The antitumor effects of adenoviral-mediated, intratumoral delivery of interleukin 23 require endogenous IL-12.

Interleukin (IL)-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. We have demonstrated previously that adenoviral-mediated, intratumoral delivery of IL-23 (Ad.IL-23) was able to induce systemic antitumor immunity. Here we demonstrate that Ad.IL-23 requires endogenous IL-12 for conferring an antitumor effect after adenoviral-mediated, intratumoral delivery. In contrast, Ad.IL-12 does not require IL-23 for its antitumor effects although endogenous IL-23 appears important for induction of systemic antitumor immunity by IL-12. However, despite the requirement for endogenous IL-12, co-delivery of IL-23 and IL-12 does not provide even an additive local or systemic antitumor effect, regardless of the dose. We further demonstrate that although the use of a single-chain IL-23 (scIL-23) results in higher level of expression and a more pronounced IL-23-mediated antitumor effect, there is still no synergy with IL-12. These results demonstrate that although significant antitumor effects are achieved by intratumoral injection of adenovirus expressing either scIL-23 or IL-12 alone and that IL-23 requires endogenous IL-12 for maximum antitumor benefit, the combined use of these cytokines provides no additive or synergistic effect.

Balb/c EGFP mice are tolerant against immunization utilizing recombinant adenoviral-based vectors encoding EGFP: a novel model for the study of tolerance mechanisms and vaccine efficacy.

Enhanced green fluorescent protein (EGFP) is a marker gene product which is readily detectable using the techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. Previous studies have demonstrated the immunogenicity of EGFP in Balb/c mice, identifying an immunodominant H2-K(d) restricted CTL epitope. To model immunological tolerance and vaccine efficiency against self-antigens, we generated a stable transgenic BALB/c mouse expressing EGFP (Balb/c EGFP) through back-crossing C57Bl/6-TG(ACTbEGFP)10sb more than ten times with Balb/c wildtype (wt) mice. High level EGFP expression was detected in the skin and heart, whereas low level expression was observed in the kidney, liver, gut, lung, and spleen. To characterize the immune reactivity to self-antigen, we immunized Balb/c EGFP and Balb/c wt mice with recombinant adenoviral-based vectors encoding EGFP (Ad-EGFP) or beta-galactosidase (Ad-betagal) as a control. Immunization utilizing the Ad-betagal vector expressing 'foreign' antigen induced robust humoral and cellular transgene-specific immunity, whereas Balb/c EGFP mice presented no reactivity following Ad-EGFP immunization against the 'self-antigen' EGFP. These findings describe the creation of a transgenic mouse line tolerant against the common protein marker EGFP, providing a novel system for the evaluation of methods of tolerance disruption and vaccine efficacy.

Ex vivo-transduced autologous skin fibroblasts expressing human Lim mineralization protein-3 efficiently form new bone in animal models.

Local gene transfer of the human Lim mineralization protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. To develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP-3 and seeded on a hydroxyapatite/collagen matrix prior to autologous implantation. Here, we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing, as determined by X-rays, histology and three-dimensional microcomputed tomography (3DmuCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3 in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.

IL-12p70 and IL-18 gene-modified dendritic cells loaded with tumor antigen-derived peptides or recombinant protein effectively stimulate specific Type-1 CD4+ T-cell responses from normal donors and melanoma patients in vitro.

