Novel chimpanzee adenovirus-vectored respiratory mucosal tuberculosis vaccine: overcoming local anti-human adenovirus immunity for potent TB protection.

Pulmonary tuberculosis (TB) remains to be a major global health problem despite many decades of parenteral use of Bacillus Calmette-Guérin (BCG) vaccine. Developing safe and effective respiratory mucosal TB vaccines represents a unique challenge. Over the past decade or so, the human serotype 5 adenovirus (AdHu5)-based TB vaccine has emerged as one of the most promising candidates based on a plethora of preclinical and early clinical studies. However, anti-AdHu5 immunity widely present in the lung of humans poses a serious gap and limitation to its real-world applications. In this study we have developed a novel chimpanzee adenovirus 68 (AdCh68)-vectored TB vaccine amenable to the respiratory route of vaccination. We have evaluated AdCh68-based TB vaccine for its safety, T-cell immunogenicity, and protective efficacy in relevant animal models of human pulmonary TB with or without parenteral BCG priming. We have also compared AdCh68-based TB vaccine with its AdHu5 counterpart in both naive animals and those with preexisting anti-AdHu5 immunity in the lung. We provide compelling evidence that AdCh68-based TB vaccine is not only safe when delivered to the respiratory tract but, importantly, is also superior to its AdHu5 counterpart in induction of T-cell responses and immune protection, and limiting lung immunopathology in the presence of preexisting anti-AdHu5 immunity in the lung. Our findings thus suggest AdCh68-based TB vaccine to be an ideal candidate for respiratory mucosal immunization, endorsing its further clinical development in humans.

Adenoviral-mediated gene transfer into bone marrow: an effective surgical technique in rat.

BACKGROUND: The role of transforming growth factor-beta 1 (TGF-ß1) in the onset of bone marrow fibrosis has been confirmed in some animal models. To further understand the genetic expression of some myeloproliferative disorders affecting marrow stem cells, however, it is necessary to develop a specific and reliable procedure to deliver modified adenoviral vectors into the bone marrow cavity. The aim of this paper is to report a surgical technique designed to deliver an adenoviral vector-mediated gene expressing TGF-ß1 into the bone marrow of rat femurs. METHODS: Forty-two Sprague-Dawley rats were used in the study. Rat femurs were exposed and the compact and trabecular bones at the proximal head removed. An intrabone marrow injection of a mutated TGF-ß1 adenoviral vector, a null adenoviral vector, or PBS was delivered into the bone. Three groups were accounted (n = 14 per group): fibrogenic and positive and negative controls. The quality of the surgical entrance was assessed by means of computerized tomography and histological changes were assessed by histochemistry. The concentration of TGF-ß1 in the bone marrow was determined by ELISA. RESULTS: The surgical technique was conducted under ideal timing (approx. 10 min) and no surgical or postsurgical complications were observed. Computerized tomography revealed no changes in the bone tissue and a clean entrance was delimited through the bone to the bone marrow. HE and Masson's trichrome staining indicated highly fibrotic areas in the profibrotic group and bone marrow lavage reported a significantly higher concentration of TGF-ß1 (p < 0.05) in that same group. CONCLUSIONS: The present study confirmed that the proposed surgical technique is an effective method to deliver adenoviral vectors into the femoral bone marrow to investigate the physiopathology of bone marrow fibrosis in rats.

Overexpression of transforming growth factor-beta1 in fetal monkey lung results in prenatal pulmonary fibrosis.

Altered transforming growth factor (TGF)-ß expression levels have been linked to a variety of human respiratory diseases, including bronchopulmonary dysplasia and pulmonary fibrosis. However, a causative role for aberrant TGF-ß in neonatal lung diseases has not been defined in primates. Exogenous and transient TGF-ß1 overexpression in fetal monkey lung was achieved by transabdominal ultrasound-guided fetal intrapulmonary injection of adenoviral vector expressing TGF-ß1 at the second or third trimester of pregnancy. The lungs were then harvested near term, and fixed for histology and immunohistochemistry. Lung hypoplasia was observed where TGF-ß1 was overexpressed during the second trimester. The most clearly marked phenotype consisted of severe pulmonary and pleural fibrosis, which was independent of the gestational time point when TGF-ß1 was overexpressed. Increased cell proliferation, particularly in α-smooth muscle actin-positive myofibroblasts, was detected within the fibrotic foci. But epithelium to mesenchyme transdifferentiation was not detected. Massive collagen fibres were deposited on the inner and outer sides of the pleural membrane, with an intact elastin layer in the middle. This induced fibrotic pathology persisted even after adenoviral-mediated TGF-ß1 overexpression was no longer evident. Therefore, overexpression of TGF-ß1 within developing fetal monkey lung results in severe and progressive fibrosis in lung parenchyma and pleural membrane, in addition to pulmonary hypoplasia.

