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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.

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

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.

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 pros and cons of purging of either bone marrow or peripheral blood stem cell preparations for autologous transplantation for cancer has been debated strongly over the past decade. Recent data implicating the role of minimal residual disease in autografted marrow in cancer relapse have renewed interest in this question. There is a considerable body of literature supporting the possibility that photosensitizer molecules in combination with light might provide a therapeutic window permitting selective elimination of malignant stem cells while sparing those of normal lineage. Molecules of this class are known to be taken up more actively by most malignant cells, and intracellular concentrations are critical in their cytotoxic effect when they are activated by light at an appropriate wavelength. The present paper reviews the observations made over the past decade on a variety of photosensitizers and their effects on hemopoietic progenitors.

The purpose of this study was to determine whether the ultraviolet (UV) radiation-induced systemic suppression of the immune response results from the release of soluble suppressor factors (TsF) by UV-induced suppressor T cells (UV Ts). Injecting a TsF-specific monoclonal antibody (B16G) significantly reduced the UV radiation-induced suppression of contact hypersensitivity (CHS). The transfer of spleen cells from the UV-irradiated, B16G-treated mice into normal recipients suppressed CHS in the recipients, indicating that while the suppression of CHS was reversed in the UV-irradiated, B16G-treated mice, suppressor cells were still present. Supernatants from cultures containing UV Ts were incubated on B16G-immunoadsorbent columns. The antibody-bound fraction (45- to 60-kDa, non-disulfide-linked proteins) suppressed CHS when injected into normal recipients. These results demonstrate that the B16G antibody reacts with TsF from UV Ts and suggest that B16G acts in vivo by inhibiting the activity of TsF. Thus, suppressor factors appear to play an essential role in the regulation of immune responses by UV Ts.

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.

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.

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) ).

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.

Uptake and release/retention of the photosensitizer, benzoporphyrin derivative, monoacid ring A (BPD; 1 - 20 (mu) g/mL) was studied using cell lines (K562, L1210) and normal, non- activated and Concanavalin A-activated murine splenocytes. Concentrations of BPD in cell lysates were determined by fluorescence (440 nm excitation, 694 nm emission). The results showed that BPD was taken up and released rapidly by all types of cells within the same time frame. Maximum of BPD depended on the type of cells and was greatest in tumor cells, lowest in normal, non-activated cells and intermediate in activated cells. In addition, the maximum uptake depended on BPD concentration in the medium, length of incubation and presence of serum. All cells, regardless of type, retained a constant proportion (20 - 30%) of the amount of BPD taken up. This proportion was independent of length of incubation, BPD concentration in the medium and presence of serum. However, due to differences in maximum amounts of BPD taken up under the same conditions, tumor cells retained more BPD than normal cells and activated cells more than non-activated. The retained BPD was able to photosensitize the cells. The results were found to be relevant to the in vivo studies.

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.

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.

We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.

Benzoporphyrin derivative (BPD) and light is a potent photosensitizer. We investigated this modality as a means to selectively eliminate clonogenic Ph(+) chronic myeloid leukemia (CML) cells. BPD at 10 ng/ml and 10.8 J/cm2 broad spectrum light eliminates from 5 to 6 logs of Ph(+) EM-2 cells. Long-term marrow culture studies of treated mixtures of normal and CML cells indicate that multipotent progenitor cell viability is retained while cells transcribing BCR-ABL are not detected. We conclude that BPD and light may offer a means of providing CML autografts potentially free of Ph(+) clonogenic cells.

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.

We have developed procedures in which the photosensitizer benzoporphyrin derivative monoacid ring A (BPD) can be covalently linked to carrier molecules of modified polyvinyl alcohol (PVA) to produce water-soluble PVA-BPD conjugates with a molecular mass in the range of 30 kd. These carriers can subsequently be covalently linked to monoclonal antibodies (MoAbs) using heterobifunctional linking agents. We describe here such a conjugate in which the MoAb (5E8) has specificity for a glycoprotein detected on human squamous cell carcinomas of the lung. We provide evidence that the conjugates produced were covalently linked and retained both their photosensitizing and antigen-binding activities. We show further that the MoAb-PVA-BPD conjugate, in the presence of 10% fetal calf serum, exhibited highly enhanced phototoxic killing of the target cell line (A549) over that exhibited by free BPD or a control MoAb-PVA-BPD conjugate. These results demonstrate, therefore, both the selectivity and specificity of this MoAb conjugate.

