Gene therapy with an improved doxycycline-regulated plasmid encoding a tumour necrosis factor-alpha inhibitor in experimental arthritis

Inhibition of tumour necrosis factor (TNF)-alpha with biological molecules has proven an effective treatment for rheumatoid arthritis, achieving a 20% improvement in American College of Rheumatology score in up to 65% of patients. The main drawback to these and many other biological treatments has been their expense, which has precluded their widespread application. Biological molecules could alternatively be delivered by gene therapy as the encoding DNA. We have developed novel plasmid vectors termed pGTLMIK and pGTTMIK, from which luciferase and a dimeric TNF receptor II (dTNFR) are respectively expressed in a doxycycline (Dox)-regulated manner. Regulated expression of luciferase from the self-contained plasmid pGTLMIK was examined in vitro in a variety of cell lines and in vivo following intramuscular delivery with electroporation in DBA/1 mice. Dox-regulated expression of luciferase from pGTLMIK of approximately 1,000-fold was demonstrated in vitro, and efficient regulation was observed in vivo. The vector pGTTMIK encoding dTNFR was delivered by the same route with and without administration of Dox to mice with collagen-induced arthritis. When pGTTMIK was delivered after the onset of arthritis, progression of the disease in terms of both paw thickness and clinical score was inhibited when Dox was also administered. Vectors with similar regulation characteristics may be suitable for clinical application

A novel hybrid promoter responsive to pathophysiological and pharmacological regulation

The aim of this study was to construct a promoter containing DNA motifs for an endogenous transcription factor associated with inflammation along with motifs for pharmacological regulation factors. We demonstrate in transfected cells that expression of a gene of interest is induced by hypoxic conditions or through pharmacological induction, and also show pharmacological repression. In vivo studies utilised electroporation of plasmid to mouse paws, a delivery method shown to be effective by bioluminescence imaging. For gene therapy, the promoter was used to drive expression of IL-1Ra in a paw inflammation model with therapeutic effect observed which was further enhanced when the promoter was additionally induced with a pharmacological activator. One of the most important observations from this study was that promoter induction by hypoxia or inflammation could be prevented by the pharmacological repressor in the absence of doxycycline. These studies demonstrate that hybrid promoters enable pharmacological adjustment to the pathophysiological level of gene expression and, importantly, that they allow termination of gene expression even in the presence of pathophysiological stimuli

Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide:establishing a reversible immune evasion mechanism in human cancer

Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand–induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell–inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer

Expression of dimeric tumour necrosis factor receptor II (dTNFR) from pGTTMIK and gene therapy application in the collagen-induced arthritis (CIA) model

<p><b>Copyright information:</b></p><p>Taken from "Gene therapy with an improved doxycycline-regulated plasmid encoding a tumour necrosis factor-alpha inhibitor in experimental arthritis"</p><p>http://arthritis-research.com/content/9/1/R7</p><p>Arthritis Research & Therapy 2007;9(1):R7-R7.</p><p>Published online 25 Jan 2007</p><p>PMCID:PMC1860065.</p><p></p> Cos 7 cells were transiently transfected with 2 μg of pGTTMIK by the calcium phosphate method. Expression of dTNFR was induced for 24 or 48 hours with doxycycline (Dox) (1 μg/ml). Values are the mean of triplicate transfections, and induction values above the black bars allow for the endogenous production of TNFR II from non-transfected (NT) cells indicated by the dotted line. Inhibition of CIA in mice treated with pGTTMIK and administered Dox. Progression of CIA both in terms of clinical score (I) and hind paw swelling (II) was inhibited by delivery of pGTTMIK (50 μg) intramuscularly with electroporation on day 28 after immunisation in mice with early disease (clinical score of less than 4). Inhibition was evident in those mice that received Dox in sweetened drinking water. Values are the mean, and vertical lines represent the standard error of the mean. Significant difference of < 0.05 and < 0.02 between the GTTMIK groups given Dox-containing and Dox-free sweetened drinking water are indicated by * and **, respectively

The Inflammatory Cytokine Tumor Necrosis Factor-Alpha Generates An Autocrine Tumor-Promoting Network In Epithelial Ovarian Cancer Cells

Constitutive expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-alpha generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNT-a., constitutive production of TNF-alpha was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-alpha production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-alpha in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-alpha was achieved. Tumors derived from TNF-alpha knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-alpha knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-alpha by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits.Wo

Regulated luciferase expression from self-contained vectors in transfected cells

<p><b>Copyright information:</b></p><p>Taken from "Gene therapy with an improved doxycycline-regulated plasmid encoding a tumour necrosis factor-alpha inhibitor in experimental arthritis"</p><p>http://arthritis-research.com/content/9/1/R7</p><p>Arthritis Research & Therapy 2007;9(1):R7-R7.</p><p>Published online 25 Jan 2007</p><p>PMCID:PMC1860065.</p><p></p> Comparison of regulated luciferase expression from pGTRTL (2 μg) (white squares) and pGTLMIK (2 μg) (black squares) in transiently transfected DBA/1 embryonic fibroblasts with the temperature-sensitive large T antigen (DTF) cells (2 × 10cells per well). Cells were grown in doxycycline (Dox)-free or Dox-containing media (concentration range of 1 pg/ml to 1 μg/ml). After 24 hours, luciferase activity was measured in cell lysates. Fold induction of luciferase expression from pGTRTL (white bars) and pGTLMIK in response to Dox was determined in DTF, A431, and 293T transiently transfected cells. Cells were transfected as in , and fold induction was calculated by dividing the Dox-induced value by the non-induced value for each transfection. Regulated expression of luciferase from pGTLMIK (1 μg) transfected into the mouse myoblast cell line C2C12 (1 × 10cells) using the amaxa system. Transfected cells were split between the wells of a six-well plate and were either non-induced or induced with Dox (1 μg/ml) for 24 hours. In all panels, luciferase measurements were standardised for protein content and each mean value or calculated value was obtained from triplicate transfections. Vertical lines in and represent standard error of the mean. RLU, relative light units

Activated pancreatic stellate cells sequester CD8+ T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. METHOD: We used tissue microarray analysis to compare immune cell infiltration of different pancreatico-biliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis), and juxtatumoral stromal (<100 μm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice, and the effects of all-trans retinoic acid (ATRA) on these processes. RESULTS: Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase(+) and CD68(+) cells, compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8(+), FoxP3(+), CD56(+), or CD20(+) cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8(+) T-cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8(+) T-cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8(+) T-cells from PDAC patients had increased chemotaxis towards activated PSCs, which secrete CXCL12, compared with quiescent PSC or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA. CONCLUSION: Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8(+) T-cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective anti-tumor immune response

Follicular lymphoma cells induce T-cell immunologic synapse dysfunction that can be repaired with lenalidomide: implications for the tumor microenvironment and immunotherapy

An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma