IL-5 is induced locally in the lung and systemically in the circulation during allergic airways eosinophilic inflammation both in humans and experimental animals. However, the precise role of local and systemic IL-5 in the development of allergic airways eosinophilia remains to be elucidated. In our current study, we demonstrate that compared with their IL-5 ϩրϩ counterparts, IL-5 Ϫ / Ϫ mice lacked an IL-5 response both in the lung and peripheral blood, yet they released similar amounts of IL-4, eotaxin, and MIP-1 ␣ in the lung after ovalbumin (OVA) sensitization and challenge. At cellular levels, these mice failed to develop peripheral blood and airways eosinophilia while the responses of lymphocytes, neutrophils, and macrophages remained similar to those in IL-5 ϩրϩ mice. To dissect the relative role of local and systemic IL-5 in this model, we constructed a gene transfer vector expressing murine IL-5. Intramuscular IL-5 gene transfer to OVA-sensitized IL-5 Ϫ / Ϫ mice led to raised levels of IL-5 compartmentalized to the circulation and completely reconstituted airways eosinophilia upon OVA challenge, which was associated with reconstitution of eosinophilia in the bone marrow and peripheral blood. Significant airways eosinophilia was observed for at least 7 d in these mice. In contrast, intranasal IL-5 gene transfer, when rendered to give rise to a significant but compartmentalized level of transgene protein IL-5 in the lung, was unable to reconstitute airways eosinophilia in OVA-sensitized IL-5 Ϫ / Ϫ mice upon OVA-challenge, which was associated with a lack of eosinophilic responses in bone marrow and peripheral blood. Our findings thus provide unequivocal evidence that circulating but not local lung IL-5 is critically required for the development of allergic airways eosinophilia. These findings also provide the rationale for developing strategies to target circulating IL-5 and/or its receptors in bone marrow to effectively control asthmatic airways eosinophilia. ( J.

SummaryEstrogen is important for breast carcinogenesis and the majority of breast cancers maintain hormone dependency. Estrogen has the ability to stimulate both breast epithelial cell growth and angiogenesis, and a well-characterized in vivo cancer model where these functional interactions can be studied is lacking. We demonstrate estrogen dependent angiogenesis, growth in vivo, and proliferation in vitro, in explants from polyoma middle T transgenic mouse mammary tumors. Thus, in addition to genetic similarities, this model also exerts a sex hormone, and angiogenic phenotype similar to human breast cancer. This immune-competent animal model offers the opportunity to study molecular events in estrogen dependent breast cancer.

It has been shown that intratumor administration of an adenovirus vector expressing IL-12 produces a potent T cellmediated response that leads to significant tumor regression in a murine breast cancer model. IP-10 and MIG are CXC chemokines that recruit mononuclear cells in vivo. In addition to their chemotactic roles, IP-10 and MIG inhibit angiogenesis. We tested whether the addition of IP-10 or MIG may both enhance the antitumor immune response of IL-12 through T cell recruitment and inhibit tumor growth through angiostasis. Adenovirus vectors expressing IP-10 or MIG and/or IL-12 were administered intratumorally in a

Although the preliminary characterization of chemokines and their receptors has been prolific, comparatively little is known about the role of chemokines in the evolution of immune responses. We speculate that the preferential recruitment of a particular immune cell population has implications for the short- and long-term features of an adaptive response. To test this hypothesis, we employed adenovirus-mediated gene transfer to express the Th1-affiliated, CXC chemokine IFN-γ-inducible protein (IP) 10 in the airways of mice undergoing a mucosal sensitization regimen known to result in a Th2-polarized allergic response. This resulted in a ∼60–75% inhibition of eosinophils in the bronchoalveolar lavage (BAL); these inflammatory changes were accompanied by enhanced IFN-γ, ablated IL-4, and, peculiarly, unaltered IL-5 and eotaxin levels in the BAL. The effect of IP-10 expression was shown to be dependent on IFN-γ, as there was no statistically significant reduction in BAL eosinophilia in IFN-γ knockout mice subjected to the IP-10 intervention. Flow cytometric analysis of mononuclear cells in the lung revealed a ∼60% reduction in the fraction of CD4+ cells expressing T1/ST2, a putative Th2 marker, and a parallel increase in the proportion expressing intracellular IFN-γ following IP-10 treatment. The effect of IP-10 expression at the time of initial Ag encounter is persistent, as mice rechallenged with OVA following the resolution of acute inflammation exhibited reduced eosinophilia and IL-4 in the BAL. Collectively, these data illustrate that local expression of the chemokine IP-10 can introduce Th1 phenomena to a Th2-predisposed context and subvert the development of a Th2 response.

SUMMARYIFN-a administration after primary tumour resection improves the survival of melanoma patients at high risk of relapse. To investigate whether this response might be due to stimulation of anti-tumour immunity, the effect of IFN-a on anti-melanoma CTL generation in MLTC was measured. IFN-a increased both allogeneic and autologous anti-melanoma CTL generation from peripheral blood lymphocytes stimulated with irradiated primary melanoma cultures. IFN-a up-regulated MHC class I expression on primary melanoma cultures, whereas IFN-g up-regulated both MHC class I and II expression. However, the effect of IFN-a on anti-melanoma CTL generation was often more potent than that of IFN-g , equalling the effect of the optimal combination of IL-2 and IL-12. Pre-treatment of primary melanoma cultures with IFN-g was sufficient for CTL generation in MLTC, whereas IFN-a needed to be present during the MLTC. While direct anti-proliferative effects of IFN-a on some tumour cells have been described, IFN-a did not inhibit proliferation of primary melanoma cultures. These results suggest that the clinical effects of IFN-a in melanoma patients may be immune-mediated.

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.

We vaccinated metastatic melanoma patients with irradiated, autologous melanoma cells genetically engineered to secrete interleukin 2 (IL-2) to investigate whether an anti-tumor immune response would be induced. Melanoma cell cultures were established from surgical specimens and were engineered to secrete IL-2 by infection with recombinant retrovirus. Twelve patients were vaccinated subcutaneously one, two, or three times with approximately 10(7) irradiated, autologous, IL-2-secreting tumor cells. Treatment was well tolerated, with local reactions at 11 of 24 injection sites and minor systemic symptoms of fever and headache after 6 injections. One patient developed anti-tumor DTH after the first vaccination and showed an increased response after the second vaccination. Anti-autologous tumor CTLs could be detected prevaccination in the peripheral blood of seven patients and their activity increased after vaccination in four patients. No UICC-defined clinical responses were seen, but three patients had stable disease for 7-15 months, one of whom has not yet progressed (15+ months). Thus, patient vaccination with autologous, genetically engineered tumor cells is feasible and safe. Anti-tumor DTH and CTLs can be induced in some patients with such a vaccine.

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 factorinduced angiogenesis, Ad-angiostatin (1 ؋ 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. Tumors in situ that are smaller than 3 mm in diameter, exist in a pre-vascular state and are limited in their ability to grow without perfusion from the blood supply.