Xenogeneic Tumor Cells Research Articles

A novel animal model for bone metastasis in human lung cancer

Metastases account for 90% of lung cancer mortalities, frequently target the skeleton and lead to rapid deterioration in quality of life. The molecular mechanism underlying bone metastases is largely unknown. Development of xenograft mouse models, such as the severe combined immunodeficient (SCID) CB-17 mouse and the non-obese diabetic (NOD)/SCID mouse, both of which lack functional B- and T-cells and are able to host allogeneic or xenogeneic tumor cells, has made great contributions in this area. However, residual natural killer (NK) cells in these models are able to significantly modify local tumor growth and metastasis. Treatment with anti-murine IL-2 receptor β chain Ab (TM-β1) antibody can abrogate NK cell activity in vivo; however, the antibody treatment may result in unexpected effects and the stability is hard to control. To overcome these shortcomings, we evaluated xenografts in NOD-scid IL2Rγ(null) immunodeficient mice that lacked mature T cells, B cells and functional NK cells. We compared the target tissue distribution of the human small cell lung cancer cell lines SBC-5 and SBC-3. Gross necropsy and whole skeletal X-ray film examination of the host mice were conducted 30 days post-tail vein injection. The SBC-5 cells colonized bone and formed lytic lesions. The cells also colonized liver, spleen and, less frequently, the pancreas, ovary and kidney. The SBC-3 cell xenografts formed easily visible tumor foci in the liver, pancreas, ovary/uterus and kidney, but not bone metastases. Our results showed that SBC-5 cells in NOD-scid IL2Rγ(null) immunodeficient mice provide a suitable xenograft model system for bone metastasis of human lung cancer. This novel animal model may therefore be used to study the molecular pathway of bone metastases and to evaluate targets for effective therapies.

Macrophage activation through phagocytosis of muramyl dipeptide encapsulated in gelatin microspheres.

Gelatin microspheres containing muramyl dipeptide (MDP) were prepared by crosslinking with glutaraldehyde. They were added to mouse peritoneal macrophages (PMs) to potentiate the tumour growth inhibitory activity. The PMs which had internalized the microspheres exhibited growth inhibitory activity to syngeneic, allogeneic, and xenogeneic tumour cells. A similar effect was observed for PMs incubated with free MDP, but the MDP encapsulated in the microspheres was more efficient in enhancing the PM activity than the free MDP. In addition, PMs were activated in much shorter periods upon incubation with the microsphere-encapsulated MDP. The duration of activity could be controlled for up to 7 days by changing the extent of crosslinking of microspheres. Dose-response experiments established that microsphere-encapsulated MDP is able to activate PMs to inhibit growth of tumour cells at concentrations approximately 2000 times lower than the free MDP present in media. The activity of PMs was also acquired on intraperitoneal injection of the microspheres, in contrast to PMs with the free MDP.

Levamisole enhances the natural cytotoxic cell activity of Japanese flounder (paralichthys olivaceus) head kidney leukocytes

Japanese flounder (Paralichthys olivaceus) head kidney (HK) leukocytes were incubated with 103 to 10‐3 ng levamisole/ml for 4, 24 or 48 h and then assayed for their natural cytotoxic activity against xenogeneic tumor cells. This activity was slightly increased after 24 h of incubation. In a second experiment, fish were fed 0, 75, 150 or 300 mg levamisole/kg diet for 10 consecutive days. The fish were then fed a commercial non‐supplemented diet and sampled 0,1,2,3,4 or 6 weeks post‐administration of levamisole. The cytotoxic activity was found to be increased with increasing levamisole dose and remained greatly enhanced until the end of the experiment. In conclusion, levamisole enhanced flounder natural cytotoxic cell activity both in vitro and in vivo and had a great lasting action when administered by feeding.

Adjuvant Effect of a Flt3 Ligand (FL) Gene-Transduced Xenogeneic Cell Line in a Murine Colon Cancer Model

Background. Flt3 Ligand (FL) has been shown to elicit antitumor responses induced by tumor antigen stimulation. Allogeneic and xenogeneic cell lines transduced with cytokine genes may be used to augment the antitumor efficacy of tumor antigens. Objective. The objective was to evaluate the augmentation of tumor lysate-induced immunity by a more clinically applicable FL gene-transduced xenogeneic cell line in combination with interleukin-2 (IL-2) in a CC-36 murine colon cancer model. Methods. Human 143B osteosarcoma tumor cells were transduced with full-length FL cDNA (143B-FL). Secretion of FL from 143B-FL was analyzed in vivo in normal BALB/c mice transplanted with 143B-FL, and expansion of dendritic cells (DC) was also analyzed in the same mice by flow cytometry. Eight-week-old, male BALB/c mice were used in a prophylactic vaccination protocol utilizing tumor lysate (CLy), 143B-FL, and soluble IL-2. Prophylactic group designations ( n = 10/group) were as follows: ten million 143B-FL cells (alone, with tumor lysate, or with tumor lysate and IL-2), IL-2 with tumor lysate, IL-2 alone, or a no treatment control. The tumor lysate (200 μg of protein) and IL-2 (100,000 IU) injections were administered intraperitoneally. Mice were challenged subcutaneously with 10 3 CC-36 tumor cells. Tumor protection and tumor burden (TB), as mean tumor diameter, were determined. Peripheral blood lymphocytes (PBLs) from the 143B-FL + IL-2 + tumor lysate vaccinated group were analyzed for cytolytic activity in 4-h chromium release assays. In addition, plasma cytokine concentrations of interleukin-12 (IL-12) and interferon gamma (IFN-γ) were assessed by ELISA. Student's t tests were used for all statistical comparisons. Results. In vivo expression of FL was observed 24 h following the inoculation of 143B-FL, and a four fold increase in DCs was observed in the peripheral blood of these mice. Mice immunized with a combination of 143B-FL, tumor lysate and IL-2 showed statistically significant protection against tumor development (10%) for 100 days after tumor challenge; incidences in other groups ranged from 40 to 100% ( P < 0.05). Moreover, this immunization protocol produced the lowest TB at 3- and 6-week time points (0, 1.6 mm) when compared to all other groups (TB between 7.2 and 15.9 mm) ( P < 0.05). In addition, PBLs from vaccinated mice showed increased cytolytic activity against CC-36 target cells. This corresponded to increased levels of IL-12 and IFN-g in the plasma of mice following vaccination. Conclusion. These data suggest that FL gene-transduced xenogeneic tumor cells may augment the immunity induced by tumor antigens and systemic IL-2 through the activation of dendritic cells and T-cell-mediated mechanisms.

Levamisole is a potent enhancer of gilthead seabream natural cytotoxic activity

Gilthead seabream ( Sparus aurata L.) head–kidney (HK) leucocytes were incubated with 10 3 to 10 −4 ng levamisole/ml for 4, 24 or 48 h and then assayed for their natural cytotoxic activity against xenogeneic tumor cells. This activity was slightly increased after 24 h of incubation. In a second experiment, fish specimens were fed 0, 75, 150 or 300 mg levamisole/kg diet for 10 consecutive days. The fish were then fed a commercial non-supplemented diet and sampled 0, 1, 2, 3, 4 and 6 weeks post-administration of levamisole. The cytotoxic activity was found increased along the experiment and remained greatly enhanced at the end. In conclusion, levamisole enhanced seabream natural cytotoxic cell activity both in vitro and in vivo and had a great and lasting action when administered by feeding.

Endogenous type I interferons as a defense against tumors

We have reviewed the experimental results which indicate that endogenous type I interferon (IFN) present either constitutively or possibly induced by the tumor plays an important role in limiting the development of transplantable tumors in mice. Thus, treatment with potent polyclonal neutralizing antibodies to IFN α/β markedly enhanced the subcutaneous growth, invasiveness and metastases of xenogeneic tumor cells (uninfected or infected with RNA or DNA viruses) in athymic nude mice; enhanced the intraperitoneal transplantability of six different syngeneic murine tumors in three strains of immunocompetent mice; and completely abrogated the resistance of allogeneic C57Bl/6 (H-2 b) or C3H (H-2 k) mice to the multiplication of Friend erythroleukemia cells (H-2 d) in the liver and spleen resulting in the death of most mice. The mechanisms by which mice respond to the injection of relatively few tumor cells appear to be multiple, to depend on the site of tumor growth, to occur early and prior to an immunologic response. Endogenous type I IFN appears to constitute an essential component of these defense mechanisms enabling the host to restrict tumor growth.

Use of xenogenized (modified) tumor cells for treatment in experimental tumor and in human neoplasia

The need to modify tumor cells in order to render them more "immunogenic" was based on the assumption that normal, nonmodified tumor cells are non- or weakly immunogenic and as such are unable to raise an efficient protective immune response. Various methods for "xenogenization" (modification of tumor cells) were suggested: induction of new foreign antigens, treatment with either chemicals or enzymes and use of mutagens. Xenogenized tumor cells by their coupling to proteins, and use of chemicals like DTIC (5-[3,3-dimethyl- 1-triazeno]-imidazole-4-carboxamide), TZC (8-carbamoyl-3-methyl-imidazo[5, 1-d]- 1,2,3,5-tetrazin-4 [3H]-one 8-carbamoyl-3-[2-chloroethyl] imidazole [5,1 -d]- 1,2,3,5-tetrazin-4[3H]-one) and antiemetic drugs, were tested in experimental models of murine leukemia. Non-tumorigenic clones, xenogenization with DNA hypomethylating agents, aryl-triazine derivatives and DTIC were evaluated for their induction of protective immune response in murine lymphoma. Murine plasmacytoma cells were used for immunization after treatment with glutaraldehyde. Viral modifications of tumor cells were evaluated for their ability to induce a protective tumor response in model systems of rat fibrosarcoma, liver metastatic rat tumor cells, lymphoid tumor cells and hamster tumor cells. In the case of human cancer, attempts were reported to use DNP-conjugated melanoma cells, mutagenic triazine compounds, an autologous colon tumor cell bacillus Calmette-Guerin (BCG) vaccine and genetically engineered vaccines for immunization. The general conclusion drawn from experimental tumor models and for human cancer is, that although modified tumor cells were found to be partially effective in experimental models, it is still necessary to provide more data in order to determine the effective use of xenogenized human tumor cells for immunotherapy.

Activation-Induced Programmed Cell Death of Nonspecific Cytotoxic Cells and Inhibition by Apoptosis Regulatory Factors

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian lymphokine-activated natural killer cells. The cytotoxic activities of NCC are enhanced by stress-activated serum factors (SASF) present in tilapia acute-phase serum. In the present study purified NCC and xenogeneic target HL-60 tumor cells and nuclei were distinguishable in mixtures determined by flow cytometry. NCC activated by target HL-60 cells undergo activation-induced programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V binding studies also demonstrated that NCC kill HL-60 cells by an apoptotic mechanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF. Pretreatment also produced more than a fourfold increase in NCC cytotoxicity (effector/target (E:T) ratio = 100:1). In the absence of SASF preincubation, the percentage of apoptotic NCC increased from 8 to 91% at E:T ratios of 1:0 and 1:1, respectively. Kinetic studies (E:T = 10:1) demonstrated that the percentage of NCC exhibiting HL-60-dependent AIPCD increased between 0.1 and 12 h and then decreased inversely with total cell necrosis over the next 60 h. Preincubation of NCC with SASF protected NCC from AIPCD for over 72 h. Crosslinkage of the NCCRP-1 receptor with monoclonal antibody (mab) 5C6 produced AIPCD between 1 and 100 μg/mL mab concentrations. Preincubation with SASF completely protected NCC from mab 5C6-dependent AIPCD. SASF-mediated protection of NCC from AIPCD was dependent upon divalent cations, as demonstrated by increases in DNA hypoploidy of 38, 67, and 88% following preincubation in the presence of 10, 100, and 1000 μM EDTA, respectively. SASF also protected NCC from glucocorticoid- (i.e., dexamethasone) induced apoptosis. Combined, these results demonstrated that NCC activity is down-regulated by AIPCD. Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Results are discussed in the context of the effects of acute stressors on innate immunity.

Enhancement of tumor killing using a combination of tumor immunization and HSV-tk suicide gene therapy.

Tumor cells genetically modified with the herpes simplex virus thymidine kinase (HSV-tk) gene in combination with ganciclovir (GCV) demonstrate a "bystander effect". Previous attempts to enhance the bystander tumor killing by combining cytokine genes with HSV-tk/GCV have met with varying results. The present study was designed to determine the effects of tumor immunization in combination with HSV-tk gene-modified tumor cells and GCV on tumor killing and to determine if the bystander tumor killing could be enhanced. Tumor-bearing mice immunized with syngeneic tumor (KBALB) prior to treatment with an i.p. injection of xenogeneic HSV-tk gene-modified tumor cells (PA-1STK) had prolonged animal survival (group 4, 56.4 days). In contrast, unimmunized tumor-bearing mice (group 2) or tumor-bearing mice immunized to the xenogeneic PA-1STK tumor cells (group 5) showed a mean survival of about 27 days after receiving an i.p. injection of PA-1STK cells and GCV. Control groups, which were either not immunized and did not receive HSV-tk cells (group 1) or immunized but treated only with GCV (group 3) showed short survival (16-18 days). Analysis of tumors for cytokine mRNA expression revealed increased TNF-alpha and IL-1alpha mRNA expression in group 4 mice. Furthermore, IL-2 mRNA expression was detectable on days 2 and 4 only in group 4 mice. Immunophenotypic analysis for tumor-infiltrating lymphocytes demonstrated an increase in macrophage (4%, p = 0.0001) and T cells (1.8%, p < 0.001) in group 4 mice with an enhanced T-cell response as compared with mice from groups 1, 2 and 3. Our results demonstrate that tumor immunization combined with HSV-tk/GCV treatment results in increased animal survival with enhanced immune response. Furthermore, the cytokine milieu observed in the present study can modulate the tumor micro-environment in vivo from one that is immunosuppressive to one that is immune-stimulatory.

Interleukin-2 and interleukin-4 display potent antitumour activity on rat medullary thyroid carcinoma cells.

Currently, surgical resection is the only treatment used for human medullary thyroid carcinoma (MTC). However, as metastases are commonly observed, we investigated the potential of adjuvant therapy with interleukin-2 (IL-2) and interleukin-4 (IL-4) in a rat model. The interleukins were delivered by means of xenogeneic tumour cells engineered to secrete IL-2 and IL-4. We found that when a mixture of MTC cells and IL-2 or IL-4 secreting cells were implanted in rats, the growth of the resulting tumours was inhibited as a function of the number of interleukin-secreting cells in the inoculum. Moreover, association of the two interleukins exerted a synergistic antitumour effect. These experimental results, showing thyroid C cell tumour rejection, are of major interest, as they show the potential therapeutic application for these two interleukins, which could be used, in particular, as postsurgical adjuvants.

The role of cytokines in mediating the bystander effect using HSV-TK xenogeneic cells.

We have previously demonstrated that genetically modified syngeneic murine tumor cells (KBALB) expressing the herpes simplex virus thymidine kinase gene (HSV-STK) can kill nearby unmodified tumor cells in the presence of ganciclovir (GCV). The killing was mediated by a 'bystander effect' as evidenced by the prolonged animal survival when syngeneic HSV-TK gene-modified tumor cells were inoculated into mice with an intraperitoneal tumor. In this study we investigated whether irradiated xenogeneic HSV-TK gene-modified tumor cells, a human colon carcinoma cell line (HCT) transfected with the HSV-TK gene, can mediate the 'bystander effect' when used in vitro and in vivo. In vitro experiments indicate that irradiated HSV-TK gene-modified xenogeneic cells (HCT) can mediate a bystander effect on the adjacent cells when the tumor population consisted of as few as 10% of the HSV-TK expressing HCT tumor cells. In vivo, animal survival experiments demonstrate that the xenogeneic gene-modified tumor cells could generate the 'bystander effect' in mice with intraperitoneal tumors as evidenced by prolonged animal survival. In addition, histologic examination of the tumors from experimental animals showed extensive tumor necrosis 3 days post HSV-TK/GCV treatment in comparison to control animals. To evaluate the cause of necrosis in vivo, we assayed for cytokines, which may be involved in mediating this process, by performing RT-PCR and immunohistochemistry on tumor RNA and tumor cells, respectively. Production of IL-1 alpha and IL-6 mRNA within the experimental tumors was observed by RT-PCR. However, mRNA expression for other cytokines including IFN-gamma, IL-2 and IL-4 was not present. Immunohistochemical analysis for IL-1 alpha protein showed reactivity within the infiltrating mononuclear cells indicating the release of this soluble factor. These results indicate that the bystander effect can be generated using irradiated xenogeneic cells both, in vitro and in vivo. Furthermore, this process is mediated by the release of cytokines such as IL-1 alpha, IL-6 which enhances the bystander effect in vivo by immunostimulation.

Analgesia induced by transplantation of encapsulated tumor cells secreting beta-endorphin.

The purpose of this study was to assess whether xenogeneic tumor cells immunologically isolated in polymer capsules could survive and continue to reduce pain when transplanted into the cerebrospinal fluid (CSF) of rats. The mouse tumor cell lines AtT-20 and gene-transfected Neuro2A, which secrete beta-endorphin, were enclosed in polymer capsules at a density of 5 x 10(6) cells/ml and transplanted into the spinal CSF space of the occipitoatlantal junction in male Sprague-Dawley rats. The analgesiometric tests (tail pinch, hot plate, and electrical stimulation) showed that the five rats with encapsulated AtT-20 or Neuro2A (eight rats) were significantly less sensitive to pain after transplantation than the eight control animals (analysis of variance; p < 0.05). The analgesia induced by encapsulated cells secreting beta-endorphin could be attenuated by the opiate antagonist naloxone, which suggested the involvement of opiate in mediating this response. Morphological study revealed that the cells in polymer capsules survived 1 month after transplantation in the CSF space. In vitro experiments with cultured capsules showed that both encapsulated AtT-20 and Neuro2A secrete peptide for 1 month. The results of this study suggest that immunologically isolated xenogeneic tumor cells can secrete opiate in the CSF space, and this method may be applied to the treatment of cancer pain.

Cell therapy with encapsulated xenogeneic tumor cells secreting β-endorphin for treatment of peripheral pain

The purpose of this study was to assess whether xenogeneic tumor cells secreting β-endorphin and immunologically isolated in polymer capsules could survive and continue to reduce pain when transplanted into the spinal cerebro-spinal fluid (CSF) space of rats. Also, a silicone container for polymer capsules was designed for the clinical application of this method of cell therapy. The mouse tumor cell lines, proopiomelanocortin gene transfected Neuro 2A which secrete β-endorphin, were enclosed in polymer capsules at a density of 5 × 10 6/mL, and transplanted into the spinal CSF space from the occipito-atlantal junction of male Sprague-Dawley rats. Three analgesiometric tests—the tail pinch test, the hot plate test, and electrical stimulation test — showed that the rats with encapsulated Neuro2A ( n = 6) were significantly less sensitive to pain after transplantation than control animals ( n = 8). The analgesia induced by the encapsulated cells secreting β-endorphin was attenuated by the opiate antagonist naloxone. Morphological study revealed that the encapsulated cells survived for 1 mo after transplantation into the CSF space. An in vitro experiment on cultured capsules (3 cm long) with a silicone container (Kaneka Medics Co) showed that the encapsulated Neuro2A (5 × 10 6 mL) could secrete peptides for 1 mo. The results of this study indicate that immunologically isolated xenogeneic tumor cells can secrete opiate in the CSF space, and that a silicone container may help the application of this method to the treatment of cancer pain.

The specificity of alternative complement pathway-mediated lysis of erythrocytes: a survey of complement and target cells from 25 species.

