Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis

The antitumor effect and mechanisms activated by murine IL-12 and IL-18, cytokines that induce IFN-gamma production, were studied using engineered SCK murine mammary carcinoma cells. In syngeneic A/J mice, SCK cells expressing mIL-12 or mIL-18 were less tumorigenic and formed tumors more slowly than control cells. Neither SCK.12 nor SCK.18 cells protected significantly against tumorigenesis by distant SCK cells. However, inoculation of the two cell types together synergistically protected 70% of mice from concurrently injected distant SCK cells and 30% of mice from SCK cells established 3 d earlier. Antibody neutralization studies revealed that the antitumor effects of secreted mIL-12 and mIL-18 required IFN-gamma. Interestingly, half the survivors of SCK.12 and/or SCK.18 cells developed protective immunity suggesting that anti-SCK immunity is unlikely to be responsible for protection. Instead, angiogenesis inhibition, assayed by Matrigel implants, appeared to be a property of both SCK.12 and SCK.18 cells and the two cell types together produced significantly greater systemic inhibition of angiogenesis. This suggests that inhibition of tumor angiogenesis is an important part of the systemic antitumor effect produced by mIL-12 and mIL-18.     

Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces local anergy to allogeneic and syngeneic tumors

After the cloning of murine cytokine synthesis inhibitory factor, it was recognized that a homologous open reading frame was encoded within the Epstein-Barr virus (human herpes virus 4). This viral protein has now been termed viral interleukin 10 (vIL-10) to reflect its protein sequence homology to "cellular" IL-10 (cIL-10, either murine or human IL-10). It is now widely accepted that vIL-10 shares many functions with cIL-10, principally, the ability to enhance survival of newly infected B cells and to diminish the production of IFN-gamma and IL-2 during ongoing immune reactions. The immunomodulatory effect of locally secreted vIL-10 and murine IL-10 (mIL-10) was examined in tumor models using CL8-1 (a BL6 melanoma cell line transfected with the H-2Kb class I gene) in syngeneic animals. Although parental BL6 tumor cells grow in immunocompetent syngeneic hosts, CL8-1 are rejected. To achieve local secretion of vIL-10, we generated vIL-10 retroviral vectors. While nontransduced CL8-1 cells (1 x 10(4)) failed to grow when injected intradermally in C57BL/6 mice, CL8-1 cells (1 x 10(4)) transduced with vIL-10 formed palpable tumors and eventually killed 80% of injected animals. Suppression of tumor rejection was also noted when CL8-1 tumors with or without vIL-10 transfection were admixed with syngeneic vIL-10-transfected fibroblasts and inoculated. Since the in vitro proliferation of the tumor was not altered after transduction with the vIL-10 gene and injection of vIL-10-transduced CL8-1 does not affect the rejection of nontransduced CL8-1 inoculated at a distant site, local vIL-10 secretion appears to suppress the process of immune rejection of the target cells in a dose-dependent manner. Similar results were observed for the H-2b MCA105 sarcoma tumor model in allogeneic BALB/c mice (H-2d). Although all animals that received nontransfected MCA105 rapidly rejected these tumors, MCA105 sarcomas transfected with vIL-10 remained palpable for up to 37 d. The local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by anti-human IL-10 monoclonal antibody or could be reversed by the systemic administration of IL-2 or IL-12. In marked contrast, mIL-10 transfection of CL8-1 significantly suppressed tumor growth and frequently led to the rejection of tumor. Similar results were obtained for the murine tumor cell lines MCA102.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunotherapy for medullary thyroid carcinoma by a replication-defective adenovirus transducing murine interleukin-2

We have evaluated the feasibility of gene transduction using replication-defective adenovirus vector as a novel therapy for medullary thyroid carcinoma (MTC), a thyroid C cell neoplasm. Replication-defective adenoviruses were constructed to express murine interleukin-2 (mIL-2) gene and Escherichia coli beta-galactosidase (beta-gal; lacZ) gene under the control of the human cytomegalovirus (CMV) promoter (AdCMVmIL2, AdCMVbeta-gal) by homologous recombination. The efficiency of transduction was evaluated using AdCMVbeta-gal at different conditions. The gene transduction efficiency was dependent on multiplicity of infection, duration of exposure to the virus, and viral concentration. The expression of functional mIL-2 in transduced tumor cells was verified both in vitro and in vivo. Two cell lines (rat MTC and mMTC) secreted large amounts of functional mIL-2 after transduction, as tested in cytotoxic T lymphocyte (CTL) L-2 cells. When AdCMVmIL2-infected mMTC cells were injected s.c. into their host animals, tumors developed in 2 of 10 animals, in contrast to 9 of 10 animals injected with AdCMVbeta-gal-infected mMTC cells and all 10 animals injected with parental mMTC cells. Moreover protected animals developed a long lasting immunity against mMTC tumor cells and their splenocytes, showing cytotoxicity to parental tumor cells, and active natural killer (NK) cell activity. BALB/c-SCID (severe combined immune deficiency) mice were also used to evaluate the function of NK cells in antitumor activities. No tumor developed in SCID mice injected with AdCMVmIL2-infected cells, whereas all animals injected with either AdCMVbeta-gal-infected or parental mMTC cells developed tumors. Our data indicate that IL-2 production by MTC cells leads to rejection in syngeneic animals and suggest that both cytotoxic T cells and NK cells may play an important role. In addition, transduction of adenoviral vectors into tumor cells produces some nonspecific antitumor effects.     

Canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity

The antitumoral activity of recombinant canarypox virus vectors (ALVAC) expressing murine interleukin 12 (IL-12) was evaluated in the syngeneic, nonimmunogenic murine mammary adenocarcinoma model (TS/A). Seven-day preestablished subcutaneous tumors (5- to 6-mm mean diameters) were injected on days 7, 10, 14, 17, 21, and 24 with the vector ALVAC-IL12 at 2.5 x 10(5) TCID50 (50% tissue culture infective dose). Total tumor regression occurred in 40 to 50% of the treated mice. Furthermore, 100% of the cured mice were protected against a contralateral subsequent challenge with the TS/A parental cells on day 28. The ALVAC-IL12 treatment is not effective in nude mice, suggesting the critical role of T cells. CD4 and CD8 T cells infiltrated the tumors treated with ALVAC-IL12 in the BALB/c model. Furthermore, in vivo depletion of CD4+ T cells totally abrogated the induction of the long-term antitumoral immune response by ALVAC-IL12. Interestingly, some tumor growth inhibition was also observed with ALVAC-betaGal treatment and a vaccinal effect was found in 33% of the treated animals, suggesting an adjuvant effect of the vector itself. Other ALVAC vectors expressing murine cytokines (IL-2, GM-CSF, IFN-gamma) were evaluated in the same model. Major antitumoral activity was observed with ALVAC-GM-CSF. However, a combination of ALVAC-GM-CSF and ALVAC-IL12 had no synergistic effect. These results suggest that in vivo gene transfer with canarypox virus expressing IL-12 may provide an effective and safe strategy for the treatment of human cancers.     

Dual role of IL-12 in early and late stages of murine collagen type II arthritis

IL-12 can promote Th1 responses, and early administration of IL-12 during immunization was shown to enhance expression of autoimmune collagen-induced arthritis (CIA). We now studied the impact of IL-12 at the stage of disease expression and during established CIA in DBA-1 mice. Accelerated onset and enhanced severity were provoked when i.p. injections of 100 ng of murine IL-12 (mIL-12) were given around the time of arthritis onset. Moreover, the onset of CIA could be ameliorated with anti-mIL-12 Abs, indicating that IL-12 is a pivotal mediator in the expression of CIA. In addition, the effect of anti-mIL-12 treatment was analyzed in established CIA. Continued treatment did not suppress established arthritis. Instead, these mice showed an impressive exacerbation of arthritis shortly after cessation of anti-mIL-12 treatment, indicative of impairment of endogenous control. Exaggerated disease was characterized by massive granulocyte influx and enhanced expression of IL-1 beta and TNF-alpha mRNA in the synovial tissue. Subsequently, we treated established collagen arthritis with recombinant mIL-12 for 7 days. Profound suppression of the arthritis score was noted, including reduced influx of cells and diminished cartilage damage. Tenfold enhanced levels of IL-10 were detected in sera of mIL-12-treated mice, and up-regulated mRNA levels of IL-10, IFN-gamma, and IL-12 were measured in synovial tissue. Finally, the anti-inflammatory effect of IL-12 on CIA could be reversed by coadministration of anti-IL-10 Abs. This study indicates that IL-12 has a stimulatory role in early arthritis expression, whereas it has a suppressive role in the established phase of collagen arthritis.     

The murine homolog of the interleukin-8 receptor CXCR-2 is essential for the occurrence of neutrophilic inflammation in the air pouch model of acute urate crystal-induced gouty synovitis

OBJECTIVE: Acute neutrophil-dependent inflammation is central to acute gout. Urate crystals induce different classes of neutrophil chemotaxins, including certain chemokines (e.g., interleukin-8 [IL-8], growth-related oncogene alpha [GROalpha]) that share CXCR-2 as a receptor. This study was undertaken to assess the role of CXCR-2 ligands in a model of acute gout. METHODS: Urate crystals were injected into subcutaneous air pouches in mice that expressed or lacked the murine CXCR-2 homolog (mIL-8RH), and the development of neutrophilic inflammation was assessed. RESULTS: In normal mice, urate crystals induced a 10-fold increase (P < 0.01) in pouch fluid leukocytes (principally neutrophils) at 4 hours. Leukocytes adhered to the pouch lining, where crystals, the mIL-8RH ligand KC/GROalpha, and cells bearing mIL-8RH were abundant. In mIL-8RH(-/-) mice, urate crystals induced a proteinaceous leukocyte-poor exudate at 4 hours, despite crystal-induced activation of resident cells (documented by KC/GROalpha expression). CONCLUSION: Chemokines that bind the IL-8 receptor CXCR-2 are essential for the development of acute neutrophilic inflammation in response to urate crystals in the subcutaneous air pouch model.     

IL-12 gene therapy protects mice in lethal Klebsiella pneumonia

IL-12 is a proinflammatory cytokine that has recently been shown to have beneficial effects in the setting of acquired host immunity. To determine the role of IL-12 in innate immunity against Gram-negative bacterial organisms, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally (i.t.), resulting in the time-dependent expression of IL-12 mRNA (p35 and p40) and protein within the lung. Passive immunization of animals with anti-IL-12 serum i.p. at the time of K. pneumoniae inoculation resulted in a 12-fold increase in K. pneumoniae CFU in lung homogenates at 48 h, as compared with animals receiving control serum. In addition, treatment of Klebsiella-infected mice with anti-IL-12 Abs significantly decreased both short and long term survival. To assess the effect of compartmentalized IL-12 overexpression on outcome in Klebsiella pneumonia, animals were treated i.t. with 5 x 10(8) PFU of a nonreplicating adenoviral vector containing a human cytomegalovirus promoter and cDNAs coding for the p35 and p40 subunits of IL-12 inserted into the E1 and E3 domains (Ad5mIL-12), respectively. In vivo transfection with Ad5mIL-12 resulted in 45% long term survival in Klebsiella pneumonia, whereas no animals with Klebsiella pneumonia receiving control adenovirus survived. Moreover, treatment with anti-IFN-gamma Abs or soluble TNF receptor:Ig construct partially and completely attenuated survival benefits observed in animals receiving Ad5mIL-12, respectively. In conclusion, endogenous IL-12 is a critical component of antibacterial host defense, and the compartmentalized overexpression of IL-12 using recombinant adenoviral gene therapy represents a safe and effective approach to deliver IL-12 to the lung in the setting of murine Klebsiella pneumonia.     

Isolated single-lung perfusion with TNF-alpha in a rat sarcoma lung metastases model

We conducted a trial of isolated lung perfusion using tumor necrosis factor (TNF) in an experimental sarcoma lung metastasis model. In an in vitro experiment, methylcholanthrene-induced sarcoma cells were incubated for 48 hours with 42 micrograms/mL of either human or murine TNF. Controls were incubated with Hank's balanced salt solution. In an in vivo experiment, 23 F344 rats were injected with 10(7) methylcholanthrene-induced sarcoma cells. On day 7, 4 animals were perfused with 210 micrograms of murine TNF, 5 animals were perfused with 420 micrograms of murine TNF, 10 animals underwent isolated lung perfusion with 420 micrograms of human TNF, and 4 animals were injected systemically with 420 micrograms of human TNF. Animals were sacrificed on day 14 and the lung nodules counted. The cells incubated with murine TNF exhibited a 21% decrease in growth (p = 0.07); cells incubated with human TNF showed a 37% decrease in growth (p < 0.05). Animals perfused with 210 micrograms/mL of murine TNF and animals treated by systemically administered human TNF showed no tumor response. Animals perfused with 420 micrograms/mL of murine TNF had 7.8 +/- 14.2 nodules on the left lung and 58.5 +/- 66.0 nodules on the right lung (p = 0.07). Animals perfused with 420 micrograms/mL of human TNF had 21.7 +/- 18.3 nodules on the left lung and 91.7 +/- 66.2 nodules on the right lung (p < 0.01). On the basis of these findings, we conclude that isolated lung perfusion with TNF can be done safely in the rat and is effective in decreasing the growth of sarcoma lung metastases.     

Potentiation of E7 antisense RNA-induced antitumor immunity by co-delivery of IL-12 gene in HPV16 DNA-positive mouse tumor

Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied, and in some cases, therapeutic effects have been demonstrated. We have previously shown that down-regulation of HPV16 E6 and E7 gene expression inhibited HPV DNA-positive C3 mouse tumor growth. Although not all of the tumor cells were transfected by pU6E7AS plasmid, complete tumor regression was achieved if the tumor size was small at the start of therapy in a syngeneic host. This suggests that some other antitumor mechanisms may be involved in addition to the direct down-regulation of HPV16 E7 oncogene expression by the antisense effect of E7AS. In the current study, we demonstrated that E7AS induces tumor cell apoptosis. More importantly, a strong antitumor immune response was elicited in the pU6E7AS-treated and tumor-regressed mice. There was no tumor growth after rechallenging the tumor-regressed mice with 1 million C3 cells. This E7AS-induced antitumor immune response was augmented by co-delivery of mIL-12 cytokine gene. The combination therapy strategy resulted in complete regression of 26 of 28 (93%) tumors. Only 12 of 31 (38%) tumors from the group treated with pU6E7AS alone and 14 of 28 (50%) tumors from the group treated with pCMVmIL-12 alone had completely regressed. Complete regression was also demonstrated in tumors located 1 cm from the treated tumors, which indicates that a systemic antitumor effect was induced by E7AS and mIL-12. Immunohistochemistry demonstrated that a significant amount of CD4+ and CD8+ cells infiltrated into tumors treated with pU6E7AS, pCMVmIL-12 and pU6E7AS+pCMVmIL-12. These data indicate that host immunity is an important factor for antisense-based gene therapy approach which can be further enhanced by combination with cytokine gene therapy.     

Delayed progression of murine AIDS in C57BL/6 mice pre-immunized with a highly antigenic 10-mer peptide encoded by the murine AIDS defective virus gag p12 gene

C57BL/6 (B6) mice were immunized with a highly antigenic 10-mer peptide (P12-10), which is encoded by the murine AIDS (MAIDS) defective virus gag p12 gene, emulsified in incomplete Freund's adjuvant (ICFA). One week later, the mice were inoculated with the MAIDS virus to see if the immunization affects progression of MAIDS. It was demonstrated that the immunization significantly delayed progression of MAIDS, although it failed to induce appreciable cytotoxic T lymphocyte (CTL) responses against the P12-10 antigen. In contrast, immunization of B6 mice with the P12-10 coupled with liposome induced substantial CTL responses but failed to protect the mice against MAIDS development. This segregation between CTL activity and in vivo protection efficacy might be worth considering when we exploit vaccines for augmenting cellular immunity mediated by CD8+ T cells.     

Cancer gene therapy using tumor cells infected with recombinant vaccinia virus expressing GM-CSF

The efficacy of a recombinant vaccinia virus (rvv-mGM-CSF) expressing murine granulocyte-macrophage colony stimulating factor (GM-CSF) for use in cancer gene therapy was evaluated. C57BL/6 mice with established B16-F10 melanoma were treated by s.c. injection of irradiated B16 cells infected with two different recombinant vaccinia virus (rvv) constructs. Mice treated with rvv-mGM-CSF vaccine survived longer (p < 0.05), were free of palpable tumors (> 4 mm) longer (p < 0.02), and had smaller mean tumor volumes (p < 0.005) compared to those treated with irradiated B16 cells infected with a control rvv (rvv-lacZ) expressing Escherichia coli beta-galactosidase or irradiated uninfected B16 cells. The vaccine appeared to be B16 tumor cell specific, because there was no therapeutic effect when heterologous but syngeneic (H-2b) colon adenocarcinoma cells, MC-38 infected with rvv-mGM-CSF were used as vaccine. In this model, rvv expressing interleukin-2 (IL-2) was ineffective. In addition, experimental lung metastasis of B16 tumor cells was significantly inhibited by rvv-mGM-CSF vaccine compared to several control vaccines when the vaccine was applied either by i.p. route (p < 0.006) or by s.c. injection (p < 0.0008). B16 cells expressing mGM-CSF after infection with rvv-mGM-CSF or transduction with a retroviral vector, were equally effective (p > 0.14) as vaccines against lung metastasis. Inhibition of metastasis was also B16 tumor cell specific. These data suggest that this approach of cancer gene therapy has a potential for use in cancer patients.     

Antitumor responses induced by transgenic expression of CD40 ligand

Because CD40 ligand (CD40L) is a co-stimulator molecule for multiple components of the immune response, we wanted to determine whether transgenic expression of the molecule would increase immune responses against a weakly immunogenic murine tumor, neuro-2a. Tumor cells were transduced with a retroviral construct containing the CD40L gene and co-injected with variable numbers of non-CD40L transduced cells into syngeneic mice. Mice injected with cells that expressed CD40L had a significant reduction in average tumor size as compared to controls (p < 0.0001). In addition, survival of the neuro-2a/CD40L mice was 48 days versus 34 days for the neuro-2a/neo controls (p < 0.02). Expression of CD40L by less than 1.5% of neuro-2a cells was sufficient for significant antitumor effects (p < 0.001). These antitumor effects protected mice from subsequent challenge with parental neuro-2a cells. The protective effects of CD40L were associated with systemic immunomodulation. In vivo depletion of CD8+ cells abrogated the CD40L-mediated antitumor effects. Analysis of spleens from CD40L-protected animals showed increased numbers of CD4+ and CD8+ cells, the majority of which co-expressed the activation marker CD25. In addition, an increased number of antigen-presenting cells (APCs) expressed the co-stimulatory molecule CD86. These experiments illustrate that transducing even a small percentage of tumor cells with CD40 ligand can create a long-lasting systemic immune response capable of impeding growth of unmodified neuroblastoma cells.     