Antigenic material was isolated from a human squamous cell bronchogenic carcinoma tissue culture line A549 that was found to react with antibodies in the serum of patients with lung cancer in an enzyme-linked immunosorbent assay (ELISA), while it did not react with sera from normal individuals. The antigen was tested with a panel of sera from a variety of patient groups by means of the ELISA. Results showed significantly higher numbers of sera from patients with lung cancer, particularly those of squamous cell origin, reacting with the antigen than of sera from 173 normal individuals or patients with breast and gynaecological cancers or melanomas.

Suppressor cell activity is induced by the addition of prostaglandin E 1 to normal mouse spleen cells (NSC). When this population is added to fresh NSC, it suppresses the primary in vitro antibody response to DNP-LPS. This suppressor cell induction requires a brain-associated thy-1 antigen-bearing cell. No role for a nylon-wool-adherent cell could be demonstrated. Suppression occurs in the absence of T cells in the responding population. The addition of indomethacin to prevent endogenous synthesis of prostaglandins suggests that prostaglandininduced suppressor activity does not require further prostaglandin synthesis in the suppressor population but does require such synthesis in the responding population.

Xeno-antiserum specific for antigenic components of bronchogenic carcinoma was raised in rabbits, by passively immunizing them to normal human lung antigens at the same time as immunization with a tumour extract from a squamous-cell carcinoma. Antiserum so raised contained minimal quantities of anti-normal antibody which could be removed by a single absorption with glutaral-dehyde-insolubilized normal lung extract. When tested by quantitative complement fixation with a panel of tumour extracts from surgical specimens, it was found that the antiserum gave positive complement fixation with all squamous-cell carcinoma extracts tested, and with some of the extracts from bronchogenic carcinomas of differing pathological types. The antiserum was essentially negative for pooled extracts from normal lung, liver and spleen but gave a weak positive reaction with an extract of pooled foetal lung tissue.

Nonadherent splenic lymphoid cells from tumor-bearing animals exhibit a poor primary in vitro plaque-forming cell (PFC) response to SRBC. This is attributable to the presence, in this population, of a T suppressor cell. This nonadherent population suppressed the in vitro anti-SRBC PFC response of normal spleen cells (NSC). This nonspecific suppressive effect was eliminated by adding indomethacin, a prostaglandin synthetase inhibitor, to the in vitro cultures. These effects were seen at indomethacin concentrations which have no direct effect on the NSC response. When exogenous prostaglandins were added to NSC cultures plus SRBC, prostaglandins PGE1 and PGE2 were shown to markedly inhibit the PFC response at a wide range of concentrations, whereas PGA1, PGF1α, and PGF2α were inhibitory only at the highest concentrations tested.

An anti-idiotypic antiserum was raised in rabbits to a monoclonal antibody (Fd-1) with specificity for one (the N epitope) of the two antigenic epitopes found on the ferredoxin (Fd) molecule. The anti-idiotypic antiserum (anti-Fd-1) was used to demonstrate that the Fd-1 idiotype was expressed at significant levels in most anti-Fd antisera raised in B10.BR mice. Examination of antisera raised in other mouse strains demonstrated that expression of this idiotype mapped to the IgH gene complex and was found in the antisera of all mouse strains examined with the Ig-1 allotype. When splenocytes from Fd-immune B10.Br mice were treated with anti-Fd-1 and transferred to irradiated syngeneic recipients, the adoptive secondary response was significantly higher in animals receiving treated cells as opposed to control animals, which received normal rabbit serum-treated cells. This response produced a net increase in antibody to both determinants, and the relative amount of Fd-1 idiotype was not significantly altered. Further studies with separated cell populations showed that the overall increase of anti-Fd antibody produced was attributable to the effects of the anti-idiotypic serum on a population(s) of T cells. Treatment of mice with the Fd-1 monoclonal antibody (which should react with anti-idiotypic cells) had an analogous effect to that of the anti-idiotype, in that mice so treated produced higher concentrations of anti-Fd antibodies when they were immunized and these antibodies exhibited net increases to both determinants. A model is presented to explain these results.

