this common disorder. Further, increased apoptosis of EC, smooth muscle cells (SMC) and immune cells has been observed in atheromatous plaques. Many pro-atherogenic factors, including oxidized low-density lipoproteins, angiotensin II and oxidative stress, can induce EC apoptosis. Such damage to the endothelium may be an initiating event in atherogenesis since EC apoptosis may compromise vasoregulation, increase SMC proliferation, SMC migration and blood coagulation. In addition, EC overlying vascular lesions have been shown to increase their expression of pro-apoptotic proteins, such as Fas and Bax, while decreasing levels of anti-apoptotic factors. Therefore, understanding EC apoptotic pathways that are altered in atherosclerosis may enable a greater understanding of disease pathogenesis and foster the development of new therapies. The present discussion outlines the biochemical characteristics of EC apoptosis and the role that altered regulation of apoptosis plays in vasculopathy.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL) trigger apoptosis by stimulating the formation of a death inducing signaling complex at the cytoplasmic terminus of their respective receptors. Photodynamic therapy (PDT) is an approved treatment for several types of cancer as well as for age-related macular degeneration and is under investigation for different cancer, ocular, autoimmune and cardiovascular indications. The effect of low dose PDT in combination with TRAIL and FasL on Jurkat lymphoma cell apoptosis was examined. Individually, TRAIL, FasL, and PDT could induce apoptosis in these cells. However, at suboptimal levels of PDT, the number of cells undergoing apoptosis was increased when recombinant FasL and/or TRAIL were added. Additive effects of these treatments were evident for different apoptosis parameters including DNA fragmentation, caspase processing and activity and caspase substrate degradation. Overall, these results provide evidence that PDT-treated cells may be more likely to undergo apoptosis when also exposed to receptor-mediated signals delivered by factors such as TRAIL or FasL. For PDT, immune cell-mediated death receptor ligation may represent a way whereby tumor cells that have withstood the direct effects of photosensitization may be eliminated.

Photodynamic therapy (PDT) is under investigation for the treatment of intimal hyperplastia in conditions such as atherosclerosis and restenosis. Although smooth muscle cells (SMCs) may be a key target for treatment, the effects of PDT on these cells are poorly characterized. In the present study, apoptosis was induced in primary human aortic SMCs by the combination of the photosensitizer verteporfin and visible light. After PDT, an increase in mitochondrial cytochrome c (cyt c) and apoptosis-inducing factor (AIF) levels were detected in the cytosol immediately and their levels increased steadily up to 2 hours. Cytosolic levels of the pro-apoptotic Bcl-2 family member Bax decreased reciprocally throughout this period, but this change did not occur before cyt c release. Confocal microscopy revealed a diffuse staining pattern of cyt c within apoptotic cells as compared to a distinct mitochondrial staining in normal cells. AIF translocated from mitochondria to the nucleus during the progression of apoptosis. After cyt c release, caspase-9 and caspase-3 processing was visible by 1 hour and caspase-6, -7, and -8 processing was apparent by 2 hours after PDT. In summary, these results demonstrate for the first time the cellular redistribution of mitochondrial AIF during SMC apoptosis, as well as the early release of cyt c and the subsequent activation of multiple caspases during PDT-induced SMC apoptosis.

Tumor cell death in vitro by photodynamic therapy (PDT) has been related to the induction of apoptosis. We measured and compared changes in apoptosis and caspase 3 activity, an effector of apoptosis, in normal and neoplastic esophageal tissues during PDT. Apoptosis index, caspase 3 cleavage activity, pro-caspase 3, p53, and bcl-2 levels were measured in normal and neoplastic tissues of patients with esophageal adenocarcinoma before, during, and after PDT with Photofrin. The apoptotic index was greater in carcinoma tissue compared to adjacent normal tissues. In concert, pro-caspase 3 immunoreactivity was absent and caspase 3-like cleavage activity was over 30-fold greater in carcinoma tissue compared to normal esophageal tissues. These parameters were unaffected by PDT. Variable changes in bcl-2 and p53 immunoreactivity were noted in normal and carcinoma tissues during PDT. Greater levels of apoptosis and caspase 3 activity are hallmarks of esophageal adenocarcinoma compared to normal esophageal tissue. These differences were unaffected by PDT. This may be due to the fact that tissues were obtained 72 h post-PDT therapy. Changes in these parameters may have occurred early after PDT therapy. An assessment of apoptosis and caspase 3 activity prior to 72 h post-PDT may provide further insight into the mechanism involved, although no sustained effects on these parameters by PDT were noted.

Photodynamic therapy (PDT) utilises light-absorbing compounds combined with directed photo-irradiation to produce clinical effects. This review updates advances in the understanding of the biochemical pathways triggered by PDT within cells, its influence upon different immune parameters and progress in the use of PDT against human immune-mediated disease. Several works have further defined the notable capacity of PDT to foster anticancer immunity.

The nuclear factor-kappa B (NF-kappaB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-kappaB principally resides within the cytoplasm in association with inhibitory kappa (IkappaB) proteins. The status of IkappaB and NF-kappaB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IkappaB proteins may be caspase substrates during apoptosis. However, the level of IkappaBbeta was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IkappaBalpha levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IkappaBalpha levels were transiently depressed after PDT. At these times, p50 and RelA NF-kappaB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a kappaB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-alpha, a well-characterized NF-kappaB activator. Productive NF-kappaB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IkappaBalpha is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

The impact of the immunomodulatory photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) and visible light on the survival and surface receptor pattern of resting and activated murine T cells was evaluated. T cells treated for 48 h with immobilized anti-CD3 monoclonal antibody upregulated expression of the interleukin-2 receptor alpha-chain (CD25), transferrin receptor (CD71), the apoptosis-regulating Fas receptor (CD95), contained a greater level of the anti-apoptotic protein Bcl-2 and accumulated significantly more BPD-MA than their unactivated counterparts. Activated T cells displayed a modestly greater susceptibility to the photodynamic induction of DNA fragmentation than resting T cells. Resting T cells treated with sub-lethal levels of BPD-MA and light did not exhibit changes in surface levels of CD3, CD4, CD8, CD28, CD45 or T cell receptor (TCR) beta-chain structures. However, levels of major histocompatibility complex (MHC) class I antigens were decreased while the density of Thy-1.2 (CD90) increased on these cells. Photodynamically treated T cells failed to express optimal CD25 levels when exposed to the mitogenic anti-CD3 antibody. Activated T cells treated with sub-lethal levels of BPD-MA and light exhibited lower CD25 levels, a temporary block in cell cycle transition, but unaltered expression of MHC Class I, CD3, CD4, CD8, CD45, CD54, CD71, CD122 (IL-2R beta-chain) or TCR beta-chain antigens 24 h afterward. Resting and activated T lymphocytes differ in susceptibility to PDT-mediated apoptosis but both types are sensitive to anti-proliferative effects the treatment exerts at sub-lethal photosensitizer levels. The marked sensitivity of activated T cells to photodynamic inactivation likely contributes to the immunomodulatory action of BPD-MA.

Treatment with the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) followed by irradiation with visible light induces apoptosis in human acute myelogenous leukaemia HL-60 cells. Photoactivation of BPD-MA induces procaspase 3 (CPP32/Yama/apopain) and procaspase 6 (Mch2) cleavage into their proteolytically active subunits in these cells. The Bcl-2 proto-oncogene product has been shown to protect cells from a number of proapoptotic stimuli. In the present study, the influence of Bcl-2 overexpression on cellular resistance to photoactivation of BPD-MA was studied. Overexpression of Bcl-2 in HL-60 cells prevented apoptosis-related events including caspase 3 and 6 activation, poly(ADP-ribose) polymerase cleavage and the formation of hypodiploid DNA produced by BPD-MA (0–200 ng ml−1) and light. However, Bcl-2 overexpression was less effective at preventing cell death that occurred after photoactivation at high levels (50–100 ng ml−1) compared with lower doses (10–25 ng ml−1) of BPD-MA. These results indicate that caspase 3 and 6 activation and their regulation by Bcl-2 may play important roles in photodynamic therapy (PDT)-induced cell killing. © 1999 Cancer Research Campaign

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae , induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.

The capacity of lymphoid dendritic cells from human cord blood or adult peripheral blood to support a mixed leukocyte reaction in cord blood and adult T cells has been compared. Cord blood dendritic cells have a limited ability to induce either adult or cord blood T cells to proliferate in response to typical concentration of phytohemagglutinin or concanavalin A. Adult blood dendritic cells, on the other hand, induce equivalent mitogen responses in cord blood and adult blood T cells. This relative deficiency can be overcome by increasing the concentration of mitogen or the numbers of dendritic cells in the culture. Neonatal primary immune responses may, in part, reflect the reduced function of dendritic cells.

Treatment with benozopophyrin derivative monoacid ring A (BPD-MA, verteporfin) and broad-spectrum fluorescent light rapidly produced apoptosis in murine P815 mastocytoma cells. Fragmentation of DNA, a fundamental characteristic of cells undergoing apoptosis, was evident within 3 h following the photodynamic treatment. Western immunoblot analysis using the specific antiphosphotyrosine monoclonal antibody 4G10 indicated that molecular species of > 200 kDa were phosphorylated on tyrosine residues during or immediately following the irradiation of cells loaded with BPD-MA. Increased tyrosine phosphorylation of a 15 kDa protein was evident by 15 min postirradiation. In the absence of light, BPD-MA did not affect the status of cellular protein tyrosine phosphorylation or cause DNA fragmentation. The protein kinase inhibitor staurosporine prevented tyrosine phosphorylation of the > 200 kDa species but did not affect tyrosine phosphorylation of the 15 kDa protein or the level of DNA fragmentation produced by the photodynamic treatment. The protein tyrosine phosphorylation events observed for P815 cells treated with cytotoxic levels of BPD-MA and light may not be directly related to the induction of the apoptotic cell death pathway.

When given to normal male mice, the photochemotherapeutic agent Photofrin® (porfimer sodium), a complex mixture of monomeric and oligomeric non-metallic porphyrins, significantly increased relative spleen weight, spleen cell numbers, DNA replication, levels of granulocyte-macrophage and erythroid progenitors and cellular responsiveness to different recombinant (r) myeloid growth factors. In contrast, monomeric hematoporphyrin had no effect on spleen weight, cellularity, erythroid progenitor levels or spleen cell cycle status. Photofrin® significantly increased spleen cell expression of the receptor (CD71) for the iron transport protein transferrin by 72 h post-injection but did not affect levels of a receptor (CD25) for the T-cell growth factor interleukin-2 (IL-2) or spleen cell responsiveness to rIL-2. Further evidence of increased splenic erythropoietic activity in Photofrin®-treated mice was provided by double color flow cytometric studies which indicated that the treatment elevated the number of nucleated spleen cells recognized by the erythroid lineage-specific monoclonal antibody TER-119. A majority of TER-119+ cells also expressed CD71 and the heat stable antigen, a marker of developing hematopoietic cells as well as mature erythrocytes. The capacity of Photofrin® to stimulate murine hematopoietic activity appears to be a property not shared by other porphyrin photosensitizers.

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.

The porphyrin photochemotherapeutic agent Photofrin stimulates hematopoietic activity within the bone marrow (BM) and spleens of normal mice. We found that the intraperitoneal (i.p.) administration of Photofrin also caused a 3-4 fold increase in peritoneal cell (PC) numbers, particularly cells bearing granulocyte surface antigens. Little granulocyte-macrophage progenitor activity was detectable within the PC population of control and Photofrin-injected mice suggesting that Photofrin had elicited an influx of inflammatory cells into the region. In contrast to cells from control animals, PC obtained 6 h to 168 h after the i.p. injection of Photofrin exhibited a consistently inferior capacity to support the growth of BM colony forming units granulocyte-macrophage (CFU-GM). When PC from control or Photofrin-treated mice were added to the BM culture system in the presence of defined growth factors there was no effect on the number of colonies formed. This finding indicated that negative regulatory elements were not responsible for the reduced hematopoietic accessory activity exhibited by PC from Photofrin-treated mice. Supernatants conditioned by PC from Photofrin-injected mice poorly supported BM CFU-GM growth in vitro and in contrast to PC supernatants prepared from control mice did not contain detectable amounts of granulocyte-macrophage colony stimulating factor (GM-CSF). The cellular changes which occur within the peritoneal cavity represent bystander events not directly related to the hematostimulatory action of Photofrin.

Transcutaneous photodynamic therapy (PDT), utilizing benzoporphyrin derivative monoacid ring A (BPD, verteporfin) and whole-body light exposure, was assessed for its capacity to modify the course of adoptively transferred experimental autoimmune encephalomyelitis (EAE) in PL mice. Using a novel cell culture technique to facilitate the induction of this neurodegenerative condition, disease signs commenced 3-4 weeks after the transfer of myelin basic protein (MBP)-reactive lymph node or spleen cells to naive syngeneic recipients. Mice administered MBP-sensitized lymph node cells preincubated with BPD followed by whole-body 690 nm light irradiation (15 J/cm2) did not display symptoms of EAE. Although almost all animals given MBP-sensitized spleen cells developed EAE, mice given BPD (1 mg/kg) and the light treatment 24, 48 or 120 h after spleen cell transfer exhibited significantly less severe disease symptoms than control animals. Mice given the photodynamic treatment 24 h after spleen cell transfer also exhibited a significantly later disease onset than the control animals. Treatment of mice with PDT 24 h prior to spleen cell transfer did not influence subsequent disease severity but modestly delayed its onset. In the absence of directed light, BPD did not influence the development of EAE. Spinal cord tissues were evaluated for the presence of T cell receptor (TCR) V alpha 4 mRNA transcripts that specifically encode for the TCR alpha-chain of MBP-reactive T cells of PL mice. Using the polymerase chain reaction, V alpha 4 TCR mRNA transcripts were present in spinal cord samples prepared from almost all control mice but in only about one-half of spinal cord samples prepared from mice treated with PDT 24 h after spleen cell transfer. These observations indicated that PDT had limited the expansion of MBP-specific V alpha 4+ T cells within the central nervous system. Transcutaneous PDT represents a new technique with which to approach the treatment of autoimmune disease.

— This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon-γ (rIFN-γ, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN-γ-activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD50= 14.4 ± 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm2) than cells (LD50= 18.2 ± 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN-γ-activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 μg/mL) took up more BPD than macrophages cultured in medium alone or with rIFN-γ. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD50= 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN-γ-activated macrophages and the greater resistance of LPS-activated macrophages, relative to medium-cultured macrophages, to photodynamic killing with BPD is uncertain. However, the increased susceptibility of macrophages, activated with the immunomodulatory cytokine IFN-γ, to treatment with BPD and light might indicate how photodynamic therapy could interfere with the development of experimental autoimmune disease, conditions in which activated macrophages are known to be involved.

We have isolated low-density, nonadherent, nonphagocytic, HLA-DR+ve cells with the morphology of dendritic cells (DCs) from the cord blood of full-term newborn infants. Relative to adult DCs, cord blood DCs were poor stimulators of the mixed leukocyte reaction when either adult or cord blood mononuclear cells (MNCs) or T lymphocytes were used as responder cells. In contrast, cord blood T cells and MNCs responded normally to allogeneic adult DCs. Cord blood DCs performed poorly as accessory cells for T-lymphocyte mitogenic responses at suboptimal concentrations of concanavalin A (Con A) and phytohemagglutinin A or at optimal concentrations of mitogen and low numbers of DCs. Addition of recombinant interleukin-2 (rIL-2) or recombinant interferon-gamma (rIFN- gamma) to cord blood DC-T-cell cultures containing a suboptimal concentration of Con A potentiated the proliferative response. In contrast, rIL-2 and rIFN-gamma exerted little effect on the proliferative response of adult T cells cultured with Con A and DCs. Flow cytometric studies showed that levels of intercellular adhesion molecule-1 (ICAM-1; CD54) and major histocompatibility complex (MHC) class I HLA-ABC and class II HLA-DR antigens on cord blood DCs were significantly lower than those on adult blood DCs. These findings suggest that the relative inefficiency of cord blood DCs in the activation of T cells may be related to their low cell surface expression of MHC and cell adhesion molecules. The demonstrated impairment of cord blood DC function could be of importance in understanding the immunologic relationship between the fetus and mother and could contribute to the susceptibility of newborns to infection.

Dermal papilla cells (DPC) control the growth character of the hair follicle through their elaboration of mitogenic factors and extracellular matrix components. Further, the dermal papilla is a primary site of androgen action in the hair follicle. Interleukin-1alpha (IL-1alpha) is prominent in skin wounding and inflammatory responses although regarded as a negative hair growth regulator. We studied the effect of IL-1alpha and the potent androgen 5alpha-dihydrotestosterone (DHT) on the expression of the androgen receptor (AR) and various factors secreted by cultured human temporal scalp DPC. IL-1alpha triggered cellular changes consistent with nuclear factor-kappaB pathway activation as well as reduced AR mRNA and protein expression levels for DHT-stimulated DPC. This cytokine also increased DPC supernatant keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations. IL-1alpha did not influence DPC supernatant levels of transforming growth factor-beta1, a negative hair growth regulator. The stimulatory effect of IL-1alpha on DPC VEGF, GM-CSF, KGF, and IL-8 expression was also evident at the mRNA level for these cytokines. IL-1alpha also increased mRNA transcript levels of protease-nexin-1, a secreted serine protease inhibitor expressed in the dermal papilla of anagen-stage hair follicles. Although DHT did not affect supernatant cytokine concentrations, the androgen altered mRNA transcript levels of several factors for DPC co-stimulated with IL-1alpha. In consideration of its in vitro activity profile, IL-1alpha may be an important modifier of dermal papilla activity as well as potentially influence androgen-regulated gene expression in DPC.

In its conventional form, photodynamic therapy (PDT) is a clinically effective technique with which to treat tumours accessible to visible light. PDT utilises light absorbing compounds which catalyse the generation of toxic oxygen species, to produce localised antitumour effects. It has become apparent over the past decade that PDT also exhibits immunomodulatory attributes. Experimental animals may possess heightened antitumour immunity after tumour ablation with PDT. In contrast, at sub-phototoxic levels of photosensitiser, in combination with whole body light irradiation, PDT lessened disease severity when applied in different models of autoimmunity. Although the behaviour of lymphocytes may be affected by treatment, the ability of PDT to down-regulate autoimmune processes appears to be related to its capacity to influence the immunostimulatory attributes of antigen presenting cells.

Pharmacia Corp, under license from Miravant Medical Technologies (formerly PDT Inc), is developing rostaporfin (SnET2, Purlytin), a light-activated cytotoxic drug developed as part of Miravant's PhotoPoint photodynamic therapy (PDT) program, for the potential treatment of wet age-related macular degeneration (AMD) [180314]. In January 2002, results of phase III trials indicated that rostaporfin had not met the primary efficacy endpoint for the wet form of AMD. At this time, a full review of the data was to be undertaken, and decisions about future development of the drug were to be made after additional analyses had been completed [435577]. The original licensing agreements included the development of rostaporfin for several ophthalmology, oncology and urology indications [289078], and for dermatological applications including certain skin cancers [267521]. However, in August 1998, Miravant reported that it no longer intended to pursue cutaneous metastatic breast cancer (CMBC), in order to focus on AMD [439372], [439384]. Also in 1998, studies in basal cell carcinoma and AIDS-related Kaposi's sarcoma were discontinued because of business considerations [439372]. Rostaporfin is activated by red light with a wavelength of 664 nm. It is injected into the patient, where it distributes and selectively binds to plasma lipoproteins, which are produced in high concentrations by hyperproliferating cells such as cancer cells. After 24 h, the targeted cells are stimulated by red light to activate the compound. This triggers the formation of toxic free radical species that destroy the cells without affecting the surrounding normal tissue [85236]. In January 2002, Credit Suisse First Boston estimated sales for Pharmacia of 40 million US dollars in 2003 and 80 million US dollars in 2004 [436118], while in the same month, Argus Research predicted peak annual sales for Pharmacia of less than 250 million US dollars[436279].

A new photosensitizer, presently designated QLT0074, may be useful for the treatment of skin conditions, particularly those mediated by T lymphocytes, with photodynamic therapy (PDT). QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. Low concentrations of QLT0074 and blue light were sufficient to induce apoptosis in peripheral blood T cells or Jurkat T lymphoma cells as indicated by expression of the apoptosis-associated mitochondrial 7A6 marker, annexin-V labeling or activation of capsase-3 and cleavage of the capsase substrate poly(ADP-ribose)polymerase (PARP). Flow cytometry studies performed following PDT showed that peripheral blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater degree than T cells with low CD25 levels. This effect of PDT was also shown by the reduction in the percentage of T cells that expressed other activation-associated markers such as very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). In the case of T cells that remained viable following PDT, CD25 expression was lower while CD54, CD95 and HLA-DR levels were unchanged. Thus, PDT with QLT0074 has selective, dose-dependent effects on T cells in vitro.

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kappaB activation and apoptosis were clearly independent although NF-kappaB was shown to counteract apoptosis mediated by PPME photosensitization.

Summary Historically, cancer has been the major therapeutic focus for PDT. The growing understanding of how PDT affects the behavior and viability of a variety of cell types has fostered the evaluation and use of PDT for non-oncologic indications, including ocular, cardiovascular, and immune diseases. The formulation, pharmacokinetics, and type of photosensitizer, the duration between its administration and light application, and the region of PDT on immune reactions. Among immune-mediated diseases, psoriasis is a promising candidate indication for PDT, since the skin lesions and the immunocytes associated with active disease are readily accessible to activating light. The beneficial effect of PDT on psoriasis may arise from a reduction in the number of pathogenic T cells, a modification of APC function, and/or an alteration of keratinocyte cytokine production within afflicted skin. Continued efforts to optimize PDT regimens will reveal the ultimate potential of this technique to effectively treat immune-mediated diseases. Likewise, there is potential to exploit the capacity of PDT to stimulate anti-tumor immunity. Both of these therapeutic areas will continue to provide opportunities to understand the underlying biological processes and develop improved treatment regimens.

PDT may be an effective treatment for certain immune-mediated disorders. The immunomodulatory action of PDT is likely a consequence of effects exerted at a number of levels including stimulation of specific cell signaling pathways, selective depletion of activated immune cells, alteration of receptor expression by immune and non-immune cells, and the modulation of cytokine availability. QLT0074, a potent photosensitizer that exhibits rapid clearance kinetics in vivo, is in development for the treatment of immune disorders. In comparison to the well-characterized and structurally related photosensitizer verteporfin, lower concentrations of QLT0074 were required to induce apoptosis in human blood T cells and keratinocytes using blue light for photoactivation. Both photosensitizers triggered the stress activated protein kinase (SAPK) and p38 (HOG1) pathways but not extracellularly regulated kinase (ERK) activity in mouse Pam212 keratinocytes. In cell signaling responses, QLT0074 was active at lower concentrations than verteporfin. For all in vitro test systems, the stronger photodynamic activity of QLT0074 was associated with a greater cell uptake of this photosensitize than verteporfin. In mouse immune models, sub-erythemogenic doses of QLT0074 in combination with whole body blue light irradiation inhibited the contact hypersensitivity response and limited the development of adjuvant-induced arthritis. QLT0074 exhibits activities that indicate it may be a favorable agent for the photodynamic treatment of human immune disease.

