Bone marrow transplant conditioning intensified with liposomal clodronate to eliminate residual host antigen presenting cells fails to ameliorate GVHD and increases PERI-BMT mortality.

BACKGROUND: Graft versus host disease (GVHD) mediated by allogeneic donor T cells may be initiated and/or exacerbated by residual host antigen presenting cells (APC) which survive the transplant conditioning regimen. We examined whether the depletion of hepatic and splenic APC could reduce the severity of hepatic GVHD after bone marrow transplantation (BMT). METHODS: Recipient mice were depleted for hepatic and splenic phagocytic APCs by i.v. injection of clodronate- (dichloromethylene diphosphonate) containing liposomes before fully allogeneic or MHC-matched, minor Ag-mismatched BMT. Severity of hepatic GVHD was scored on histological sections 2, 3, 4, or 9 weeks after BMT. RESULTS: No differences in the severity of GVHD were observed between APC-depleted mice and control mice. APC-depleted mice had increased peritransplant mortality due to sepsis. Bacterial clearance assays showed that APC-depleted mice were unable to efficiently clear bacteria, although nondepleted, transplanted mice were able to clear bacteria as quickly as naive control mice. CONCLUSIONS: Residual host phagocytic APC do not appear to play a role in the induction of GVHD after BMT. They are, however, essential for prevention of sepsis in the transplant host.

Local low-dose IL-2 therapy.

Interleukin-2 (IL-2) is a powerful drug for treating cancer. However, it is only powerful if it is properly applied. That is, IL-2 should be applied at the tumor site, because at the transition of normal and malignant tissue are the tumor infiltrating cells. These should be activated by IL-2. Local application implies that IL-2 can be used in relatively low doses. It is becoming clear that even a single injection of IL-2 can cure cancer. IL-2 can also enhance the therapeutic effects of irradiation and Cisplatin. Locally applied IL-2 therapy is virtually non-toxic.

Interleukin 2 (IL-2) therapy: potential advantages of locoregional versus systemic administration.

There has been an apparent discrepancy between the results obtained with IL-2 based immunotherapy in animal tumour models, including veterinary cancer patients, and human cancer patients. We argue that this is due to differences in the therapeutic regimens used to treat human and veterinary cancer patients. The main differences are systemic therapy and surgical removal of the primary tumour in case of human cancer patients, whereas these treatment modalities are not used in IL-2 treated veterinary cancer cases. We have developed a treatment protocol, in which IL-2 is applied locally, that has been successful against transplanted tumours as well as spontaneous tumours of varying origin and immunogenicity. In view of immunobiological considerations we conclude that local treatment of cancer with IL-2 makes more sense than systemic treatment, as usually applied in the clinic, and that surgical removal of tumours may be detrimental to successful IL-2 therapy.

Enhancement of graft-versus-tumor activity and graft-versus-host disease by pretransplant immunization of allogeneic bone marrow donors with a recipient-derived tumor cell vaccine.

Allogeneic bone marrow transplantation (BMT) can be accompanied by a beneficial T cell-mediated antitumor immune response known as graft-versus-tumor (GVT) activity. However, BMT donor T cells are not exposed to target antigens of GVT activity until transfer to the host, where tumor antigen presentation may be suboptimal. This study tested in a murine model the hypothesis that immunization of MHC-matched allogeneic donors with a recipient-derived tumor cell vaccine would substantially increase GVT activity and extend survival of BMT recipients with preexisting micrometastatic tumor. C3H.SW and C57BL/10 mice were immunized against a C57BL/6-derived fibrosarcoma or leukemia, and they were used as BMT donors. Recipients were H-2-matched, minor histocompatibility antigen-mismatched C57BL/6 mice with previously established micrometastatic tumors. Donor immunization led to a significant increase in GVT activity that was T cell dependent and cell dose dependent. In some settings, donor immunization also prolonged survival of recipients with preexisting micrometastatic tumors. However, donor immunization significantly increased the incidence of fatal graft-versus-host disease such that long-term survival was uncommon. In vitro cytotoxicity assays indicated that donor immunization induced both tumor-selective and alloreactive cytolytic T-cell populations. In vivo cross-protection assays showed that a substantial portion of the GVT effect was mediated by alloreactive cells not specific for the immunizing tumor. In conclusion, immunization of allogeneic BMT donors with a recipient-derived whole tumor cell vaccine substantially increases GVT activity but also exacerbates graft-versus-host disease.

