Purpose: Delineation of the long-term follow-up data on a series of patients with malignant mesothelioma, who received a single intrapleural dose of a nonreplicative adenoviral (Ad) vector encoding the herpes simplex virus thymidine kinase ''suicide gene'' (Ad.HSVtk) in combination with systemic ganciclovir. Experimental Design: This report focuses on the 21 patients receiving ''high-dose'' therapy, defined by an intrapleural dose of vector (z1.6 Â 10 13 viral particles), where transgene-encoded tk protein was reliably identified on immunohistochemical staining. In 13 patients, the vector was deleted in the E1 and E3 regions of the Ad; in the other eight patients, the vector had deletions in the Ad genes E1 and E4. Safety, immunologic responses, transgene expression, and clinical responses were evaluated. Results: Both the E1/E3-deleted vector and the E1/E4-deleted vector were well tolerated and safe, although production of the E1/E4 vector was more difficult. Posttreatment antibody responses against the tumors were consistently seen. Interestingly, we observed a number of clinical responses in our patients, including two long-term (>6.5 year) survivors, both of whom were treated with the E1/E4-deleted vector. Conclusions: Intrapleural Ad.HSVtk/ganciclovir is safe and well tolerated in mesothelioma patients and resulted in long-term durable responses in two patients. Given the limited amount of gene transfer observed, we postulate that Ad.HSVtk may have been effective due to induction of antitumor immune responses. We hypothesize that approaches aiming to augment the immune effects of Ad gene transfer (i.e., with the use of cytokines) may lead to increased numbers of therapeutic responses in otherwise untreatable pleural malignancies.

Immunotherapy against a variety of malignancies, including pleural-based malignancies, has shown promise in animal models and early human clinical trials, but successful efforts will need to address immunosuppressive factors of the tumor and host, particularly certain cytokines and CD4(+) CD25(+) regulatory T cells (Treg). Here, we evaluated the cellular and cytokine components of malignant pleural effusions from 44 patients with previously diagnosed mesothelioma, non-small cell lung cancer (NSCLC), or breast cancer and found significant differences in the immune profile of pleural effusions secondary to mesothelioma vs. carcinoma. Although a high prevalence of functionally suppressive CD4(+) CD25(+) T cells was found in carcinomatous pleural effusions, mesothelioma pleural effusions contained significantly fewer CD4(+) CD25(+) T cells. Activated CD8(+) T cells in pleural fluid were significantly more prevalent in mesothelioma than carcinoma. However, there is clear patient-to-patient variability and occasional mesothelioma patients with high percentages of CD4(+) CD25(+) pleural effusion T cells and low percentages of CD8(+) CD25(+) pleural effusion T cells can be identified. Mesothelioma pleural effusions contained the highest concentrations of the immunosuppressive cytokine transforming growth factor (TGF)-beta. Thus, the contribution of cellular and cytokine components of immunosuppression associated with malignant pleural effusions varies by tumor histology and by the individual patient. These results have implications for the development of immunotherapy directed to the malignant pleural space, and suggest the need to tailor immunotherapy to overcome immunosuppressive mechanisms in tumor environments.

Current evidence suggests that tumor necrosis factor ␣ (TNF␣) and the family of interferons (IFNs) synergistically regulate many cellular responses that are believed to be critical in chronic inflammatory diseases, although the underlying mechanisms of such interaction are complex, cell-specific, and not completely understood. In this study, TNF␣ in a time-dependent manner activated both janus tyrosine kinase 1 and Tyk2 tyrosine kinase and increased the nuclear translocation of interferon-regulatory factor-1, STAT1, and STAT2 in human airway smooth muscle cells. In cells transfected with a luciferase reporter, TNF␣ stimulated ␥-activated site-dependent gene transcription in a time-and concentration-dependent manner. Using neutralizing antibodies to IFN␤ and TNF␣ receptor 1, we show that TNF␣-induced secretion of IFN␤ mediated ␥-activated site-dependent gene expression via activation of TNF␣ receptor 1. In addition, neutralizing antibody to IFN␤ also completely abrogated the activation of interferon stimulation response elementdependent gene transcription induced by TNF␣. Secreted IFN␤ acted as a negative regulator of TNF␣-induced interleukin-6 expression, while IFN␤ augmented TNF␣-induced RANTES (regulated on activation normal T cell expressed and secreted) secretion but had little effect on TNF␣-induced intercellular adhesion molecule-1 expression. Furthermore TNF␣, a modest airway smooth muscle mitogen, markedly induced DNA synthesis when cells were treated with neutralizing anti-IFN␤. Together these data show that TNF␣, via the autocrine action of IFN␤, differentially regulates the expression of proinflammatory genes and DNA synthesis. TNF␣1 is now considered to be one of the most pleiotropic cytokines in mediating inflammatory and immune responses in chronic lung diseases. In vivo studies using selective inhibitors of TNF␣ activity demonstrate that TNF␣ plays a major role in antigen-induced airway inflammation (leukocyte infiltration) and airway hyper-responsiveness in animal models of asthma (1, 2). Others who used receptor knock-out mice confirmed the importance of both TNF␣ receptors (TNFRs), TNFR1 and TNFR2, in the abnormal airway changes induced by allergen challenge in sensitized animals (3-5). A potential site for TNF␣ deleterious action in the lungs is airway smooth muscle (ASM), a primary effector tissue thought to only regulate bronchomotor tone (6). In human cultured ASM cells that retain physiological responsiveness and express both TNF␣ receptors (7), TNF␣ alters proinflammatory gene expression that in turn may play an important role in the pathogenesis of allergic asthma. In previous reports, we showed that TNF␣ increased expression of ICAM-1, IL-6, and RANTES by selectively activating TNFR1, although TNFR2 was also involved in TNF␣-induced RANTES secretion (8 -10). TNFR1-associated gene expression has been involved in the development of bronchial hyper-responsiveness (6,11,12).TNF␣ also cooperates with other cytokines such as interferon ␥ (IFN␥) to regulate the expression of cytokines (IL-1 an...

Purpose: Transforming growth factor (TGF)-␤ blockade has been proposed as an anticancer therapy; however, understanding which tumor patients might benefit most from such therapy is crucial. An ideal target of such inhibitory therapy might be malignant mesothelioma (MM), a highly lethal, treatment-resistant malignancy of mesothelial cells of the pleura and peritoneum that produces large amounts of TGF-␤. The purpose of this study was to explore the possible therapeutic utility of TGF-␤ blockade on MM.Experimental Design: To evaluate this hypothesis, we tested the effects of a soluble TGF-␤ type II receptor (sTGF-␤R) that specifically inhibits TGF-␤1 and TGF-␤3 in three different murine MM tumor models, AB12 and AC29 (which produce large amounts of TGF-␤) and AB1 (which does not produce TGF-␤).Results: Tumor growth of both established AB12 and AC29 tumors was inhibited by sTGF-␤R. In contrast, AB1 tumors showed little response to sTGF-␤R. The mechanism of these antitumor effects was evaluated and determined to be primarily dependent on immune-mediated responses because (a) the antitumor effects were markedly diminished in severe combined immunodeficient mice or mice depleted of CD8 ؉ T cells and (b) CD8 ؉ T cells isolated from spleens of mice treated with sTGF-␤R showed strong antitumor cytolytic effects, whereas CD8 ؉ T cells isolated from spleens of tumor-bearing mice treated with of control IgG2a showed no antitumor cytolytic effects.Conclusions: Our data suggest that TGF-␤ blockade of established TGF-␤-secreting MM should be explored as a promising strategy to treat patients with MM and other tumors that produce TGF-␤.

In normal lung epithelial cells, cellular division is an ordered, tightly regulated process involving multiple checkpoints that assess extracellular growth signals, cell size, and DNA integrity. In contrast, neoplastic lung cells develop the ability to bypass several of these checkpoints, particularly at the G1/S and G2/M boundaries. We used genomic profiling to compare gene expression levels in early stage lung adenocarcinomas and non-neoplastic pulmonary tissue in order to comprehensively identify alterations in the process of cell cycling. RNA extracted from node negative, poorly differentiated lung adenocarcinomas (15 patients) and non-neoplastic pulmonary tissue (5 patients) was hybridized to oligonu-cleotide microarray filters containing 44,363 genes. Ontological classification was used to extract genes involved with cell cycle progression. Further analysis discovered a subset of differentially expressed genes for further study. Of the 624 cell cycle genes on the microarray filters, 40 genes were predicted to be differentially expressed in lung adeno-carcinomas. Alterations in several genes (i.e., cyclin B1, cyclin D1, p21, MDM2) are consistent with published data in the literature. We also identified 19 novel genes that have neither been described in non-small cell lung cancer (i.e., cdc2, cullin 4A, ZAC, p57, DP-1, GADD45, PISSLRE, cdc20) nor in any other tumors (i.e., cyclin F, cullin 5, p34). These results identified several potential cell cycle genes altered in lung cancer.