Immunization with adenovirus at the large intestinal mucosa as an effective vaccination strategy against sexually transmitted viral infection.

The large intestinal mucosa contains immunological structures that may potentially serve as a site for induction of mucosal immunity against infections. Adenovirus (Ad), which is effective in gene transfer to epithelia, may be an ideal antigen delivery system for vaccination at the large intestinal mucosa. To investigate this potential, we immunized mice with recombinant replication-deficient Ad through a single intracolorectal (ICR) administration. Effective transfer of encoded genes was found in both the epithelial layer and lamina propria of the colorectal mucosa. Dendritic cells were able to transfer antigen to the draining lymph nodes, where antigen-specific CD8(+) T cells were primed. Functional antigen-specific CD8(+) T cells and IgA-specific antibodies were detected during the effector phase in the large intestine. Compared to other immunization routes (intranasal, subcutaneous), ICR immunization induced stronger colorectal immune responses and more potent protection against rectal challenge with pathogenic viruses. Further, this immunization strategy provided vaginal protection, more potent than that induced by vaccination in the nose or skin. Therefore, large intestine mucosal immunization using Ad represents an effective vaccination strategy against virus infection at both rectal and vaginal mucosal tissue sites.

TGF-beta receptor II in epithelia versus mesenchyme plays distinct roles in the developing lung.

Transforming growth factor (TGF)-beta signalling plays important roles in regulating lung development. However, the specific regulatory functions of TGF-beta signalling in developing lung epithelial versus mesenchymal cells are still unknown. By immunostaining, the expression pattern of the TGF-beta type II receptor (TbetaRII) was first determined in the developing mouse lung. The functions of TbetaRII in developing lung were then determined by conditionally knocking out TbetaRII in the lung epithelium of floxed-TbetaRII/surfactant protein C-reverse tetracycline transactivator/TetO-Cre mice versus mesenchyme of floxed-TbetaRII/Dermo1-Cre mice. TbetaRII was expressed only in distal airway epithelium at early gestation (embryonic day (E)11.5), but in both airway epithelium and mesenchyme from mid-gestation (E14.5) to post-natal day 14. Abrogation of TbetaRII in mouse lung epithelium resulted in retardation of post-natal lung alveolarisation, with markedly decreased type I alveolar epithelial cells, while no abnormality in prenatal lung development was observed. In contrast, blockade of TbetaRII in mesoderm-derived tissues, including lung mesenchyme, resulted in mildly abnormal lung branching and reduced cell proliferation after mid-gestation, accompanied by multiple defects in other organs, including diaphragmatic hernia. The primary lung branching defect was verified in embryonic lung explant culture. The novel findings of the present study suggest that transforming growth factor-beta type II receptor-mediated transforming growth factor-beta signalling plays distinct roles in lung epithelium versus mesenchyme to differentially control specific stages of lung development.

Three-dimensional computed tomography imaging in an animal model of emphysema.

Emphysema is a major health problem and novel drugs are needed. Animal disease models are pivotal in their development, but the validity and sensitivity of current tools for the evaluation of drug efficacy is limited. The usefulness of micro computed tomography (CT) as an innovative tool to assess emphysema in a mouse model was investigated. Serial CT scans were performed in bi-weekly intervals in Smad3 knockout (KO) mice, which spontaneously develop airspace enlargement. Lung density was quantified in two- and three-dimensional images and correlated to mean linear intercept and lung compliance. CT scans of Smad3 KO lungs revealed a significant decrease in lung density at age 8 weeks and a further progression at age 14 weeks with respect to age-matched wild-type (WT) animals. Emphysema could be reliably assessed with both the two- and three-dimensional approach, but the three-dimensional approach was superior, due to normalisation to lung volumes and less variability. Lung compliance by week 14 was 0.053+/-0.005 and 0.034+/-0.002% of maximum volume.cmH(2)O(-1) for KO and WT mice, respectively, reflecting significant physiologically relevant emphysema. Small animal computed tomography imaging and density quantification in a reconstructed three-dimensional image is a useful tool for quantifying emphysematous changes in an animal disease model. It adds significant information to conventional assessment.