Interleukin-2: hope in cases of cisplatin-resistant tumours.

To establish whether or not local low-dose recombinant interleukin-2 (rIL-2) therapy might result in therapeutic benefit for ovarian cancer patients treated with cisplatin, the antitumour effects of rIL-2 and of combined treatment with cisplatin and rIL-2 in a mouse ovarian tumour (MOT) model were studied. In addition, some possible mechanistic aspects underlying the observed antitumour responses were analysed. MOT cells were injected i.p. into syngeneic, immunocompetent, female C3HeB mice. Tumour-bearing mice received i.p. treatment with cisplatin, rIL-2 or both. The MOT tumour appeared to be hardly responsive to treatment with cisplatin only or rIL-2 only. In contrast, combined local treatment with low doses of cisplatin (1 and 5 mg/kg body weight) and rIL-2 (60000 U/day) resulted in an effective antitumour response in MOT-bearing mice. Complete rejection of the i.p. (local) tumour occurred in up to 60% of the cases. In vitro studies showed that cisplatin and rIL-2 do not have cumulative direct toxic effects on MOT cells. Mice cured after combined treatment with cisplatin and rIL-2 were not able to reject a rechallenge with tumour cells, indicating that these mice had not developed immunity to the tumour. Analysis of tumour-associated leucocytes, however, showed that combined treatment with cisplatin and rIL-2 did result in enhanced non-specific cytolytic activity of peritoneal leucocytes. We have thus demonstrated that, in the MOT model, combined local treatment with low doses of cisplatin and of rIL-2 is far more effective than therapy with cisplatin alone. Non-specific cytotoxicity of leucocytes appears to be involved in antitumour responses induced by combined treatment with cisplatin and rIL-2. These results suggest that, in human ovarian carcinoma, much better results may be obtained with the combined treatment of cisplatin and low (non-toxic) doses of rIL-2 than with cisplatin only. This may also apply to cisplatin-resistant ovarian carcinoma.

Analgesics in mice used in cancer research: reduction of discomfort?

During the last decades, an increase is apparent in the use of analgesics for laboratory animals in situations where this was previously considered unnecessary. Mice with advanced tumours often show clear signs of discomfort which may be a result of chronic pain or a result of general ill-being. The syngeneic murine tumour model most frequently used in our experiments was used to investigate whether this discomfort can be reduced with an analgesic. Twenty DBA/2 mice bearing SL2 lymphoma were given 0.5 mg/kg buprenorphine (Temgesic) in food gel twice daily, 20 tumour-bearing mice were given control food gel at the same times. Indicators of well-being were monitored daily. These included behavioural parameters such as exploration, grooming, and posture; food and water consumption and fur quality. All mice showed a clear increase of discomfort with time: explorative behaviours and grooming decreased, while sitting in hunched posture increased. Food and water consumption and fur quality also decreased. Major significant differences between the buprenorphine treated group and the control group were not apparent. In conclusion, we could not document a positive effect or buprenorphine on discomfort in mice as evaluated by our scoring system. It remains possible that pain itself was not the primary cause of the discomfort in mice bearing these tumours, or that the analgesic effect of buprenorphine was insufficient under these circumstances.

Local low-dose interleukin-2 induces systemic immunity when combined with radiotherapy of cancer. A pre-clinical study.

Tumor recurrence and outgrowth of metastases limit the therapeutical effect of radiotherapy. We have tested whether these problems can be overcome by supplementing radiotherapy with locoregional interleukin-2 (IL-2) treatment. The SL2 lymphoma and the M8013 mammary carcinoma were used. Mice bearing a 10-day-old s.c. tumor were locally irradiated and were treated daily with IL-2 peritumorally for 5 or 10 days. Low-dose IL-2 therapy improved local response (LR) and increased disease-free survival (DFS) in both tumor models following either single-dose irradiation or fractionated irradiation. For example, 93% of SL2-bearing mice treated with single-dose irradiation and 10 days of IL-2 experienced long-term DFS, compared with 17% for irradiation alone (p < 0.0001). Additionally, treatment of one tumor with irradiation +IL-2 led to anti-tumor effects in a second, untreated tumor in 80% of SL2-bearing mice. LR was increased to 100% and DFS to 70% when the second, non-irradiated tumor was also treated with peritumoral IL-2. We conclude that supplementing local radiotherapy with low doses of IL-2 results in increased local tumor control and regression of distant, non-irradiated tumors. This type of radioimmunotherapy is a promising new approach for the clinic.

Priming of the antitumor response promotes efficacy of interleukin-2 therapy.

We have studied the effect of active specific immunization (ASI) on the antitumor response induced by locoregional, low-dose interleukin-2 (IL-2) therapy. On day 0, mice were injected i.p. with viable, syngeneic tumor cells and with irradiated tumor cells (ASI). Low-dose IL-2 treatment was given i.p. for 5 consecutive days. ASI led to extended survival in two out of seven models tested. In these two models, enhanced efficacy was observed when both ASI and IL-2 were administered. In the five models in which ASI had no therapeutic value, ASI + IL-2 treatment was no more effective than IL-2 therapy. In the SL2 lymphoma model, use of ASI prior to IL-2 therapy given as early as days 1-5 led to at least 60% cure, whereas IL-2 therapy without ASI was only effective when administered after day 9. In the P815 mastocytoma model, however, ASI, IL-2, and the combination caused negative (suppressive) effects when administered on days 6-10. IL-2 administered on days 6-10 was therapeutically effective in this model when mice were treated with cyclophosphamide on day 6. In both the SL2 and the P815 tumor models, cured mice were specifically immune. The positive and negative effects observed were not due to the increased number of cells injected (non-specific inflammation) nor to possible antigenic alteration of the ASI cells by irradiation, as ASI with fragmented tumor cells was also effective in inducing synergy. Investigations into the underlying mechanism indicated that CD4+ cells play an important role. In total, the results indicate that ASI may be a good supplement to locoregional IL-2 treatment if care is taken to alleviate immunosuppressive activities.

Anti-tumor effects of local irradiation in combination with peritumoral administration of low doses of recombinant interleukin-2 (rIL-2).

The aim of this work was to improve radiotherapy results by immune stimulation. We tested the effects of a combination of radio- and immunotherapy, i.e., local low dose recombinant interleukin-2 (rIL-2) treatment, in two murine tumor models. Syngeneic tumors (SL2 lymphoma or M8013 mammary carcinoma) were induced subcutaneously on one or both flanks of mice. Irradiation was given either as a single dose (20 Gy) or fractionated (25 Gy) in 2 weeks. One or two cycles of rIL-2 were given concurrent with or subsequent to radiotherapy. One cycle of rIL-2 consisted of peritumoral injections administered on 5 consecutive days. Treatment effects were measured in terms of local tumor response and disease-free survival (DFS). The combined treatment modality was significantly better than treatment with either irradiation alone or rIL-2 alone. When tumors were inoculated on both flanks of the mice, combined radioimmunotherapy of one of the tumors also resulted in regression of the contralateral untreated tumor, indicating that a systemic anti-tumor immune reaction was induced. Additional rIL-2 injections did not enhance radiation toxicity. In conclusion supplementing irradiation with locally administered low doses of rIL-2 results in better local anti-tumor responses and DFS rates than either treatment alone without enhanced treatment toxicity. Furthermore, the local treatment induces a systemic anti-tumor reaction, influencing the growth patterns of a second, untreated tumor.

Three histopathological types of retinoblastoma and their relation to heredity and age of enucleation.