Enhancement of interleukin-4-mediated tumor regression in athymic mice by in situ retroviral gene transfer

Intratumoral grafting of genetically engineered cells that produce interleukin-4 (IL-4) has been shown to produce tumor regression as well as prolong survival of mice harboring intracerebral gliomas. We sought to determine whether retroviral-mediated gene delivery into tumor cells in situ resulted in enhanced tumor regression by IL-4. Two mouse fibroblast lines were obtained: they both secreted similar levels of IL-4 but one produced a retrovirus vector bearing the IL-4 gene (CRE-MFG-IL-4 cells), whereas the other did not (NIH3T3-IL-4 cells). In mixed transplantation assays in the subcutaneous flanks of athymic mice, CRE-MFG, IL-4 cells were more effective than NIH3T3-IL-4 cells in inhibiting the growth of rat C6 glioma cells (p < 0.005, ANOVA). Subcutaneous tumors injected with fibroblasts that produced a control retrovirus vector without producing IL-4 (CRE-MFG-LacZ cells) did not inhibit subcutaneous tumor growth. An intracranial assay was used to evaluate survival of athymic mice harboring intracranial gliomas. Three days after implanting rat C6 glioma cells into the right frontal lobes of athymic mice, NIH3T3-IL-4 cells (n = 10) or CRE-MFG-IL-4 cells (n = 10) were stereotactically inoculated into the tumor bed. The average survival of mice treated with CRE-MFG-IL-4 cells was 38 days (+/- 2.4, SE), whereas that of mice treated with NIH3T3-IL-4 cells was 31 days (+/- 0.8, SE) (p < 0.005, ANOVA; p < 0.001, log-rank analysis).(ABSTRACT TRUNCATED AT 250 WORDS)     

Keratinocyte growth factor (KGF/FGF-7) has a paracrine role in canine prostate: molecular cloning of mRNA encoding canine KGF

BACKGROUND: Recent studies have demonstrated the usefulness of gene-modified tumor cells for immunotherapy. Using the tumorigenic murine fibrosarcoma, MCA 106, we investigated the effects of localized interferon-gamma (IFNg) secretion on tumorigenicity and on long-term memory. METHODS: The murine IFNg (MuIFNg) gene was introduced into tumor cells. High and low IFNg-secreting clones were isolated. C57BL/6 mice were injected subcutaneously (s.c.) with either parental (P), high or low IFNg-secreting (H- or L-IFNg) cells, and tumor growth was assessed weekly. Spleens were harvested on different days postinjection (p.i.) to assess in vitro cytolytic activity. In parallel, tissues from injection sites were stained with macrophage-, CD4-, and CD8-detecting antibodies. Mice were injected s.c. with H-IFNg MCA106 tumor. After 150 days the animals were rechallenged s.c. with MCA106P in one leg and with irrelevant syngeneic tumor in the other. RESULTS: Both P- and L-IFNg cells had similar growth, whereas the H-IFNg cells never grew. Only splenocytes from the H-IFNg animals showed in vitro CTL activity persisting until day 30 p.i. Histological data revealed a macrophage and CD4+ infiltrate much earlier in the H-IFNg group compared with the P group. Only the irrelevant, syngeneic tumor grew in animals previously injected with H-IFNg cells, whereas both P and irrelevant syngeneic tumors grew in controls. CONCLUSIONS: Transduction of MCA106 cells with the MuIFNg gene diminished in vivo tumorigenicity in proportion to the amount of IFNg secreted. Immunization with H-IFNg cells elicited a host response characterized by macrophages and CD4+ cells. Long-term tumor-specific memory was seen after immunization with H-IFNg cells.     

