Induction of transplantation immunity by dansylated tumor cells

Tumor cells were coupled with fluorecent dansyl group in aqueous medium by dansyl chloride-cycloheptaamylose complex (CDC) without destruction of the cells. C57BL/6 mice and Donryu rats pretreated respectively with dansylated EL4 leukemia cells and with dansylated Yoshida sarcoma cells acquired transplantation immunity to the corresponding tumor cells. Serum and spleen cells obtained from EL4 immune mice showed cytotoxicity to EL4 cells but not to other allogeneic leukemia cells. Hapten-specific cytotoxicity of immune serum and spleen cells was not observed in the present immune system.     

Selective tumor kill of cerebral glioma by photodynamic therapy using a boronated porphyrin photosensitizer

The prognosis for patients with the high-grade cerebral glioma glioblastoma multiforme is poor. The median survival for primary tumors is < 12 months, with most recurring at the site of the original tumor, indicating that a more aggressive local therapy is required to eradicate the unresectable "nests" of tumor cells invading into adjacent brain. Two adjuvant therapies with the potential to destroy these cells are porphyrin-sensitized photodynamic therapy (PDT) and boron-sensitized boron neutron capture therapy (BNCT). The ability of a boronated porphyrin, 2,4-(alpha, beta-dihydroxyethyl) deuteroporphyrin IX tetrakiscarborane carboxylate ester (BOPP), to act as a photosensitizing agent was investigated in vitro with the C6 rat glioma cell line and in vivo with C6 cells grown as an intracerebral tumor after implantation into Wistar rats. These studies determined the doses of BOPP and light required to achieve maximal cell kill in vitro and selective tumor kill in vivo. The data show that BOPP is more dose effective in vivo by a factor of 10 than the current clinically used photosensitizer hematoporphyrin derivative and suggest that BOPP may have potential as a dual PDT/BNCT sensitizer.     

Induction of antitumor immunity by interleukin-2 gene-transduced mouse mammary tumor cells versus transduced mammary stromal fibroblasts

BACKGROUND: Tumor cell-targeted cytokine gene transfer has been used to generate tumor cell vaccines, but this approach is limited by the need to establish and implant live tumor cells. PURPOSE: The purpose of this study was to determine if stromal fibroblasts could be used as an alternative vehicle for delivery of the cytokine interleukin-2 (IL-2) into the tumor microenvironment. We attempted to establish the feasibility of (a) genetic immunotherapy in a mammary tumor system and (b) engineering stromal fibroblasts as well as tumor cells. We compared the effects of tumor cell-mediated and stromal fibroblast-mediated local IL-2 expression on the generation of antitumor immune responses. METHODS: Retroviral vectors containing a human IL-2 gene were used to transduce a mouse mammary tumor line, 4TO7, and an immortalized but nontumorigenic fibroblast line established from syngeneic mammary fatpads. Expression of the IL-2 gene in transduced cells was determined by measuring IL-2 secretion, by RNA-polymerase chain reaction, and by immunochemistry. Groups of 5-12 BALB/c mice were injected with either 4TO7 cells or various doses of IL-2-secreting 4TO7 cells (4TO7-IL-2); tumor growth was monitored. To test whether local IL-2 expression by transduced cells could influence the growth of unmodified tumor cells, we determined tumor development in groups of mice treated with 4TO7 cells co-injected with either 4TO7-IL-2 cells or IL-2-secreting fibroblasts. RESULTS: 4TO7-IL-2 cells induced active immunity able to reject the immunizing tumor and to resist challenge with parental 4TO7 cells on the contralateral side. Mice pretreated with 4TO7-IL-2 were significantly protected compared with untreated control animals or mice pretreated with irradiated 4TO7 cells. The immunity induced by 4TO7-IL-2 cells did not protect against challenge with another subline, 4T1, which was derived from the same spontaneously arising mammary tumor as 4TO7. Co-injection of 4TO7 cells with 4TO7-IL-2 cells reduced tumorigenicity, whereas co-injection of 4TO7 cells with IL-2 secreting fibroblasts did not. CONCLUSION: Our results suggest that induction of anti-tumor immune response by local IL-2 production is most effective when the helper cytokine is secreted by the tumor cell. IMPLICATION: Our studies caution against the use of IL-2 gene-transduced syngeneic stromal cells as an alternative strategy of gene therapy for cancer. However, they may allow study of the mechanisms of tumor antigen recognition and the possible involvement of co-stimulatory signals for effective tumor vaccination by gene-modified cells.     

Measurement of tumor vascular damage in mice with 99mTc-MIBI following photodynamic therapy

The clinical perfusion agent 99mTc-MIBI was used to monitor changes in tumor vascular perfusion (TVP) induced by Photofrin (PII)-mediated photodynamic therapy (PDT). BALB/c mice bearing an EMT-6 tumor on each hind thigh were given an intravenous injection of 1, 2 or 5 mg kg-1 PII. Twenty-four hours later, one tumor was illuminated (600-650 nm, 200 mW cm-2, 400 J cm-2) while the other served as a control. At various time intervals after PDT (0, 2 and 24 h) mice received an intravenous injection of 99mTc-hexakismethoxyisobutylisonitrile (MIBI) (0.18 MBq g-1) and were sacrificed 2 min later. The light-treated and the untreated tumors were then dissected, the radioactivity was counted and the percentage of the injected dose per gram of tumor (%ID g-1) was calculated as a measure of TVP. We observed that TVP is drug dose dependent, develops progressively with time post-PDT and is inversely related to PDT efficacy. Our data show that early tumor retention of 99mTc-MIBI is a simple method to assess TVP and vascular damage induced by PDT.     

Historical development of photodynamic therapy

Photodynamic therapy is based on the accumulation of photosensitizing drugs in tumours and subsequent activation by visible light, leading to the release of singlet oxygen in photochemical reactions. Besides the treatment of precancerous lesions and malignant tumours in superficial sites, new experimental indications, such as psoriasis, are being investigated. The development of new photosensitizing agents for topical application and appropriate light sources has led to increasing interest in this promising treatment modality among dermatologists. This historical review deals with the scientific investigations of photodynamic therapy and diagnosis that started with the experiments of Oscar Raab at the end of the nineteenth century.     

Vascular effects of photodynamic therapy

Vascular damage and blood flow stasis are consequences of photodynamic therapy (PDT) of solid tumors using many photosensitizers. Microvascular stasis and resulting hypoxia are effective means to produce cytotoxicity and tumor regression. The observation of blood flow stasis after photodynamic therapy results from a combination of damage to sensitive sites within the microvasculature and the resulting physiological responses to this damage. A generalized hypothesis for the mechanisms leading to vessel stasis begins with perturbation and damage to endothelial cells during light treatment of photosensitized tissues. Endothelial cell damage leads to the establishment of thrombogenic sites within the vessel lumen and this initiates a physiological cascade of responses including platelet aggregation, the release of vasoactive molecules, leukocyte adhesion, increases in vascular permeability, and vessel constriction. These effects from damage combine to produce blood flow stasis.     

Light distribution by linear diffusing sources for photodynamic therapy

The distribution of the light emitted by linear light diffusers commonly employed in photodynamic therapy (PDT) has been investigated. A device is presented which measures the angular distribution of the exiting light at each point of the diffuser. With these data the fluence rate in air or in a cavity at some distance from the diffuser can be predicted. The results show that the light is scattered from the diffuser predominantly in the forward direction. Experiments and calculations show that the fluence rate in air and in a cavity of scattering tissue at some distance from the diffuser has a maximum near the tip of the diffuser, instead of near the middle. However, the fluence rate resulting from an interstitial diffuser in a purely scattering tissue phantom shows a maximum in the bisecting plane of the diffuser as would be predicted when the diffuser emitted light isotropically. The scattering nature of the tissue is expected to cancel the anisotropy of the diffuser.     

Advances in ophthalmological photodynamic therapy

Photodynamic therapy is a new experimental therapeutic technique which is attracting increasing attention. Its biopharmacological basis of action is the specific interaction of a photosensitizing compound with the cellular elements of pathological lesions. The photosensitizer is thought to enter specifically into the pathologic cells, where it accumulates. The lesion is then irradiated with a sensitizing laser-beam of specific wave-length to activate the photosensitizer, which then becomes a generator of free oxygen radicals. These radicals destroy the sensitizer-harboring pathological cells. The advantage of specifically destroying pathological lesions without affecting surrounding normal tissue is obvious. Recently, many experimental studies have been conducted to test the usefulness of photodynamic therapy for ocular disorders, mainly advanced age-related macular degeneration and uveal melanoma. Results so far are encouraging.     

Clinical and preclinical photodynamic therapy

Photodynamic therapy (PDT) is a treatment modality that utilizes a photosensitizing drug activated by laser generated light, and is proving effective for oncologic and nononcologic applications. This report provides an overview of photosensitizers, photochemistry, photobiology, and the lasers involved in photodynamic therapy. Clinical and preclinical PDT studies involving Photofrin and various second generation photosensitizers are reviewed.     

Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy

A major goal of tumor immunotherapy is the induction of tumor-specific T cell responses that are effective in eradicating disseminated tumor, as well as mounting a persistent tumor-protective immunity. We demonstrate here that a genetically engineered fusion protein consisting of human/mouse chimeric anti-ganglioside GD2 antibody and human interleukin-2 is able to induce eradication of established B78-D14 melanoma metastases in immunocompetent syngeneic C57BL/6J mice. This therapeutic effect is mediated by host immune cells, particularly CD8+ T cells and is associated with the induction of a long-lived immunity preventing tumor growth in the majority of animals when challenged up to four months later with B78-D14 cells. This effect was tumor-specific, since no cross-protection against syngeneic, ganglioside GD2+ EL-4 thymoma cells was observed. Furthermore, this tumor-specific protection can be transmitted horizontally to naive, syngeneic SCID mice by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of cytokines provides a means to elicit effective immune responses against established tumors in the immunotherapy of neoplastic disease.     

Interstitial photodynamic therapy with tetra(m-hydroxyphenyl)chlorin: tumor versus striated muscle damage

In this study, we have investigated the changes in the number of individual presynaptic boutons in the neocortex of rats and correlated them with cognitive performance. Brown Norway x Fischer 344 F1 hybrid rats, aged from one to 24 months, were used. Using synaptophysin as a marker for presynaptic boutons, we found that in the parietal II region of the neocortex an age-related decrease in the density of immunostained punctae representing presynaptic boutons occurred. Regression analysis showed that this decline in the number of presynaptic boutons correlates with ageing (r=0.495, P<0.05). Interestingly, we found that this age-related depletion of presynaptic boutons was more intense in the deeper cortical lamina, such as laminae V and VI (mean decrease of 18%), than in the superficial laminae (mean decrease of 8% in laminae I-IV). Using the Morris water maze test, we observed that young rats acquired the task at twice the speed of aged animals (48.9 +/- 9.0 s and 91.0 +/- 4.9 s for young and aged animals, respectively). Furthermore, at the end of the training period, the aged cohort still showed significantly higher escape latencies in the Morris water maze. The present findings support the concept that the decline in cognitive performances in ageing is related to the loss of synapses in the cerebral cortex.     

Heteroclitic immunization induces tumor immunity

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.     

Induction of protective immunity against murine secondary hydatidosis

Significant protective immunity against secondary hydatidosis in mice was achieved by immunization with a preparation of surface molecules of E. granulosus protoscoleces with Freund's incomplete adjuvant (PSEx-IFA). Study of PSEx-IFA immunogenicity demonstrates that glucidic epitopes evoked mainly IgM responses while peptidic epitopes evoke mainly IgG responses; however both types of epitopes elicit both types of responses. Analysis of the possible association between susceptibility or resistance to infection and antibody responses after challenge was also performed. Cyst fluid antigen (CFAg) specific antibody titres on month eight after challenge correlated with number and size of cysts. On the other hand no correlation was observed between protection and PSEx specific IgG titres either on the day of challenge or one month later. Nonetheless, immunoblot analysis revealed that some PSEx molecules were recognized on day 30 after challenge only by sera from immunized mice.     

Enhancement of the efficiency of photodynamic therapy of tumours by t-butyl-4-hydroxyanisole

The effects of photodynamic therapy (PDT) alone and in combination with 3(2)-t-butyl-4-hydroxyanisole (BHA) on Ehrlich ascites carcinoma (EAC) cells have been investigated. BHA, a widely used food antioxidant, administered to the cells prior to light exposure is found to cause concentration-dependent alterations of the haematoporphyrin derivative (HpD)-based PDT. BHA (0.15 mM) causes a small (about 10%) inhibition in the rate of HpD-photosensitized injury of EAC cells. In contrast, upon increasing the concentration of BHA from 0.15 to 0.5 mM, a 1.3-fold enhancement in HpD-PDT efficiency is achieved. The cytotoxic effect on the cells treated with HpD-PDT and a higher concentration of BHA (0.5 mM) is additive. When BHA (0.5 mM) is given immediately after HpD-PDT, the combination is found to be three to four times more effective than when BHA is added to EAC cells before phototherapy. In this treatment regimen BHA acts synergistically with HpD-PDT. Such a difference in the action of BHA on the efficiency of HpD-PDT might be explained by the ability of BHA to inhibit the HpD-photosensitized destruction of some biomolecules. An enhancing action of BHA on the intensity of HpD-photosensitized death of tumour cells is also observed in vivo. Even a single dose of BHA (0.6 mM kg-1, 15 min after irradiation) causes (in an additive manner) an approximately two-fold increase in the efficiency of HpD-PDT of mice bearing Ehrlich ascites tumour (intraperitoneal transplantation). The results obtained indicate that the potentiating effect of BHA on the HpD-PDT could be caused by the impairment of the mitochondrial respiration, since there is a good correspondence between the concentration of BHA that increases the efficiency of PDT and the concentration that inhibits the oxygen consumption and dehydrogenase activity of EAC cells. The influence of BHA on the efficiency of PDT does not depend on the nature of the photosensitizer used; the effects with chlorin-e6 trimethyl ester are similar to that seen for HpD.     

Induction of glycosuria and hyperglycemia by topical corticosteroid therapy

A patient with psoriasis is described who had an abnormal response to the glucose tolerance test without other evidence of diabetes and then developed postprandial hyperglycemia and glycosuria during a period of topical administration of a corticosteroid cream, halcinonide cream 0.1%, under occlusion. A second patient with a similar glucose tolerance test result showed postprandial hyperglycemia when treated similarly with betamethasone valerate cream 0.1%. Two additional patients with midly abnormal responses to glucose tolerance tests showed no evidence of altered glucose metabolism when treated with halcinonide cream in a similar manner.