The histopathology of 61 eyes was studied with special attention to the morphology of the retina adjacent to the main tumour. Three retinal types were distinguished. Retina type 1 (RT-1, 28 specimens) contained a single tumour that was sharply demarcated from surrounding normal retina. In retina type 2 (RT-2, 29 specimens) large parts of the retina were affected and the main tumour mass gradually blended with the adjacent pathological retina. Retina type 3 (RT-3, four specimens) was characterised by a retina almost entirely affected by diffuse tumour growth. RT-1 correlated significantly with early enucleation (0-3 years) both in hereditary and non-hereditary cases. RT-2 was seen in eyes enucleated later (2-5 years). The progressing tumour may release growth factors in the intraocular space that stimulate the cells of the adjacent retina and lead to multiple new primary tumours in the adjacent retinal area. RT-3 was only present in non-hereditary cases with late enucleation (at 2-5 years). Hereditary retinoblastoma cases are usually detected early. Therefore in hereditary cases RT-1 is significantly more common than RT-2. In 25 eyes of the 44 patients with unilateral sporadic retinoblastoma, multifocal tumours of the retina were observed. Such cases should not mistakenly be classified as hereditary cases on the basis of the histological pattern of multifocality of the tumour process.

The success of locoregional, low-dose recombinant interleukin-2 therapy in tumor-bearing mice is dependent on the time of rIL-2 administration.

The extremely different administration schedules that are used in testing recombinant interleukin-2 (rIL-2) therapies for cancer may account for the extreme variation in efficacy reported in various studies in animal models. A major point may be the variation of the time interval between tumor transplantation and rIL-2 therapy. We hypothesized that administration of rIL-2 before the immune system has mounted a specific cellular reaction against the tumor (associated antigens) might result in lesser efficacies than later rIL-2 administration. This hypothesis was tested in DBA/2 mice bearing a syngeneic SL2 lymphoma. When 7000 IV/day rIL-2 was administered to tumor-bearing mice for 5 consecutive days starting on day 1, 3, 4, 5, or 6 after tumor inoculation, the survival curve of the mice did not significantly differ from that of diluent-treated mice. In contrast, a significant difference was observed when treatment was begun on day 7, 8, 9, 10, or 12 (p < or = 0.004). rIL-2 therapies begun on day 9 or 10 were most effective, curing up to 80% of mice treated, despite there being an enormous burden of disseminated tumor present at that time (1-4% of the total body weight). When rIL-2 was administered for fewer than 5 consecutive days, beginning on day 10, the efficacy of the therapy dropped radically (p < or = 0.055). Involvement of a specific anti-tumor reaction was also tested. All mice that were cured of the tumor as a result of rIL-2 therapy proved to be specifically immune to the SL2 tumor. Furthermore, day 10-14 administration of rIL-2 was completely ineffective in CD4(+)-cell depleted mice (p = 0.0116 vs. rIL-2 therapy in non-depleted mice). Together, this implies that this form of rIL-2 therapy is mediated by tumor-specific T-cells. As a whole, these results indicate that T-cell mediated rIL-2 therapy of cancer in animal models is sensitive to the time when the rIL-2 is administered and to the length of time for which the rIL-2 is given. This should be taken into account when planning new therapy protocols and when analyzing published data.

Ocular squamous cell carcinoma in Simmental cattle in Zimbabwe.

In Zimbabwe, ocular squamous cell carcinoma (OSCC) was frequently observed in 5 breeding herds of Simmental cattle, a Bos taurus breed originating from Switzerland. In these herds, initial signs of OSCC were already noticeable in cattle about 3 years old. Gradually, OSCC prevalence increased, and 36 to 53% of cattle over 7 years old had 1 or more tumors. More tumors developed in Simmental cattle with periorbital white skin than in cattle with periorbital pigmented skin. Other breeds of cattle (eg, Friesian) also are partly white-faced and live in Zimbabwe in a comparable environment; yet, OSCC prevalence was lower in those breeds.