Sera from 20 species of mammals were tested for their ability to lyse erythrocytes from 18 species of mammals and birds by the alternative complement pathway. Erythrocytes were not lysed by homologous complement, with one minor exception, but all erythrocytes tested were lysed by at least one complement source, and all sera tested except that of the horse lysed at least one type of erythrocyte. Control experiments indicated that lysis was via the alternative complement pathway and that antibodies were not involved. Complement from the various species could be ranked from most active to least active, and erythrocytes could be ranked from most susceptible to least susceptible. There was an inverse correlation between complement activity and erythrocyte susceptibility. The ranking of the orders of placental mammals, from strongest to weakest complement, was carnivore > artiodactyl (ruminants and swine) > primate = armadillo > rodent > rabbit > horse. Opossum serum had activity that placed it in the centre of this range. Ferret complement, the most potent tested, lysed all erythrocytes tested except for homologous erythrocytes, with APCH50 titres as high as 4000. Although the overall reactivity pattern was clear, there were several striking exceptions. For example, the only complement source which lysed ferret erythrocytes was sera of the mouse. The amount of sialic acid present on erythrocytes of 14 mammals was determined, and was, in general, directly correlated with resistance to alternative complement pathway lysis, although there were prominent exceptions to this correlation, involving erythrocytes of the horse, burro and human. All 20 types of complement were also tested for their ability to lyse antibody-coated human tumour cells, under conditions in which both the classical and alternative complement pathways were functional. The data obtained suggest that alternative pathway activation is, in some cases, a major factor determining the effectiveness of a particular complement source in the lysis of xenogeneic tumour cells.

Cell-mediated immunity to chemically xenogenized tumors—VI. The effect of cell treatment with retroviral env antisense oligonucleotides

The occurence of aberrant, retrovirus-related proteins is a common finding in immunogenic clones of the triazene-xenogenized L5178Y lymphoma line (L5178Y/DTIC). In clone D-cells, newly expressed 80 kDa antigens related to xenotropic murine leukemia virus env gene products induce specific humoral and cell-mediated responses and possess biologic activity in vivo. To further clarify the relationship between immunogenic properties of clone D and retroviral gene expression, tumor cells were treated in vitro with antisense oligonucleotides complementary to xenotropic and/or polytropic env sequences of murine leukemia virus. The cells were then assayed for expression of antigens recognized by humoral and cell-mediated responses with specificity for clone D. The results demonstrated that inhibition of env mRNA translation adversely affected the expression of immunogenic determinants in the xenogenized tumor cells.

Induction of lymphokine-activated killer cells of equine origin: specificity for equine target cells

The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibit potent cytolytic activity against an equine tumor cell line, EqT8888. Cytolytic activity against the EqT8888 cells required 3 days of incubation with IL-2, was mediated primarily by T-cells, and was not restricted by major histocompatibility complex antigens. Though LAK activity could only be demonstrated using equine-derived target cells, xenogeneic targets could be lysed in a lectin-mediated cytotoxicity assay. The xenogeneic targets also failed to block LAK cell-killing of the EqT8888 cells in a cold-target competition assay. These results indicate that LAK cells in the horse appear to utilize a species-specific recognition mechanism during target cell lysis.

Immunomodulating effects of ozone on macrophage functions important for tumor surveillance and host defense.

Ozone (O3) is a toxic gaseous pollutant that has been implicated in laboratory studies as a potential lung carcinogen or cocarcinogen in mice. To begin to assess the role of altered macrophage (M phi) responses as a possible mechanism by which O3 may influence carcinogenesis, we examined the effects of repeated in vivo O3 exposure on pulmonary M phi functional and biochemical activities deemed important in tumor surveillance, and host defense in general. Rabbits were exposed by inhalation to 1 ppm O3 for 3 d (2 h/d) and the lungs were lavaged immediately (t0) and 24 h (t24) after exposure. Results demonstrate that O3 reduced M phi viability and increased the number of neutrophils collected immediately after exposure. Effects of O3 on M phi movement were as follows: random migration was depressed immediately after the final exposure and chemotactic migration increased after 24 h. M phi-mediated cytotoxicity toward xenogeneic tumor cells in vitro was significantly depressed, compared to control, immediately and 24 h after O3 exposure. Release of cytotoxic factors deemed important for mediating tumor cell destruction was also assessed. Spontaneous and stimulated production of tumor necrosis factor, as measured by cytotoxicity toward LM cells (a clone of L-929 mouse fibroblasts), was unaffected by exposure to O3. Zymosan-stimulated production of superoxide anion radical (.O2-) was depressed at t0 and increased at t24; however, no significant effects on H2O2 production by resting or zymosan-stimulated M phi were observed at either time interval. Inhaled toxicants such as O3, which can compromise M phi functions important in tumor surveillance, could potentially alter host susceptibility to pulmonary cancer. Results of this study have important implications for human health, and demonstrate the need for further studies examining the carcinogenic/cocarcinogenic potential of O3.

The improved effects of specific active immunotherapy on a rat fibrosarcoma by antitumor drugs.

We have tried to find out if the combination of a xenogenized tumor cell vaccine and antitumor drugs is able to induce a synergistic increase in the antitumor therapeutic effect. The degree of increase in the LTD50 (50% lethal tumor dose) is expressed numerically, as a quantitative index designed to compare degrees of transplantation resistance to tumor cell challenge. A LTD50 was achieved by an intradermal (i.d.) immunization with xenogenized tumor cells when challenged with tumor cells implanted intraperitoneally 2 weeks after the immunization: this LTD50 value was 527,000 times higher than that of the non-immunized group. When we combined this type of immunization with appropriate doses of bleomycin (BLM) or cyclophosphamide (CY), which are able to augment antitumor immunity, the LTD50 was 723,000-1,190,000 times higher than that of the non-immunized group. This increase in the LTD50 is definitely higher than that achieved by a single immunization with irradiated tumor cells (x 33,000) and combined with either BLM (x 93,000) or CY (x 140,000). We also studied the therapeutic effect of a tumor cell vaccine combined with antitumor drugs BLM or CY in tumor-bearing rats. We observed a synergistic effect caused by BLM or CY after i.d. immunization with xenogenized tumor cells: this showed a significant increase when compared with the therapeutic effects obtained by chemotherapy alone (P less than 0.01). Nevertheless, there was no evidence that the above antitumor effects is superior to the effect achieved by irradiated tumor cells.

IL-2 reverses the inhibition of cytotoxic T-cell responses induced by 5-(3,3′ dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) in vitro

One of the major limitations in the use of triazene compounds for inducing increased immunogenicity of tumor cells in vivo (i.e. chemical xenogenization) is the profound immunodepressive activity of these drugs. The present study analysed the inhibitory effects of DTIC on various T-dependent immune responses in mice in an attempt to determine the mechanism of action and appropriate treatments for reverting the immune damage produced by the agent. Results obtained show that treatment with DTIC in vivo produced: (a) inhibition of spleen cell proliferation; (b) reduced IL-2 production in response to allogeneic stimuli; (c) reduction of the generation of IL-2R + CD8 + cells in allogeneic MLC; (d) inhibition of allo-CTL generation. The addition of IL-2 to MLC on day 2 of the co-culture restored full allogeneic CTL responses. These data suggest that exogenous IL-2 could be used to counteract DTIC-induced depression of T-cell reactivity, which is presumably involved in hosts' responses against autochthonous xenogenized tumor cells.

Modulation of rat bronchoalveolar lavage cell function by the intratracheal delivery of interferon-gamma.

To determine if there is a rationale for compartmentalized immunostimulation, we have carried out a series of experiments to evaluate whether intratracheal delivery of interferon-gamma (rRatIFN-gamma) can activate rat bronchoalveolar lavage cells (BAL) for in vitro expression of tumoricidal activity against xenogeneic P815 tumor cells and enhanced in vitro microbicidal activity against the intracellular protozoan Toxoplasma gondii. Treatment of rat alveolar macrophages in vitro activates them for both of these activities as well as enhanced production of superoxide anion. We found that a single intratracheal dose of 5,000-10,000 units of rRatIFN-gamma activated rat BAL for both microbicidal and tumoricidal activity. To determine the duration of activation, microbicidal activity was determined 1, 2, 3, 5, and 7 days after a single intratracheal dose of 5,000 units of IFN. Enhanced microbicidal activity was maintained through day 3 but returned to control levels by day 5. Alveolar macrophages always accounted for the majority of cells in the lavage populations. However, intratracheal IFN caused an increase in the number of polymorphonuclear leukocytes and lymphocytes in the lavageable cells and, although these cells were always in the minority, they may have contributed to both the tumoricidal and microbicidal activity of the lavage cells. These studies demonstrate that local administration of an immunostimulant can activate pulmonary defense cells and may be a feasible route of drug delivery for prophylaxis against pulmonary infections.