Allium sativum (garlic) treatment for murine transitional cell carcinoma

BACKGROUND: Currently, immunotherapy with Bacillus Calmette-Guerin (BCG) is the most effective treatment for superficial bladder carcinoma, but treatment-related toxicity may limit its use in some patients. Alternative treatments are needed for patients who fail to respond to BCG immunotherapy. Allium sativum (AS), or garlic, is known to have a broad range of biologic activities, including immune stimulation and reported antitumor activity. For these reasons, the authors conducted a series of experiments designed to explore the possible therapeutic effects of AS in the MBT2 murine bladder carcinoma model. METHODS: C3H/HeN mice were randomized prior to initiation of each experimental protocol. Mice received 1 x 10(3) MBT2 cells in 0.1 mL RPMI-1640, administered subcutaneously into the right thigh, on Day 0 of the experiment. AS was injected at the site of tumor transplantation on Day 1 and at 2- to 7-day intervals up to Day 28. To evaluate the effects of oral AS in this model, treatment was initiated 30 days prior to tumor inoculation and continued for 30 days after tumor inoculation. Animals in all experiments were followed for tumor incidence, tumor growth, and survival. RESULTS: In the initial experiments, subcutaneous AS significantly reduced tumor volume compared with the saline control (P < 0.05). Unfortunately, treatment-related death was also observed, requiring reduction in the total dose of AS. Animals that received 5 weekly immunizations of AS (5 mg, 5 mg, 1 mg, 1 mg, and 1 mg; cumulative dose = 13 mg) had significantly reduced tumor incidence, tumor growth, and increased survival when compared with animals that received the saline control. No treatment-related deaths were observed with this treatment schedule. To determine whether systemic AS administration might be effective, orally administered AS was tested at doses of 5 mg, 50 mg, and 500 mg per 100 mL of drinking water. Mice that received 50 mg oral AS had significant reductions in tumor volume (P < 0.05) when compared with animals that received the saline control, and mice that received 500 mg oral AS had significant reductions in both tumor volume and mortality (P < 0.05). CONCLUSIONS: The significant antitumor efficacy of subcutaneous and oral AS warrants further investigation and suggests that AS may provide a new and effective form of therapy for transitional cell carcinoma of the bladder.     

Effect of homologous interleukin-1, interleukin-6 and tumor necrosis factor-alpha on the core body temperature of mice

Lipopolysaccharide (LPS) and homologous cytokines were tested for their effect on core temperature in mice using battery-operated telemetric devices placed in the peritoneal cavity. One microgram LPS injected intraperitoneally (i.p.) induced a biphasic effect on core body temperature (Tc), a rapid decrease in Tc with a peak around 30-45 min followed by a prolonged rise around 150-300 min. When a higher dose of LPS (5 microg) was used, the hypothermia was increased in magnitude and lasted much longer, and no fever was observed. Both the decrease and the increase in Tc caused by LPS were prevented by pretreating the mice with indomethacin, a cyclooxygenase inhibitor, but not by a nitric oxide synthase inhibitor. Mouse interleukin-1beta (mIL-1beta, 100 ng, i.p.) induced changes resembling those to LPS, a short-lived decrease in Tc, followed by a small increase. When 1 microg mIL-1beta was injected a profound hypothermia lasting more than 3 h was observed. Mouse IL-6 (1 microg) failed to alter core temperature after either intravenous (i.v.) or i.p. administration. Human IL-6 was also ineffective. Recombinant mouse tumor necrosis factor-alpha (mTNFalpha) also failed to alter the core temperature of mice when injected at a dose of 1 microg (i.p. or i.v.). However, a higher dose of mTNFalpha (5 microg i.p.) caused a short-lived decrease in Tc, followed by a small increase. Similar results were obtained with LPS and the cytokines in C57Bl/6J mice, except that mIL-1beta was ineffective in this strain. These results indicate that the endocrine, neurochemical and behavioral responses to IL-1, IL-6 and TNFalpha administration cannot be explained by changes in Tc, although they may contribute to them. They also suggest that IL-1beta may account for the fever observed following LPS, but that these cytokines are probably not the only factors involved in LPS-induced changes in Tc.     

Age incidence of meningococcal infection England and Wales, 1984-1991

Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells. Rats inoculated with 4 x 10(4) 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (beta-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3-5 x 10(6) thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10(6)) resulted in a lower frequency of tumor regression (5 of 13 rats). To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10(6) beta-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranaside staining revealed maximal staining of beta-galactosidase within the tumor 7-14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4- chloro-3-indolyl-beta-D-galactopyranaside positive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenovirus-mediated expression of melanoma antigen gp75 as immunotherapy for metastatic melanoma

Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57BI/6 mice challenged i.v. with 10(5) B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 +/- 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells +/- i.p. Ad.IL2 before B16 cell challenge and Ad.beta gal-treated mice had similar numbers of metastases as controls (P > 0.1). In marked contrast, preimmunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 +/- 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 +/- 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen.