[Medline record in process]
Inflammatory bowel disease (IBD) is characterized by altered immunoregulation and augmented intestinal synthesis of nitric oxide. The purpose of this study was to determine the effects of exogenous IL-4, introduced by a recombinant human type 5 adenovirus (Ad5) vector, on the tissue injury associated with an experimental model of colonic immune activation and inflammation. Colitis was induced in rats by the intrarectal administration of trinitrobenzene sulfonic acid (TNB) dissolved in 50% ethanol, and control rats received saline via the same route. 1 h later, all rats were randomized into two groups. The first group was injected intraperitoneally (ip) with 3.0 x 10(6) plaque forming units (PFUs) of Ad5 transfected with murine interleukin-4 (Ad5IL-4) and the second group was injected ip with the same amount of Ad5 expressing the Escherichia coli Lac Z gene (Ad5LacZ). One-half of the colitic and control rats were injected again with 3.0 x 10(6) PFUs of Ad5IL-4 or Ad5LacZ on day 3 of the 6-d study. When introduced once or twice via the peritoneal route into control rats, Ad5LacZ was localized to the serosal lining of the peritoneal cavity, the diaphragm and the liver on day 6. One or two injections of Ad5IL-4 into rats also produced measurable levels of circulating IL-4. TNB-colitis in both Ad5LacZ-treated groups was associated with pronounced elevations in serum IFN-gamma, and mucosal ulceration of the distal colon. Myeloperoxidase and inducible nitric oxide synthase II (NOS II) synthetic activity were also increased by 30- and fivefold, respectively, above control levels in the distal colon. However, two injections of Ad5IL-4 into colitic rats caused the overexpression of IL-4, and significantly inhibited tissue damage, serum and colon IFN-gamma levels and myeloperoxidase activity in the distal colon. In addition, NOS II gene expression and NOS II nitric oxide synthesis was significantly inhibited. No therapeutic effect was observed in rats injected once with Ad5IL-4. Thus, IL-4, introduced by Ad5, is therapeutic during acute inflammation in the rat colon. The therapeutic effect of IL-4 was associated with an inhibition of inducible nitric oxide expression and a reduction in nitric oxide synthesis.
PMID: 9389741, UI: 98052579
[Medline record in process]
Adenovirus (Ad) vectors are being intensively studied as vehicles for cancer gene therapy. We have been exploring the benefits of direct intratumoral injection of Ads expressing cytokines for immunotherapy. Our previous work demonstrated that therapy using a vector expressing interleukin-12 (AdmIL-12.1) produced regressions in approximately 80% of treated tumors supporting further preclinical investigations. Recent reports have shown that immunity to Ad can be a major limiting factor in Ad-mediated gene transfer. As most animal studies with Ad vectors have involved nonimmune hosts, it remains difficult to predict how effective these treatments will be in humans, where the majority of individuals have had previous exposure to Ad. To address this question, we compared the effectiveness of the AdmIL-12.1 cancer therapy in naive and Ad-immune mice. We found that both groups responded equally well to treatment and that the response to AdmIL-12.1 in both groups resulted in the generation of CTL reactive against tumor antigen indicating that antitumor immunity was achieved. Peak transgene expression in the tumor was only reduced by 2.4 fold in Ad-immune animals compared with nonimmune mice. It was also observed that in naive animals, the virus disseminated from the site of the tumor following injection and by 72 h substantial transgene expression was detected in peripheral organs, most notably the liver. Transgene expression in the liver of Ad-immune animals was reduced by greater than 1000-fold relative to that in naive mice. These results strongly support the clinical utility of Ad-based cancer gene therapy and suggest that Ad immunity may be advantageous in that it is not a complete block to gene transfer in the tumor and it greatly reduces virus dissemination.
PMID: 9415313, UI: 98077021
Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.
PMID: 9380730, UI: 98021464
J Clin Invest 1997 Aug 15;100(4):768-776
Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.
PMID: 9259574, UI: 97406007
Mol Med 1997 Aug;3(8):519-529
BACKGROUND: The pleiotropic cytokine interleukin-6 mediates its multiple effects at the cell level through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130). A soluble form of gp80 (sIL-6R or gp55) is found released from the surface of cells and appears to possess interleukin-6 (IL-6) agonist activity. Increases in circulating levels of sIL-6R have been reported in different pathological conditions but the precise role of this protein in vivo remains unknown. MATERIALS AND METHODS: The cDNA encoding the extracellular domain of the rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned into the E1 region of an adenovirus vector under the control of the hCMV promoter (Ad5.sIL-6R). RESULTS: Infection of different human or rodent cell lines with Ad5.sIL-6R leads to extended production of recombinant sIL-6R protein into the culture media. The kinetics of transgene expression depends both on the cell type and the species. sIL-6R produced in this manner is biologically active as it confers responsiveness of human hepatoma cells (HepG2) to rat IL-6 stimulation. Adenovirus vectors have been shown to be highly effective for transient delivery of cytokines in vivo. Antibodies against recombinant rat soluble IL-6R were generated and an ELISA developed that allowed us to quantify sIL-6R concentrations. The sIL-6R expressing adenovirus vector has been instilled intratracheally into rats and induced an increase in lung sIL-6R concentration from Day 1 up to Day 10. We demonstrate the potency of our system to deliver in vivo or in vitro soluble cytokine receptors in a prolonged but transient manner.
PMID: 9307980, UI: 97453240
Hum Gene Ther 1997 Jul 20;8(11):1355-1363
Evaluation of the potential role of dendritic cells (DCs) as adjuvants for tumor vaccination has focused primarily on techniques that load DCs with peptide tumor antigens. Our aim has been to optimize the induction of antitumor immunity by enhancing the ability of DCs to present tumor-associated antigens endogenously to the afferent lymphatic system in the appropriate major histocompatibility complex (MHC)-restricted context. We have used replication-defective adenovirus vectors (Ads) to transduce DCs with various genes, including tumor antigen genes. We found that 90% of murine bone marrow derived-DCs could be infected with an Ad vector expressing the beta-galactosidase gene and still retain their physiologic and phenotypic characteristics. Furthermore, we demonstrated that transgene expression was detectable in the spleen for at least 3 days following intravenous injection of Ad-transduced DCs. Using a polyoma middle T (PymT) transgenic murine mammary carcinoma model, we have shown that a single injection of 10(5)-4 x 10(6) DCs transduced with an Ad vector expressing PymT provided complete and specific protection against tumor cell challenge in 100% of vaccinated animals. Immunization against the PymT tumor by injection with the PymT expressing Ad vector alone resulted in varying degrees of effectiveness, was highly dependent upon the route of administration, and led to significant hepatic toxicity that was not seen in mice immunized with DC transduced with the Ad vector. Our results suggest that: (i) DCs can be very efficiently modified by ex vivo Ad transduction to express tumor-specific antigens, (ii) such modified DCs appear nontoxic and stimulate a potent antitumor response.
PMID: 9295130, UI: 97439574
J Leukoc Biol 1997 Jun;61(6):695-702
PMID: 9184564, UI: 97328006
Chest 1997 Jun;111(6 Suppl):128S-129S
PMID: 9184560, UI: 97328002
Hum Gene Ther 1997 Mar 20;8(5):545-553
Daily systemic administration of hematopoietic growth factors can be associated with dose-limiting systemic side effects. To overcome this, we have investigated hematopoietic cytokine gene transfer to the marrow cavity of dogs by direct intramarrow injection of adenoviral vectors. In marrow culture, replication-deficient (E1-deleted) adenoviral vectors were able to transduce marrow stromal cells, demonstrating 30-fold greater expression than from other marrow cell types. High-level (ng/ml) cytokine production from transduced stromal cells persisted for 14 days in culture. Because adenovectors could efficiently transduce marrow stromal cells in culture, we investigated if stromal cells could also be transduced in vivo following direct intramarrow vector injection. Adenovectors with genes for interleukin 6 (IL-6) and Lac Z (beta-galactosidase) were injected directly into the marrow cavity of dogs resulting in protein expression localized to within the treated marrow. To evaluate this approach further in dogs, we constructed a vector expressing biologically active canine granulocyte-macrophage colony stimulating factor (GM-CSF). 293 cells infected with ADGM-CSF demonstrated prevalent GM-CSF mRNA by Northern blot and 135 +/- 30 ng/ml of protein as measured by enzyme-linked immunosorbent assay (ELISA). In vitro bioactivity of protein expressed was confirmed by canine GM colony-forming assay (CFU-GM). In vivo high-level protein production was noted in supernatants of marrow aspirates 72 hr following direct intramarrow administration of ADGM-CSF (baseline mean +/- SEM, 27 +/- 22 ng/ml, 72-hr sample 921 +/- 461 ng/ml). A localized myeloid expansion of marrow and significant peripheral leukocytosis (neutrophilia) were noted in all ADGM-CSF-treated dogs. Peripheral blood changes lasted for up to 3 weeks in dogs following single intramarrow injection. Thus, adenoviral cytokine expression from the marrow of a single large bone (ilium) led to compartmentalized expression of growth factor and an increase of hematopoiesis sufficient to cause peripheral blood changes in a large animal model.
PMID: 9095406, UI: 97249520
Gene Ther 1997 Feb;4(2):140-149
Interleukin-10 (IL-10) is a potent anti-inflammatory/immune cytokine and has received growing attention for its therapeutic potential. To aid therapeutic studies of IL-10 in vivo, a replication-deficient adenoviral vector expressing mouse IL-10 was constructed and characterized. The transgene protein IL-10 was shown to markedly inhibit endotoxin-induced tumor necrosis factor alpha (TNF alpha) production by mouse and rat macrophages in vitro. Intramuscular injection of this vector in mice resulted in efficient expression of transgene mRNA in the muscle and active release of IL-10 protein into the bloodstream. To investigate the therapeutic potential of IL-10 using this vector, endotoxemia was induced by intraperitoneal injection of a sublethal dose of endotoxin. Expression of TNF alpha and IL-6 mRNA in the lung, spleen and heart and the circulating levels of these cytokines markedly increased in endotoxemia. This endotoxin-induced TNF alpha and IL-6 up-regulation was however suppressed in mice expressing IL-10 after intramuscular gene transfer. While cytokine gene expression was inhibited to varying degrees in different organs, a maximal reduction was seen in the lung, thus also indicating the efficacy of systemic IL-10 gene product at multiple tissue sites. Finally, we provided evidence that only when present in abnormally high concentrations in the circulation following intraperitoneal gene delivery, IL-10 by itself had some toxic effects of transient nature, primarily manifested by acute phase reaction and hemostatic disturbance. Thus, our studies demonstrate the usefulness of adenoviral vectors for therapeutic applications of IL-10 in vivo.
PMID: 9081704, UI: 97226863
Am J Pathol 1997 Jan;150(1):59-66
We have previously reported that transfer to rat lung of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene leads to high expression of GM-CSF between days 1 and 4 and granulation tissue formation followed by an irreversible fibrotic response starting from day 12 onward. In the current study, we investigated the underlying mechanisms. We found that GM-CSF overexpression did not enhance production of tumor necrosis factor-alpha in a significant manner at any time after GM-CSF gene transfer. However, the content of transforming growth factor-beta 1 in bronchoalveolar lavage fluid was markedly induced at day 4 and appeared to be maximal around day 7 and remained high at day 12. Macrophages purified from bronchoalveolar lavage fluid 7 days after GM-CSF gene transfer spontaneously released significant quantities of transforming growth factor-beta 1 protein in vitro. After peak transforming growth factor-beta 1 production was the emergence of alpha-smooth muscle actin-rich myofibroblasts. Accumulation of these cells was most prominent at day 12 within the granulation tissues and they were still present in fibrotic areas between days 12 and 24 and diminished markedly afterward. Thus, we provide the first in vivo evidence that tumor necrosis factor-alpha may be dissociated from participation in a fibrotic process in the lung and GM-CSF may play a more direct role in pulmonary fibrogenesis at least in part through its capability to induce transforming growth factor-beta 1 in macrophages and the subsequent emergence of myofibroblast phenotypes. This GM-CSF transgene lung model is useful for a stepwise dissection of both cellular and molecular events involved in pulmonary fibrosis.
PMID: 9006322, UI: 97158981
J Immunol 1996 Dec 1;157(11):5076-5084
RANTES (regulated on activation, normal T cell expressed and secreted) is a member of the C-C superfamily of chemokines and is reported to function as a potent chemoattractant for monocytes, eosinophils, and a subpopulation of CD4+ T cells. Using a recombinant human type 5 adenovirus containing the murine RANTES cDNA (Ad5E3 mRANTES), which is capable of expressing biologically active cytokine upon infection, we initiated a study to characterize the biologic functions of RANTES cytokine in vivo. Intratracheal administration of Ad5E3 mRANTES targeted transient RANTES expression to the bronchial epithelium of the lung in Sprague-Dawley rats. Bronchoalveolar lavage fluids (BAL) collected at 24 h had increased chemotactic activity vs controls as measured in a murine CD4+ T cell Boyden chamber microchemotaxis assay. There was a dramatic increase in the number of cells (macrophage, monocytes, and neutrophils) recovered from BAL samples taken from Ad5E3 mRANTES-treated animals at 24 h, with a >50-fold increase in monocytes, indicating a proinflammatory effect for this cytokine in vivo. This effect on monocytes was transient, decreasing by 7 days, with evidence of increased eosinophils and lymphocytes at this time. Histologic examination of lung sections at 24 h revealed greatly increased numbers of mononuclear cells, primarily monocytes, within the lungs of Ad5E3 mRANTES-treated animals, with increased extravasation of monocytes around blood vessels, indicating an ongoing process of peripheral blood monocyte recruitment. This study provides further evidence for RANTES to be a monocyte chemoattractant in vivo.
PMID: 8943417, UI: 97098740