TGF-beta, Smad3 and the process of progressive fibrosis.

Transient adenovirus-mediated gene transfer of active TGF-beta1 (transforming growth factor-beta1) induces severe and progressive fibrosis in rodent lung without apparent inflammation. Alternatively, transfer of IL-1beta (interleukin 1beta) induces marked tissue injury and inflammation, which develops into progressive fibrosis, associated with an increase in TGF-beta1 concentrations in lung fluid and tissue. Both vector treatments induce a fibrotic response involving myofibroblasts and progressive matrix deposition starting at the peri-bronchial site of expression and extending over days to involve the entire lung and pleural surface. Administration of the TGF-beta1 vector to the pleural space induces progressive pleural fibrosis, which minimally extends into the lung parenchyma. The mechanisms involved in progressive fibrosis need to account for the limitation of fibrosis to specific organs (lung fibrosis and not liver fibrosis or vice versa) and the lack of effect of anti-inflammatory treatments in regulating progressive fibrosis. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signalling pathways involving the ALK5 receptor and signalling molecules Smad2 and Smad3. Transient gene transfer of either TGF-beta1 or IL-1beta to Smad3-null mouse lung provides little evidence of progressive fibrosis and no fibrogenesis-associated genes are induced. These results suggest that mechanisms of progressive fibrosis involve factors presented within the context of the matrix that define the microenvironment for progressive matrix deposition.

Induction of a fibrogenic response in mouse colon by overexpression of monocyte chemoattractant protein 1.

BACKGROUND AND AIMS: Monocyte chemoattractant protein 1 (MCP-1) is increased transmurally in inflammatory bowel disease (IBD). Although MCP-1 is considered to play an important role in fibrotic disease in other organs, the role of MCP-1 in gut fibrosis is unknown. We investigated the fibrotic potential of MCP-1 in the gut by overexpressing this chemokine in the mouse colorectal wall. METHODS: Intramural gene transfer by direct injection of adenovector into the mouse rectal wall was established. C57BL/6 and Rag2(-/-) (B and T cell deficient) mice received 2.5 x 10(9) plaque forming units of an adenovector encoding murine MCP-1 (AdMCP-1) or control virus (AdDL70) via intramural injection. Mice were killed at various time points and tissues were obtained for histopathological and biochemical analysis. RESULTS: AdMCP-1 significantly increased collagen production in the colorectum and this was associated with significant elevation of transforming growth factor beta (TGF-beta) and tissue inhibitor of metalloproteinase (TIMP-1) protein. Transmural collagen deposition was observed after AdMCP-1 administration, and was accompanied by CD3+ T cells, F4/80+ macrophages, and vimentin+ cell infiltrates. Collagen was differentially distributed, with type I deposited in the muscularis mucosa and muscularis propria and type III in the submucosa and myenteric plexus. AdMCP-1 failed to induce collagen overproduction in immunodeficient Rag2(-/-) mice. CONCLUSION: These findings suggest that MCP-1 can induce fibrosis in the gut and that this process involves interaction between T cells and vimentin positive fibroblasts/myofibroblasts, as well as the subsequent upregulation of TGF-beta and TIMP-1 production. This model provides a basis for considering MCP-1 in the pathogenesis of strictures in IBD.

The gene transfer of soluble VEGF type I receptor (Flt-1) attenuates peritoneal fibrosis formation in mice but not soluble TGF-beta type II receptor gene transfer.