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleen of normal or tumor-bearing mice, the TsF produced by tumour-specific, or hapten-specific T-cell hybridomas, and with polyclonal whole human TsF isolated from tonsillar tissue. This pan-reactivity inherent to the B16G antibody suggests that it recognizes some common, shared epitope of the TsF molecule. In this study, we have used B16G as a probe to isolate a TsF-producing T-cell hybridoma, S-50, from CBA mice (H-2k) that is specific for the Lupus-associated antigen, RNP-Sm. The TsF bound specifically to RNP-Sm and inhibited the production of anti-RNP-Sm antibody cell cultures from MRL-lpr mice. SDS-PAGE analysis of purified S-50 TsF revealed a B16G-reactive band with a molecular weight of 43 kd.

T-cell lines were derived from ferredoxin nonresponder B10.D2 mice that share an idiotype expressed by a monoclonal antibody (Fd-B2) with specificity for one of the two major antigenic determinants (the C determinant) of the antigen. The T-cell line and T-cell clones derived from it release interleukin 2 not only in the presence of anti-Fd-B2 idiotype antibody but in the presence of ferredoxin. The line was shown to be major histocompatibility complex-restricted in that it would respond to the anti-idiotype and antigen only in the context of presentation by cells of the H-2d haplotype. This observation also establishes that the nonresponder status of H-2d animals cannot be attributed to a lesion at the level of antigen presentation. Analysis of the fine specificity of one idiotypic clone showed that it responded only to the anti-idiotype or products of the antigen containing the C determinant, since enzymatically degraded peptides devoid of this determinant did not stimulate these cells. Furthermore, it was found that presentation of both the antigen and the anti-idiotype to the specific clone could be blocked by the Fd-B2 monoclonal antibody.

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2 d antiserum (raised in congenic mice) were all able to block killing when 51 Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2 d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of 51 Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2 d serum but not by the anti-P815 serum. The implications of these observations are discussed.

A monoclonal antibody (Fd-B2) to ferredoxin, which bears an idiotype scarcely expressed in any of a wide variety of mouse strains, is able to markedly enhance the response to ferredoxin of both high-responder and intermediate-responder strains. A rabbit anti-idiotype serum to Fd-B2 also specifically enhances the response to ferredoxin in such mice. Most remarkably, the treatment of nonresponder T cells by either the idiotype (Fd-B2) plus complement or anti-idiotype antiserum plus complement causes them to be responsive in adoptive transfer experiments. The two responding populations (idiotype-treated and anti-idiotype-treated) can then be combined to reconstitute the nonresponsive state. When the nonresponders are treated with either Fd-B2 idiotype plus complement or anti-idiotype plus complement and subsequently respond, the idiotype of the anti-ferredoxin antibody produced does not bear the Fd-B2 idiotype. We interpret the results as being consistent with a model in which the unresponsive state for ferredoxin is a state of high network connectivity of the ferredoxin-specific T cells.

In a previous publication a monoclonal antibody (B16G) which appeared to recognize T suppressor cells and a T-suppressor factor (TsF) in the spleens of DBA/2 mice was described. B16G appears to be directed to a public specificity of DBA/2 TsF and therefore has been shown to inhibit a variety of immunological reactions. The present study involves preliminary characterization of the material with which B16G reacts. It was found that the B16G-reactive protein (putative TsF) could be absorbed and eluted specifically from a B16G immunoadsorbent column. Material eluting from the B16G column reacted with B16G in an ELISA and appeared to run as two or more bands of 40–45 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted material was biologically active (i.e., suppressive) in the standard assay (mixed leukocyte reaction of DBA/2 splenocytes with B10.BR targets), and its suppressive activity was abrogated by the addition of B16G to the mixed leukocyte reaction cultures. Sephadex G-150 chromatography of the B16G-reactive material showed that under these conditions, its native molecular mass was between 80–90 kDa, indicating that it might occur as a dimer under natural conditions.

Mice were immunized with antigen (Rabbit Fab' fragments) attached to syngeneic, or f 1 (semi-syngeneic) irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers showed that the response towards antigen on syngeneic or F 1 cells, was significantly lower than that towards the same antigen on allogeneic cells. By subsequent in vitro incubation of immune spleen cells with antigen followed by plaque assay, it was found that those spleen cells exhibiting lowered plaque forming cell numbers initially, (i.e., those mice immunized with antigen on syngeneic or F 1 cell surfaces) showed, after incubation, a response equal to or greater than those cells which initially (before in vitro incubation) demonstrated a larger response (i.e. those mice immunized with antigen on allogeneic cells).

The properties of a rabbit antiserum (anti-AML) raised to a purified protein from membranes of human acute myelogenous leukemia (AML) cells is described. Bone marrow and peripheral blood leukocytes (PBL) from either normal individuals or patients with either myeloproliferative or other disorders were analyzed in a fluorescence-activated cell sorter (FACS IV) after labeling with anti-AML, normal rabbit serum (NRS), or antiserum raised to normal human membrane antigens. Of 40 cell samples from patients with acute myelogenous leukemia, 39 reacted strongly with the anti-AML antiserum. Similarly, all of 19 specimens from patients with chronic granulocytic leukemia reacted with the anti-AML. When 42 bone marrow or PBL samples from patients with a variety of lymphoproliferative disorders were examined, 2 specimens reacted with the antiserum, both from individuals with diagnoses of acute lymphocytic leukemia (ALL). None of the 14 normal bone marrow or PBL donor specimens tested reacted with the antiserum. It was also found that essentially all samples from patients in clinical remission from AML had high numbers of cells reactive with the anti-AML. When cells from such individuals were labeled and sorted on the FACS IV, it was found that cells fluorescing strongly with the anti-AML contained cells of both myeloid and lymphoid origin. The implications of these results are discussed.

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt- 12+ , whereas the syngeneic effector cells were found to be predominantly Lyt- 2+ . The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt- 1+ cell is essential in this suppression.

Spleen cells from C57Bl/6, BALB/c and CBA/J mice bearing primary methylcholanthrene induced sarcomas and C3H/HeJ mice bearing primary spontaneous mammary tumors gave suppressed responses to mitogens. Those spleens also contained a population of suppressor cells which inhibited the mitogen responses of normal spleen cells. Immunoglobulin rich fractions of sera from all these mice except the BALB/c tumor bearers, contained a suppressive factor which inhibited the mitogen response of normal syngeneic spleen cells.

The NH 2 -terminal heptapeptide and the COOH-terminal pentapeptide of performic acid-oxidized ferredoxin from Clostridium pasteurianum have been shown to encompass the two major haptenic regions of this molecule. These peptides were conjugated to succinylated bovine serum albumin (S-BSA) to yield an immunologically bivalent hapten-carrier conjugate (N + C-S-BSA). Peptides were also synthesized which contained the NH 2 -terminal and COOH-terminal haptenic peptides linked by a bridge of five amino acids (N-5-C), these two peptides linked by 10 amino acids (N-10-C), and one containing two COOH-terminal peptides linked by 12 amino acids (C-12-C). The ability of these preparations to elicit various immunological responses was tested. In O-Fd-sensitized guinea pigs, N + C-S-BSA, N-5-C, and N-10-C elicited immediate and delayed skin reactions; N-5-C and N-10-C inhibited the migration of macrophages; N + C-S-BSA and N-10-C stimulated the proliferation of lymphocytes from these sensitized animals, however, N-5-C and C-12-C did not. In animals sensitized to N + C-S-BSA, skin reactions were elicited by O-Fd, S-BSA, and the NH 2 -and COOH-terminal peptides alone. In these animals, lymphocyte proliferation was stimulated significantly by either O-FD or S-BSA. The N-5-C peptide was found to be nonimmunogenic by the schedule used here. However, the N-10-C peptide was found to be strongly immunogenic, and, in animals sensitized to N-10-C, skin reactions and MIF were elicited by N-10-C and 0-Fd, and lymphocyte proliferation was stimulated by N-10-C and O-Fd, but not by C-12-C. The implications of these results in relation to the bicellular mechanism of the immune response are discussed.