We have explored the effect of photodynamic therapy (PDT) with verteporfin on the induction and expression of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB) in normal mice and IL-10-deficient mice. Our results indicate that DNFB sensitized mice given PDT with verteporfin and whole body red light irradiation exhibited a significant reduction in CHS compared with control animals. Administration of rIL-12 reversed the effect(s) of PDT as did treatment of mice with anti-IL-10-neutralizing Ab. Knockout mice deficient in IL-10 were found to be resistant to the inhibitory effects of PDT. In vitro proliferative responses using spleen cells from DNFB-sensitized and PDT-treated mice showed a significantly lower response to DNBS as compared with cells from DNFB-sensitized mice or DNFB and PDT-treated IL-10-deficient mice. Finally, naive mice exposed to PDT exhibited an increase in skin IL-10 levels, which peaked between 72 and 120 h post-PDT. Together these data support the role of IL-10 as a key modulator in the inhibition of the CHS response by whole body PDT.

Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in endothelial cells remain undefined. In the current study, apoptosis was induced in primary human umbilical venous endothelial cells by the photosensitizer verteporfin and light. Release of mitochondrial cytochrome c into the cytosol was detectable immediately and accumulated over 2 hours after treatment while cytosolic levels of the proapoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another proapoptotic Bcl-2 family member, Bid, was observed by 2 hours after treatment. Although Bid cleavage has been shown to occur as an upstream event responsible for inducing cytochrome c release, we demonstrate that Bid cleavage can also occur after cytochrome c release. Activation of caspases 2, 3, 6, 7, 8, and 9 occurred following the release of cytochrome c, and cleavage of downstream substrates was observed. In summary, endothelial cell death involves the cellular redistribution of Bax and cytochrome c, followed by the activation of multiple caspases which manifest the apoptotic phenotype.

Photodynamic Therapy (PDT) is accepted for treatment of superficial and lumen-occluding tumors in regions accessible to activating light and is now known to be effective in closure of choroidal neovasculature in Age Related Macular Degeneration. PDT utilizes light absorbing drugs (photosensitizers) that generate the localized formation of reactive oxygen species after light exposure. In a number of systems, PDT has immunomodulatory effects; Photodynamic Immune Modulation (PIM). Using low- intensity photodynamic regimens applied over a large body surface area, progression of mouse autoimmune disease could be inhibited. Further, this treatment strongly inhibited the immunologically- medicated contact hypersensitivity response to topically applied chemical haptens. Immune modulation appears to result from selective targeting of activated T lymphocytes and reduction in immunostimulation by antigen presenting cells. Psoriasis, an immune-mediated skin condition, exhibits heightened epidermal cell proliferation, epidermal layer thickening and plaque formation at different body sites. In a recent clinical trial, approximately one-third of patients with psoriasis and arthritis symptoms (psoriatic arthritis) displayed a significant clinical improvement in several psoriasis-related parameters after four weekly whole-body PIM treatments with verteporfin. The safety profile was favorable. The capacity of PIM to influence other human immune disorders including rheumatoid arthritis is under extensive evaluation.

Conventional photodynamic therapy (PDT) utilizes light-absorbing compounds that have anti-cancer activity upon visible light irradiation. PDT has also been utilized for the treatment of certain immune conditions. To further understand the action of PDT upon immune cells, DBA/2 mouse thymocytes were treated with the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) and/or an apoptosis-inducing anti-Fas (APO-1, CD95) monoclonal antibody. Nanomolar levels of BPD-MA in combination with nonthermal visible light irradiation rapidly induced apoptosis as gauged by DNA fragmentation assays. Thymocytes were modestly more sensitive to PDT-induced apoptosis than mature splenic T cells. BPD-MA and light or the anti-Fas antibody decreased CD4 + CD8 + cell numbers while relatively sparing CD4 − CD8 − , CD4 + CD8 − , and CD4 − CD8 + thymocytes. In combination, anti-Fas antibody and PDT augmented activity levels of the apoptosis-related protease caspase-3, cleavage of the caspase-3 substrate poly(ADP) polymerase, and the proportion of cells exhibiting DNA fragmentation and further impacted CD4 + CD8 + thymocyte survival. Although CD4 + CD8 + thymocytes had the greatest sensitivity to photodynamic depletion, BPD-MA was taken up by the other major thymocyte subsets with equal or greater avidity. Since CD4 + CD8 + thymocytes are selectively impacted by PDT and anti-Fas antibody can act in concert with PDT to further cytotoxicity, thymocytes may be useful for the identification of factors that govern immune cell susceptibility to this form of phototherapy.

In recent years, intense research has been directed towards understanding molecular mechanisms involved in viral pathogenesis. It is now known that many viruses manipulate host defense mechanisms to prevent apoptosis in order to maximize viral replication. Towards the end of their replication cycle, certain viruses direct the synthesis of proteins that induce apoptosis or cell lysis thereby facilitating viral release from the cell. The present review summarizes the current understanding of interactions between viral proteins and the host cell death machinery.

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other pro-apoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1–2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2–3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.

Treatment of mice with certain photosensitizers combined with exposure to visible light limits the development of theimmunologically-mediated contact hypersensitivity (CHS) response against topically-applied chemical haptens. Understandingof the inhibitory action of photosensitizers upon the CHS response is incomplete. Benzoporphyrin derivative monoacid ring A(BPD-MA, verteporfm), a photosensitizer with immunomodulatory activity, strongly depressed CHS responses to the haptendinilrofluorobenzene (DNFB). However, if mice were administered 1 ig of a recombinant preparation of the pro-inflammatorycytokine interleukin-12 (rIL-12), full-fledged CHS responses to DNFB ensued in animals treated with BPD-MA and light. Incontrast, when rIL-12 was given in combination with an anti-IL-12 antibody the restorative effect ofrlL-12 on the CHS responseof PDT-treated mice was blocked. Evaluation of the cytokine status of spleen and draining lymph node cells showed for DNFBpainted animals, that the release of the immunosuppressive cytokine IL-lO was increased by PDT and rIL-12 counter-acted theincrease in IJ,.lO liberation associated with PDT. These studies indicate that IL-lO formation is upregulated and the availabilityofIL-12 may be limited in mice treated with PDT. These features may contribute to deficient CHS responses observed with PDTKey words: antigen presentation, benzoporphyrin derivative, contact hypersensitivity, cytokines, immune modulation,immunosuppression, interferon-y, interleukin-4, interleukin-lO, interleukin-12, photodynamic therapy, photosensitizers.Abbreviations: APC, antigen presenting cells; BPD-MA, benzoporphyrin derivative monoacid ring A, verteporfm; CHS,contact hypersensitivity; DC, dendritic cell; DNFB, 2,4-dinitrofluorobenzene; IFN-'y, interferon-y; IL, interleukin; LC,Langerhans cells; PDT, photodynamic therapy; r, recombinant.

Four structural analogs of benzoporphyrin derivative (BPD), a potent anti-tumor photosensitizer, were evaluated for their capacity to influence the immunologically-mediated contact hypersensitivity (CHS) response against the hapten 2,4-dinitrofluorobenzene (DNFB). Immunocompetent hairless strain mice received BPD monoacid ring A (BPD-MA, verteporfin) and returned to normal housing conditions or treated with 690 nm red light (transcutaneous photodynamic therapy, PDT). Unexpectedly, we found that mice given BPD-MA exhibited significantly reduced CHS ear swelling responses to DNFB upon antigenic challenge, whether or not they had been treated with PDT. A significant reduction in the CHS response to DNFB was observed when BPD-MA or PDT was given 48 or 24 h prior to, on the same day, or 24 or 72 h after DNFB sensitization. However, the magnitude of the CHS response was unaffected if these treatments were given 96 h after DNFB sensitization, 24 h before challenge with DNFB. Significantly reduced CHS responses also occurred in Balb/c mice given BPD-MA with or without PDT. Mice given BPD-MA but retained in total darkness throughout the experimental period generated full-fledged ear swelling responses to DNFB indicating that CHS suppression with BPD-MA was light dependent. BPD monoacid ring B (BPD-MB) strongly reduced the CHS response of Balb/c mice kept under ambient light while BPD diacid ring A (BPD-DA) and BPD diacid ring B (BPD-DB) also lowered the CHS response but were less effective than the monoacid forms. Other photosensitizers including Photofrin, tin etiopurpurin, and zinc phthalocyanine did not alter the CHS response of Balb/c mice maintained under ambient light. The ability of different BPD analogs to inhibit the CHS response in mice held under ambient light conditions appears related to the potent photosensitizing activity of these compounds.

The impact of bensoporphyrin derivative monoacid ring A, and visible light was determined for mouse splenic dendritic cells (DC), potent antigen-presenting cells (APC) of the immune system. It was discovered that sub-lethal doses of BPD-MA and light significantly altered the surface receptor pattern of DC as well as diminishing the capacity of these cells to activate allogeneic T cells. Treatment of highly purified DC with BPD-MA and 690 nm wavelength light decreased DC expression of major histocompatibility (MHC) Class I and II antigens, leukocyte common antigen CD45, intercellular adhesion molecule-1 (ICAM-1, CD54), the co- stimulatory molecules CD80 and CD86, CD95 as well as integrin CD11c. In contrast, DC expression of leukocyte function-associated-1 (LFA-1, CD11a), CD11b, CD18, CD40, and the DC DEC-205 receptor increased after the treatment. Changes in receptor levels occurred rapidly. DC MHC Class I and ICAM-1 expression declined to 40 percent of control levels by 2 hours post-PDT. DC treated with BPD-MA and light were poor stimulators of allogeneic T cells in the mixed leukocyte reaction. BPD-MA, in the absence of light, had no effect on the immunostimulatory properties of these cells. The changes in DC receptor expression pattern produced by BPD-MA and light were comparable to those produced by ultraviolet B light, a treatment known to alter the immunostimulatory characteristics of DC. Photodynamic therapy with BPD-MA represents an innovative approach for the modification of immune reactivity.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The rat monoclonal antibody LR-1 was initially described to be reactive with an antigen present on murine splenic B lymphocytes. However, flow-cytometric analyses of cells obtained from thymus, bone marrow, spleen, and lymph nodes showed that LR-1 stained approximately 95, 95, 60–70 and 20% of cells present within these tissues in normal DBA/2 mice. The marker recognized by LR-1 was present on peripheral erythrocytes and splenic dendritic cells, and activation with lipopolysaccharide A further increased expression of this antigen by splenic B cells. This particular tissue and cellular distribution was similar to that delineated with monoclonal antibodies reactive with heat-stable antigen (HSA). Dual-labelling studies were conducted to compare the reactivity patterns of LR-1 and the HSA-reactive monoclonal antibody J11d and indicated that both antibodies recognized splenocytes bearing B cell (IgM) or erythroid (TER-119, CD71) but not T cell (CD4, CD8) markers. Splenocytes exposed to phosphoinostol-specific phospholipase C showed marked reduction in LR-1 binding, indicating that this antibody recognized a glycosylphosphatidylinositol-anchored cell surface protein, consistent with the known structure of HSA. Mixing of LR-1 with the HSA-specific antibodies Jlld or M1/69 provided flow-cytometric profiles indistinguishable from those obtained with either antibody alone. However, LR-1 inhibited M1/69 binding to splenocytes by 83%, while J11d reduced Ml/69 binding to these cells by only 18%. This finding suggested that LR-1 and Ml/69 recognize identical splenic HSA epitopes, while LR-1 and J11d bind distinct antigenic determinants of spleen HSA. Western blot analysis of splenocyte, thymocyte, bone marrow cell and erythrocyte detergent extracts revealed that LR-1 reacted with glycoforms of HSA of known molecular weights (30–55 kD). Thus, LR-1 recognizes HSA, the murine analogue of human CD24, and will be a useful reagent with which to investigate the role of HSA in the immune response and hematopoiesis.

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.

The porphyrin photosensitizer, Photofrin® porfimer sodium (Photofrin®), has been widely studied for its capacity to evoke destruction of malignant tissues. In addition to its photosensitizing properties, Photofrin® may exert myelostimulatory effects in normal and immunosuppressed mice in the absence of activating light. In the present set of experiments, we examined the effect of Photofrin® upon the immunohematopoietic axis of sublethally irradiated DBA/2 mice. Administration of Photofrin® (10 mg/kg) I and 4 days following irradiation (4 Gy) significantly enhanced the recovery of spleen cellularity, spleen and bone granulocyte/macrophage progenitors (colony-forming units, CFU-GM) and peripheral blood leukocytes levels. Proliferative responses to the T-cell mitogen concanavalin A by spleen cells prepared from Photofrin®-treated mice were significantly less than those of cells from irradiated control mice 8 and 15 days post-irradiation. Photofrin® given 1, 4 and 7 days following irradiation elevated splenic CFU-GM 3 to 4-fold 10 days post-irradiation relative to the irradiated controls and mice given only two injections of Photofrin®. In contrast to the effect of two injections, multiple (three or four) injections of Photofrin® did not elevate bone marrow CFU-GM above control levels beyond 8 days post-irradiation. In addition, splenic CFU-GM levels in animals receiving three or four injections of Photofrin® were no different than those of the irradiated controls later than 10 days post-irradiation. These findings indicate that prolonged exposure to Photofrin® in sublethally irradiated mice may induce regulatory factors which dampen the enhanced myelopoietic recovery stimulated by only two injections of the drug.

Photodynamic therapy (PDT) using benzoporphyrin derivative (BPD, Verteporfin) and whole body irradiation, can affect the course of adoptively transferred experimental allergic (autoimmune) encephalomyelitis (EAE) in PL mice. Murine EAE is a T cell-mediated autoimmune disease which serves as a model for human multiple sclerosis. Using a novel disease induction protocol, we found that mice characteristically developed EAE within 3 weeks of receipt of myelin basic protein (MBP)-sensitized, in vitro-cultured spleen or lymph node cells. However, if animals were treated with PDT (1 mg BPD/kg bodyweight and exposed to whole body 15 Joules cm2 of LED light) 24 hours after receiving these cells, disease onset time was significantly delayed. PDT-treated mice developed disease symptoms 45 +/- 3 days following cell administration whereas untreated controls were affected within 23 +/- 2 days. In contrast, application of PDT 48 or 120 hours following injection of the pathogenic cells had no significant effect upon the development of EAE. Experiments are in progress to account for the protective effect of PDT in this animal model. These studies should provide evidence on the feasibility of PDT as a treatment for human autoimmune disease.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Porphyrins are a group of organic compounds involved in a wide spectrum of fundamental biological processes. Non-metallic, naturally occurring and synthetic porphyrin derivatives may produce cytotoxic effects in malignant or normal tissues exposed to visible light. Supra-clinical doses of the photosensitizing porphyrin, Photofrin are hematostimulatory when administered to normal and immunosuppressed inbred mice. To determine if a non-photosensitizing metalloporphyrin has similar hematostimulatory activity, we have synthesized iron (III) hematoporphyrin chloride (FeHp) and administered it to sub-lethally irradiated mice. FeHp (10 mg/kg) given 1 and 4 days or 1, 4 and 7 days following sub-lethal (7 Gy) whole body irradiation significantly increased spleen colony forming units of progenitor cells of the granulocyte-macrophage lineage (CFU-GM) 14 days post-irradiation, relative to irradiated controls. In addition, total splenocyte numbers were significantly increased 17 days post-irradiation in mice that had received FeHp 1 and 4 days post-irradiation. When FeHp was given 24 hours prior to irradiation and again 48 hours or 48 and 96 hours post-irradiation, significant increases in splenic CFU-GM and spleen cell numbers, relative to control mice, were observed 15 days post-irradiation. A non-metallic photosensitizing monomeric fraction of Photofrin, deuteroporphyrin IX, 2,4 (4,2) hydroxyethyl vinyl (HVD) was compared to Photofrin for its ability to influence the hematopoietic recovery of irradiated mice. Only Photofrin but not HVD given in 3 doses (10 mg/kg) 1, 4 and 7 days following irradiation (4.8 Gy) significantly enhanced the recovery of spleen cellularity and splenic CFU-GM. In addition, Photofrin significantly increased bone marrow CFU-GM 7 and 10 days following the sub-lethal dose of gamma-radiation. The mechanism by which certain porphyrins augment hematopoiesis in the mouse is unknown. However, the identification of FeHp as a non-photosensitizing monomeric porphyrin with hematostimulatory activity in vivo, indicates that further study of metalloporphyrins is warranted and may reveal their clinical potential within the context of therapeutically-induced immunosuppression.

Adjuvant arthritis (AA) and type II collagen (CII)-induced arthritis (CIA) in the rat serve as models of chronic human arthritis. Adoptive transfer of AA was observed in 21 of 25 Lewis rats given concanavalin A (Con A)-treated spleen cells prepared from animals immunized with Mycobacterium butyricum in mineral oil (complete Freund's adjuvant, CFA). No arthritic changes were noted in rats given spleen cells obtained from donors that had received incomplete Freund's adjuvant (IFA, 0/22), type I collagen in IFA (CI-IFA, 0/6) or CII-IFA (0/28). Administration of spleen cells from IFA, CI-IFA or CII-IFA-injected animals did not modify the development of CIA when these rats were subsequently challenged with CII-IFA. However, partial protection against induction of AA was provided by the transfer of spleen cells prepared from rats immunized with CII-IFA (6/11) but not by those obtained from rats injected with IFA (1/15) or CI-IFA (0/3). Rats that did not develop clinically evident arthritis following the administration of spleen cells prepared from CFA-injected rats were also resistant to AA induction by CFA. Pre-treatment of rats with a synthetic peptide, corresponding to amino acids 180-188 of the Mycobacterium 65 kD heat shock protein (65 kD HSP), significantly delayed the onset of AA, but not that of CIA. Disease-specific resistance to AA, provided by spleen cells prepared from rats injected with CII-IFA and by pre-treatment with the 65 kD HSP 180-188 peptide, may result from the induction of protective tolerance to arthritogenic epitopes present in the Mycobacterium and CII preparations.

This report describes the effects of the porphyrin photosensitizers, Photofrin ® and benzoporphyrin derivative (BPD) on the immunohematopoietic system of normal and immunosuppressed DBA/2 mice in the absence of activating light. Photofrin ® (10 and 25 mg/kg) significantly increased in vitro colony formation by cells of the granulocyte-macrophage lineage in the spleen and bone marrow. Splenic hypercellularity, splenomegaly and elevated levels of blood leukocytes were observed in these mice 7 days following Photofrin ® injection. Evidence that Photofrin ® influenced the lymphohematopoietic compartment was suggested by a significant increase in blood lymphocytes and a population of spleen cells identified by a monoclonal antibody (LR-1) reactive with mouse splenic B lymphocytes. Proliferative reponses of spleen cells from Photofrin ® -treated mice to sub-optimal concentrations of Con A were greater than that observed for controls. However, spleen cell responses to LPS were unaltered by Photofrin ® administration. In contrast, BPD (10 mg/kg) did not alter any of the immunohematopoietic parameters studied. When Photofrin ® was administered to mice treated with the myeloablative agent 5-FU there was a significant acceleration in the recovery of total blood leukocyte and spleen cell numbers, relative to the controls. These studies demonstrate that, in addition to its previously documented activities as a photosensitizer, Photofrin ® can exert stimulatory effects upon murine hematopoiesis.

The effects of the photosensitizers, PhotofrinTM and benozoporphyrin derivative monoacid ring A (BPD) on a variety of hematopoietic cell functions have been studied, both in the presence and absence of light activation. A marked increase in hematopoiesis was observed in the bone marrow and spleens of DBA/2 mice administered high dose Photofrin but not BPD. This was manifested in an increased relative spleen weight, nucleated spleen cell number and circulating white blood cell concentration 7 days following Photofrin injection. We have shown that BPD and light doses just below phototoxic ranges stimulate the growth of human colony forming committed myeloid progenitors as well as pluripotent stem cells grown in long term marrow culture. Studies on the effect of BPD on the function of T lymphocytes in the absence of light has also demonstrated a stimulatory effect. The dose range in which this is observed is considerably broader than that observed with light activation. The mechanisms involved in this stimulatory effect have been studied and are discussed.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The proinflammatory cytokine interleukin-20 (IL-20) may exert the majority of its activity in the skin. We examined the effect of various treatments including several forms of phototherapy on IL-20 expression using cultured normal human epithelial keratinocytes (NHEK). Broadband UVB light, recombinant (r) IL-1 and rIL-8 increased, while hydrocortisone reduced, NHEK supernatant IL-20 levels. Elevation of NHEK IL-20 mRNA and maximal supernatant IL-20 levels occurred with a UVB light dose (40 mJ cm(-2)) that reduced cell viability by approximately 50%. While this UVB light dose also elevated supernatant IL-1 alpha and IL-8 levels, antibody neutralization studies indicated that neither of these cytokines was directly responsible for this increase in IL-20 expression. However, the elevation in IL-20 levels was fully inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB-203580, suggesting involvement of this stress signaling pathway in this UVB light response. Photodynamic therapy (PDT) with the photosensitizer lemuteporfin, UVA light, cisplatin, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) or recombinant interferon-gamma (rIFN-gamma) either had little effect or decreased NHEK supernatant IL-20 levels. Reduced IL-20 levels paralleled the cytotoxic actions of PDT, UVA light or cisplatin and the antiproliferative effect of rIFN-gamma. Neither rIL-20 supplementation nor anti-IL-20 antibody treatments affected cell viability indicating that soluble IL-20 did not affect the short-term survival of UVB light-irradiated NHEK. Stimulation of IL-20 expression in keratinocytes by UVB light suggests that this cytokine might participate in skin responses to this ever-present environmental factor and potentially has a role in UV light-associated dermatoses.

The photosensitizer benzoporphyrin-derivative monoacid ring A (BPD-MA, verteporfin), in combination with visible light irradiation, a clinical procedure termed photodynamic therapy (PDT), has immunomodulatory activity in various mouse models. We studied the impact of BPD-MA and light upon DBA/2 mouse splenic dendritic cells (DC), a potent antigen-presenting cell (APC) type. DC treated with nanomolar amounts of BPD-MA and 690 nm wavelength light had a reduced capacity to stimulate the proliferation of alloreactive T cells. Treatment with BPD-MA and light reduced DC levels of major histocompatibility (MHC) Class I and II antigens, intercellular adhesion molecule-1 (ICAM-1, CD54), the costimulatory B7-1 (CD80) and B7-2 (CD86) molecules, leucocyte common antigen CD45, the apoptosis-regulating Fas (CD95) receptor and the integrin CD11c. In contrast, DC expression of leucocyte function-associated-1 (LFA-1, CD11a), Mac-1 (CD11b), integrin beta2 chain (CD18) and the DEC-205 receptor increased, while CD40 levels were relatively unchanged 24 h after the treatment. MHC Class I and ICAM-1 levels decreased to 40% of control levels within 2 h following the photodynamic treatment. In the absence of light, BPD-MA did not affect DC receptor levels. Changes in DC receptor levels produced by BPD-MA and red light were similar to those produced by ultraviolet B light irradiation. The photodynamic treatment of activated splenic B cells, a separate APC class, had little effect upon receptor expression, except that MHC Class II levels were moderately decreased 24 h later. Changes in DC receptor expression may contribute to the immunomodulatory action of PDT.

Iodine balance was studied in immature rainbow trout (Salmo gairdneri) held at 12 °C in water of iodine content 1.85 μg/L and fed daily 1% of body weight with food containing 0.48 μg I/g. Iodide flux into the plasma pool was 148 ng I∙h−1∙100 g−1 of which