Peritoneal fibrosis formation is a consequence of inflammation/injury and a significant medical problem to be solved. The effects of soluble VEGF receptor type I (sFlt-1) gene transfer on experimental peritoneal fibrosis were examined and compared with soluble transforming growth factor-beta (TGF-beta) receptor type II (sTGF beta RII) gene transfer. Male C57BL/6 mice were injected with 1.5 x 10(8) plaque-forming unit of adenovirus encoding active TGF-beta (AdTGF beta) intraperitoneally. Some mice had been treated with sTGF betaRII or sFlt-1 plasmid injection into skeletal muscle with electroporation 4 days before virus administration. Mice were euthanized at day 14 after virus administration. AdTGF beta induced significant elevation of serum active TGF-beta, caused significant inflammatory response [weight loss, elevation of serum amyloid-P (SAP) and IL-12, increased expression of monocyte chemoattractant protein-1 (MCP-1) mRNA], and induced marked thickening of the peritoneum and collagen deposition. Gene transfer of sFlt-1 reduced the collagen deposition approximately 81% in mesenteric tissue. Treatment with sFlt-1 decreased ICAM-1 and MCP-1 mRNA expression significantly. Significant negative correlation between serum sFlt-1 and placental growth factor level was observed, whereas there was no significant negative correlation between sFlt-1 and VEGF. On the other hand, sTGF beta RII treatment enhanced the AdTGF beta-induced inflammation (significant elevation of SAP, TNF-alpha, and IL-12 levels and upregulation of ICAM-1 and MCP-1 mRNA expressions) and failed to prevent collagen deposition. These observations indicate that sFlt-1 gene transfer might be of therapeutic benefit in peritoneal fibrosis.

Disruption of CD40-CD40 ligand pathway inhibits the development of intestinal muscle hypercontractility and protective immunity in nematode infection.

In our previous studies, we demonstrated that during Trichinella spiralis infection, T helper (Th) 2 cells contribute to the development of intestinal muscle hypercontractility and worm expulsion from the gut via STAT6. In addition, we have linked the altered muscle contractility to the eviction of the parasite and thereby to the host defense. However, the initial events linking infection to the development of muscle hypercontractility are poorly understood. In this study, we examined the contribution of CD40-CD40 ligand (CD40L) interaction in the development of intestinal muscle hypercontractility, in monocyte chemoattractant protein-1 (MCP-1) production, and in the Th2 response in CD40 ligand-deficient (CD40L -/-) mice infected with T. spiralis. Expulsion of intestinal worms was substantially delayed in CD40L -/- mice compared with the wild-type mice after T. spiralis infection. Consistent with delayed worm expulsion, there was a significant attenuation of intestinal muscle contractility in CD40L -/- mice. Infected CD40L -/- mice also exhibited marked impairment in the production of MCP-1, IL-4, IL-13, IgG1, IgE, and mouse mucosal MCP 1 (MMCP-1), and in goblet cell response. These results demonstrate that CD40-CD40 ligand interaction plays an important role in MCP-1 production, Th2 response, intestinal muscle hypercontractility, and worm expulsion in nematode infection. The present data suggest that the early events leading to the generation of Th2 response include CD40-CD40 ligand interaction, which subsequently influences the production of Th2 cytokines, most likely via upregulation of MCP-1.

Proinflammatory properties of IL-4 in the intestinal microenvironment.

IL-4 is involved in type 2 T helper cell (Th)2-type immune responses and, in some cases, can promote Th1 responses. However, the proinflammatory potential of IL-4 alone is unclear. In this study, we examined the ability of IL-4 to induce colitis after its overexpression in the colon using an adenoviral vector (Ad5) and compared results with those obtained after overexpression of IL-12, a cytokine implicated in several models of colitis. Overexpression of IL-4 or IL-12 caused a fatal colitis within 24 h in 60% of animals and was dose and strain dependent. IL-12-induced colitis was accompanied by the local expression of IFN-gamma and TNF-alpha but not IL-4 mRNA and protein. Conversely, IL-4-induced colitis was accompanied by the local expression of IL-4 and TNF-alpha but not IFN-gamma mRNA and protein. The Ad5-IL4-induced colitis did not persist beyond 3 days and was present in recombinase activation gene-2 (RAG-2)-/- mice but not in STAT6-/- mice. Acute lethal colitis induced by Ad5IL12 was T cell mediated and IFN-gamma receptor (IFN-gamma R) dependent. Furthermore, TNF-alpha was found to be important in the pathogenesis of Ad5IL-4 and Ad5IL-12-induced colitis. Results of this study indicate that IL-4 alone can act as a proinflammatory cytokine in the gut of normal mice, inducing a rapid onset and short-lived colonic injury while maintaining a Th2-type cytokine profile that functions via a local T cell-independent mechanism involving TNF-alpha.

Effects of adenoviral-mediated gene transfer of interleukin-10, interleukin-4, and transforming growth factor-beta on the survival of axotomized retinal ganglion cells.

Nitric oxide, synthesized by reactive microglia and astrocytes has been implicated in promoting neuronal degeneration observed in many diseases and insults of the central nervous system. We have recently shown that inducible nitric oxide synthase is expressed by retinal glial cells following optic nerve transection and that inhibition of nitric oxide synthesis enhances the survival of injured retinal ganglion cells. Anti-inflammatory cytokines including interleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growth factor-beta (TGF-beta) have been shown to prevent inducible nitric oxide synthase expression, and inhibit nitric oxide synthesis by microglia and astrocytes in culture. In the present study, we examined the effects of adenoviral mediated gene transfer of anti-inflammatory cytokines on the survival of axotomized retinal ganglion cells. Intraocular administration of adenoviral vectors encoding interleukin-10 (Ad.IL-10) and interleukin-4 (Ad.IL-4) enhanced the survival of axotomized retinal ganglion cells at 14 days after axotomy. Adenoviral vectors encoding TGF-beta (Ad.TGF-beta) had no effect on retinal ganglion cell survival. Separate animals were pretreated by injection of Ad.IL-10 or Ad.IL-4 into the superior colliculus (s.c.), the major target of ganglion cells, 7 days prior to axotomy. S.c. administration of Ad.IL-10 or Ad.IL-4 significantly increased ganglion cell survival compared with intraocular injection. IL-10 and IL-4 gene transfer also reduced the density of infiltrating ED1 positive monocytes in the nerve fiber layer at 14 days postaxotomy. Ad.TGF-beta increased the density of ED1 positive monocytes infiltrating the nerve fiber layer after axotomy. Vectors encoding IL-10 or IL-4 also decreased nitrotyrosine immunoreactivity in the inner retina at 7 days postaxotomy, suggesting that these cytokines protect retinal ganglion cells from peroxynitrite formation that results from nitric oxide synthesis by activated glial cells. The present study has implications for the treatment of CNS injury and diseases that involve reactive microglia and astrocytes. Our results suggest that interleukin-10 and interleukin-4 may help prevent neurodegeneration caused by the activation of glial cells after CNS injury.

Oestradiol enhances tumour regression induced by B7-1/IL-2 adenoviral gene transfer in a murine model of breast cancer.

The majority of breast cancers are oestrogen dependent and although current treatment strategies have improved, approximately 50% of the patients will develop metastasis. New treatments that result in long-term systemic immunity are therefore being developed. We have previously shown that adenoviral gene transfer of B7-1/IL-2 to murine breast cancer induces a high rate of complete tumour regression and systemic immunity. Since oestrogens not only affect breast cancer but also have been shown to modulate immune function and secretion of immune-regulatory cytokines, we explored whether administration of oestradiol altered the immune response induced by an adenoviral vector expressing B7-1/IL-2. An oestrogen-dependent murine breast cancer tumour was used in ovariectomised mice, supplemented either oestradiol or placebo. We report the somewhat unexpected finding that intratumoral injection of adenovirus expressing B7-1/IL-2 induces complete tumour regression in 76% of oestradiol-supplemented mice, while only 18% of the tumours regressed in the oestrogen-depleted group. Cured mice in both groups exhibited a similar CTL response against the tumour antigen. However, intratumoral IFN-gamma levels, 2 days after B7-1/IL-2 injection, were significantly higher in mice treated with oestradiol compared to placebo. This may be one mechanism explaining the higher response rate of tumours in oestradiol-replenished mice.

Intratumoral administration of an adenovirus expressing a kinase dead form of ErbB-2 inhibits tumor growth.

ErbB-2 is amplified or overexpressed in a number of different cancers including breast, ovarian, lung, prostate and stomach. This overexpression leads to enhanced receptor dimer formation and stabilization allowing the receptor to remain in an active state. The clinical consequences of ErbB-2 overexpression include increased tumor aggressiveness, poor prognosis, decreased patient survival and resistance to chemotherapy. As a result, a variety of different strategies are being examined to inhibit its function or expression. In this study, we explored the efficacy of a type 5 recombinant adenovirus encoding a kinase dead form of ErbB-2, AderbB-2 Delta tk, as a potential therapeutic agent for breast cancer using a murine breast model expressing constitutively active ErbB-2. Co-expression in tumor cells of the kinase dead form of ErbB-2 inhibits receptor activity and induces the death of cells expressing constitutively active ErbB-2. In addition, AderbB-2 Delta tk exhibits antitumor activity in both immune-competent and immune-deficient animals with increased antitumor activity in the immune-competent animals. The results suggest both immune and non-immune mechanisms contribute to the antitumor efficacy of this vector.

Budesonide enhances repeated gene transfer and expression in the lung with adenoviral vectors.

The immune response with generation of neutralizing antiviral antibodies is an obstacle to effective repeated adenoviral gene transfer. Different immunosuppressive drugs facilitate repeat administration of adenovectors, but the clinical utility is uncertain because of systemic side effects. We investigated the use of topical corticosteroid in improving gene expression after repeated injection of adenovectors into mouse lungs. Using a vector expressing murine interleukin-6 (mIL-6) as a marker cytokine for gene expression, we show that budesonide given around exposure to adenovirus to the lung significantly maintained high levels of expressed transgene protein in bronchoalveolar lavage fluid (BALF) after as many as four consecutive injections of virus at two weekly intervals (p = 0.02 versus saline). Differences between treatment groups were most obvious 4 and 6 wk after the initial exposure to adenovirus (equivalent to three and four total exposures). In Week 4, transgene mIL-6 concentration was 2,327 +/- 955 pg/ml in budesonide compared with 336 +/- 246 pg/ml in saline-treated mice (p = 0.001). However, budesonide did not significantly protect transgene expression beyond Week 8 (four prior exposures). The improved transgene expression in budesonide-treated compared with saline-treated animals was associated with a reduction, but not prevention of neutralizing antiviral antibodies (BALF p < 0.001, serum p = 0.04). We conclude that budesonide can be valuable in gene therapy of the lung where repeated transient gene transfer is necessary.

Adenoviral vector expressing murine angiostatin inhibits a model of breast cancer metastatic growth in the lungs of mice.

Angiostatin, an internal fragment of plasminogen, has been shown to inhibit the process of angiogenesis or neovascularization. In this study, we have expressed the cDNA for murine angiostatin under the control of the human cytomegalovirus promoter from a human type-5 adenovirus and shown that this vector produces a protein which retains biological activity. Angiostatin expression was determined by Northern blot analysis and Western immunoblotting. Ad-angiostatin, but not a control vector Ad-dl70, significantly reduced the viability of infected human umbilical cord vein endothelial cells (HUVEC) in vitro. In an in vivo model of basic fibroblast growth factor-induced angiogenesis, Ad-angiostatin (1 x 10(9) pfu) could inhibit endothelial cell migration and the formation of capillaries within a Matrigel plug which had been implanted for one week subcutaneously into C57BL/6 mice. Endothelial cells in these plugs had an altered, rounded, phenotype with dark picnotic nuclei indicative of apoptosis, which was confirmed using transmission electron microscopy. In contrast, endothelial cells from bFGF alone or in combination with the control vector-treated plugs retained the long spindle shape characteristic of endothelial cells. Intranasal delivery of Ad-angiostatin into the lungs of FVB/n mice demonstrated comparable cellular infiltration in the recovered bronchoalveolar lavage fluid with no signs of abnormal pathology as compared to PBS or control vector-treated animals. In a pulmonary metastatic breast cancer model, the delivery of Ad-angiostatin (1 x 10(9) pfu) to the lung significantly delayed tumor growth as measured by the number of visible surface tumor nodules. This study has demonstrated that the specific targeting of tumors to inhibit angiogenesis using an adenovirus expressing angiostatin, may deliver localized concentrations of protein having a greater impact on inhibition of tumor growth.

Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis.

IL-1beta is one of a family of proinflammatory cytokines thought to be involved in many acute and chronic diseases. Although it is considered to participate in wound repair, no major role has been attributed to IL-1beta in tissue fibrosis. We used adenoviral gene transfer to transiently overexpress IL-1beta in rat lungs after intratracheal administration. The high expression of IL-1beta in the first week after injection was accompanied by local increase of the proinflammatory cytokines IL-6 and TNF-alpha and a vigorous acute inflammatory tissue response with evidence of tissue injury. The profibrotic cytokines PDGF and TGF-beta1 were increased in lung fluid samples 1 week after peak expression of IL-1beta. Although PDGF returned to baseline in the third week, TGF-beta1 showed increased concentrations in bronchoalveolar lavage fluid for up to 60 days. This was associated with severe progressive tissue fibrosis in the lung, as shown by the presence of myofibroblasts, fibroblast foci, and significant extracellular accumulations of collagen and fibronectin. These data directly demonstrate how acute tissue injury in the lung, initiated by a highly proinflammatory cytokine, IL-1beta, converts to progressive fibrotic changes. IL-1beta should be considered a valid target for therapeutic intervention in diseases associated with fibrosis and tissue remodeling.

IL-12 gene transfer alters gut physiology and host immunity in nematode-infected mice.

Immune responses elicited by nematode parasite infections are characterized by T helper 2 (Th2) cell induction. The immunologic basis for changes in intestinal physiology accompanying nematode infection is poorly understood. This study examined whether worm expulsion and associated goblet cell hyperplasia and muscle contractility share a similar immune basis by shifting the response from Th2 to Th1 using interleukin-12 (IL-12) overexpression. We used a single administration of recombinant adenovirus vector expressing IL-12 (Ad5IL-12) in Trichinella spiralis-infected mice. Ad5IL-12 administered 1 day after infection prolonged worm survival and inhibited infection-induced muscle hypercontractility and goblet cell hyperplasia. This was correlated with upregulated interferon-gamma (IFN-gamma) expression and downregulated IL-13 expression in the muscularis externa layer. We also observed increased IFN-gamma production and decreased IL-4 and IL-13 production from in vitro stimulated spleen and mesenteric lymph node cells of infected Ad5IL-12-treated mice. These results indicate that transfer and overexpression of the IL-12 gene during Th2-based nematode infection shifts the immune response toward Th1 and delays worm expulsion. Moreover, the immune response shift abrogated the physiological responses to infection, attenuating both muscle hypercontractility and goblet cell hyperplasia. These findings strongly indicate that worm expulsion, muscle hypercontractility, and goblet cell hyperplasia share a common immunologic basis and may be causally linked.

Combined treatment of a murine breast cancer model with type 5 adenovirus vectors expressing murine angiostatin and IL-12: a role for combined anti-angiogenesis and immunotherapy.

In this study, we used intratumor delivery of adenoviral vectors to induce a selective anti-tumor response by combining the potent angiogenesis inhibitor murine angiostatin (adenovirus (Ad)-angiostatin) with the powerful immune simulator and angiostatic cytokine murine IL-12 (Ad-IL-12). In a murine model of breast carcinoma, intratumor injection of Ad-angiostatin delayed mean tumor growth, as compared with control virus with an initial regression of tumor growth, in 65% of treated animals. However, all treated animals eventually succumbed to the tumors. Mice injected with Ad-IL-12 alone responded with an initial regression in 20% of treated animals, with only 13% developing a total regression. Coinjection of the vectors resulted in 96% of the treated animals developing an initial regression, with 54% undergoing a total regression of the tumor. These mice were resistant to tumor rechallenge and developed a strong CTL response. Frozen tumor sections were stained for microvessel density using an Ab against murine CD31, an endothelial cell marker. Automated image analysis revealed the mean microvessel density following the administration of Ad-angiostatin and Ad-IL-12 alone or in combination was significantly reduced compared with the control-treated tumor. In summary, we have shown that a short-term course of antiangiogenic therapy combined with immunotherapy can effectively shrink a solid tumor and vaccinate the animal against rechallenge. The rationale for this therapy is to limit the tumor size by attacking the vasculature with angiostatin, thereby allowing IL-12 to mount a T cell-specific response against the tumor AG:

Proteoglycans decorin and biglycan differentially modulate TGF-beta-mediated fibrotic responses in the lung.

Transforming growth factor (TGF)-beta is a key cytokine in the pathogenesis of pulmonary fibrosis, and pharmacological interference with TGF-beta can ameliorate the fibrotic tissue response. The small proteoglycans decorin and biglycan are able to bind and inhibit TGF-beta activity in vitro. Although decorin has anti-TGF-beta properties in vivo, little is known about the physiological role of biglycan in vivo. Adenoviral gene transfer was used to overexpress active TGF-beta, decorin, and biglycan in cell culture and in murine lungs. Both proteoglycans were able to interfere with TGF-beta bioactivity in vitro in a dose-dependant manner. In vivo, overexpression of TGF-beta resulted in marked lung fibrosis, which was significantly reduced by concomitant overexpression of decorin. Biglycan, however, had no significant effect on lung fibrosis induced by TGF-beta. The data suggest that differences in tissue distribution are responsible for the different effects on TGF-beta bioactivity in vivo, indicating that decorin, but not biglycan, has potential therapeutic value in fibrotic disorders of the lung.