Performic acid oxidized ferredoxin from C. pasteurianum (O-Fd) was capable of eliciting both immediate and delayed hypersensitive skin reactions in guinea pigs sensitized with O-Fd. Two haptenic peptides from O-Fd (the NH2-terminal heptapeptide and the COOH-terminal octa- or tetrapeptide) were also capable of initiating both immediate and delayed reactions when they were administered together to sensitized guinea pigs. When either peptide was administered alone, delayed reactions were observed more frequently than immediate reactions. Both O-Fd and the haptenic peptides were capable of inhibiting the migration of peritoneal exudate cells taken from O-Fd-sensitized guinea pigs. Conjugates of succinylated BSA (S-BSA) were made with a combination of both haptenic peptides (N + C-S-BSA), with the COOH-terminal tetrapeptide (C-S-BSA) and with the NH2-terminal heptapeptide (N-S-BSA). These conjugates caused inhibition of migration of peritoneal exudate cells from O-Fd-sensitized animals. While O-Fd was capable of stimulating 3H-thymidine uptake in lymph node cells taken from O-Fd-sensitized animals, the haptenic peptides (either together or alone) were not. When the three conjugates were tested in this way it was found that, at the doses tested, the N + C-S-BSA stimulated 3H-thymidine whereas the C-S-BSA and the N-S-BSA did not.

Photodynamic therapy (PDT) is a clinically effective cancer treatment. For human promyelocytic leukemia HL-60 cells, cleavage of pro-caspase-3 (CPP32/Yama/apopain) into its proteolytically active subunits rapidly follows the photodynamic treatment of these cells with cytotoxic levels of the photosensitizer benzoporphyrin derivative monoacid ring A and visible light. Cleavage of a recently identified cytosolic 45 kDa protein, DNA fragmentation factor (DFF), is required for endonuclease activation leading to DNA fragmentation. In the present study, DFF was rapidly processed following PDT. Overexpression of the anti-apoptotic Bcl-XL gene in HL-60 cells prevented PDT-induced caspase activation, DFF cleavage and DNA fragmentation. These results demonstrate for the first time an example of chemotherapeutic drug-induced activation of DFF and its regulation by Bcl-XL.

Adhesion is a primordial cell function that, among others, regulates inflammation, metastasis, and tissue repair. To understand how these events could be affected by photodynamic therapy (PDT), we studied the effects of PDT on human foreskin fibroblast (HFF) adhesion to bovine collagen type I, human vitronectin or fibronectin. PDT, using benzoporphyrin derivative monoacid ring A (verteporfin) as the photosensitizer, inhibited cell adhesion in a drug dose-dependent manner, with no significant difference among matrices. The drug dose that killed 90% of cells within 20h post-treatment inhibited HFF adhesion by 55%–68%. However, 45min following PDT, a time period corresponding to that of the adhesion assay, HFF membrane integrity remained unaltered. In addition, cell surface expression of integrins was not modified for at least 2h following PDT. Western blots of cell lysates, using the anti-phosphotyrosine 4G10 monoclonal antibody, revealed that PDT prevented the adhesion-induced phosphorylation of 110–130kDa proteins. Immunoblots of cell lysates immunoprecipitated with antibodies to focal adhesion kinase suggested that its phosphorylation was suppressed by PDT. These results demonstrate that PDT inhibits cell adhesion and affects integrin signalling without modifying cell membrane integrity or integrin expression.

Previous studies using synthetic immunogenic molecules containing two haptenic peptides (ther NH2-terminal heptapeptide and the COOH-terminal pentapeptide of oxidized ferredoxin (O-Fd) from C. pasteurianum) have shown that both peptides individually are capable of initiating lymphocyte transformation and inhibiting migration in populations of lymphocytes from O-Fd-sensitized guinea pigs. While migration inhibition could readily be stimulated by single haptenic peptides, lymphocyte treasformation appeared to be more easily induced by molecules containing two or more haptenic peptides (these could be identical or different) (Kelly, B., Levy, J.G. and Hull, D., Eur. J. Immunol., 1973. 3:574). If lymphocyte transformation is a T cell-mediated phenomenon, these observations indicate the possibility of T cell-T cell interaction. The two haptenic peptides (designated "N" and "C") were synthesized and conjugated to succinylated bovine serum albumin (S-BSA), forming the conjugates N-S-BSA and C-S-BSA, respectively. These conjugates were labeled to high specific activity with 125iodine and were used in an "antigen suicide" procedure to treat guinea pig lymph node cell preparations previously sensitized to O-Fd and keyhole limpet hemocyanin (KLH). Cell populations exposed to either 125I-labeled C-S-BCA demonstrated decreased lymphocyte transformation in the presence of O-Fd but not in the presence of KLH. These results indicate specific cell inactivation by the radioactive peptide conjugates of those cells responsible for initiating cell transformation. Experiments performed by mixing 125I-labeled N-S-BSA-treated cells with 125I-labeled C-S-BSA-treated cells were successful in partially restoring the response to O-Fd and suggest possible synergy between N and C determinant binding cells in the cellular immune response to O-Fd. Evidence from B cell depletion studies suggests that this is a T cell-T cell interaction.

The ferredoxin (Fd) molecule is a small non-mammalian immunogenic protein containing 55 amino acid residues with only two major antigenic determinants located with the NH 2 -terminal heptapeptide and the COOH-terminal pentapeptide. Selective enzyme cleavages of Fd with either trypsin or carboxypeptidase. A result in the inactivation of the antigenic determinants by the removal of a tripeptide at the NH 2 -terminal and two amino acid residues at the COOH-terminal, effectively leaving 52 and 53 amino acid fragments respectively, each containing a single antigenic determinant. Fd digested with both enzymes yielded a 50 amino acid peptide with both determinants inactivated. Purity of these digests was assessed using monoclonal antibodies in standard and antigen-blocking ELISAs. The doubly digested peptide had virtually no reactivity with anti-Fd sera, reconfirming that the central cysteine-rich region is serologically silent. It was found that the sum of the reactivities of the N- and C-determinant-bearing peptides as equal to that of the native Fd and that the ratio of the reactivities could be used to assess determinant selectivity in the response to Fd in congenic recombinant strains of mice. This method was used in mapping the determinant selectivity in the antibody response to the MHC of mice to the left of the I-B subregion. Use of the B10.HTT strain indicated that separate genes mapping to the same subregion code for the magnitude of the antibody response and the determinant selectivity of the response.

A low molecular weight fraction from chitinase digested cell walls of T. mentagrophytes containing both polysaccharide and peptide moieties was found to have immunological reactivity at both the cellular and humoral level. This fraction (UM2(a)) was further degraded by treatment with either a combination of pronase and carboxypeptidase A or with trypsin. Peptides were separated from the carbohydrate-rich fraction by ultrafiltration. The carbohydrate-rich fraction retained the ability to induce both immediate and delayed skin reactions in sensitized guinea pigs and to stimulate the proliferation of sensitized lymphocytes in vitro. The peptide moieties retained reactivity in that they caused delayed reactions and lymphocyte proliferation but were unable to induce immediate or Arthus reactions in sensitized animals. Tryptic peptides from UM2(a) were purified by ion exchange chromatography. A high proportion of these peptides demonstrated immunological activity at both the cellular and humoral level since they were capable of inducing delayed reactions and/or lymphocyte transformation, as well as being capable of blocking the complement fixation reaction between UM2(a) and specific antiserum.