Although CD4(+) Type-1T helper (Th1) cells secreting interferon-gamma (IFN-gamma) appear to play an essential role in promoting durable antitumor immunity, we have previously shown that patients with cancer exhibit dysfunctional Th1-type responses against epitopes derived from tumor antigens, such as MAGE-A6. Here, we engineered human dendritic cells (DCs) to secrete high levels of the IFN-gamma-inducing cytokines, interleukin (IL)-12p70 and IL-18, via recombinant adenoviral infection to generate an in vitro stimulus capable of promoting previously deficient patient Th1-type responses. Dendritic cells co-infected with Ad.IL-12 and Ad.IL-18 (DC.IL-12/18) were more effective at stimulating MAGE-A6-specific Th1-type CD4(+) T-cell responses than DCs infected with either of the cytokine vectors alone, control Ad.Psi5 virus or uninfected DCs. Furthermore, we show that DC.IL-12/18 loaded with recombinant MAGE-A6 protein (rMAGE) and used as in vitro stimulators promote Th1-type immunity that is frequently directed against multiple MAGE-A6-derived epitopes. The superiority of DC.IL-12/18-based stimulations in melanoma patients was independent of disease stage or current disease status. Based on these results, we believe this modality may prove clinically useful as a vaccine platform to promote the recovery of tumor antigen-specific, Th1-type CD4(+) T-cell responses in patients with cancer.

Sequential delivery of interferon-alpha gene and DCs to intracranial gliomas promotes an effective antitumor response.

Effective presentation of tumor antigens by dendritic cells (DCs) is considered to be essential for the induction of antitumor T-cell responses. Apoptotic and necrotic tumors have been noted to be a robust antigen source for DCs. Because glioma cells undergo apoptosis after transfection with the type I interferon (IFN) gene and type I IFNs promote the stimulatory activity of DCs, we hypothesized that transfection of glioma cells with type I IFN genes and provision of DCs would promote particularly effective antitumor activity by both facilitating apoptosis of glioma cells and the presentation of the glioma antigens, thereby inducing specific immune responses against glioma cells. We have previously reported the proof of this hypothesis in vitro and in a subcutaneous tumor model. Here we report an extension of this approach in intracranial (i.c.) gliomas using adenoviral IFN-alpha (Ad-IFN-alpha) vector. Mice bearing day-5 i.c. GL261 glioma received sequential intratumoral (i.t.) delivery of Ad-IFN-alpha and bone marrow-derived syngeneic DCs. This treatment prolonged survival in that nine of 17 animals survived long term (> 60 days versus 0 of 10 control animals). Specific CTL activity was demonstrated following this regimen in the cervical lymph nodes, and the therapeutic efficacy was dependent upon CD8+ cells. Furthermore, these animals were protected against subsequent re-challenge with GL261 gliomas. DCs injected i.t. survived in the tumor and migrated into cervical lymph node. In vitro migration assays revealed the ability of DCs to migrate toward the tumor, suggesting that i.t. injected DCs migrate through the glioma. Taken together, this combination of gene therapy and cellular immunotherapy may be an effective future strategy for treating human gliomas.

Efficient bone formation by gene transfer of human LIM mineralization protein-3.

LIM mineralization protein (LMP) is a novel positive regulator of the osteoblast differentiation program. In humans, three different LMP splice variants have been identified: LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 (hLMP-1) induces expression of genes involved in bone formation, including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro, ectopic bone formation in vivo, and is therapeutic in animal models of posterior thoracic and lumbar spine fusion. To examine the osteoinductive properties of the LMP-3 in vitro and in vivo, we have generated plasmid and adenoviral vectors expressing codon-optimized hLMP-3. Here we demonstrate that gene transfer of hLMP-3 induces expression of the bone-specific genes osteocalcin, osteopontin, and bone sialoprotein and induced bone mineralization in preosteoblastic and fibroblastic cells. We also demonstrate that hLMP-3 is able to induce bone mineralization and the expression of the bone-specific genes, BMP-2, OSX, RunX2, and alkaline phosphatase in human mesenchymal stem cells in a dose-dependent manner. Finally, we demonstrate that direct gene transfer of hLMP-3 into murine skeletal muscle results in ectopic bone formation more efficiently than BMP-2. These results demonstrate that hLMP-3 gene transfer can be used to promote bone formation in cell culture and in vivo as or more efficiently than BMP-2, thus establishing feasibility and efficacy of direct gene delivery of hLMP-3 to produce bone in vivo. These results suggest that gene transfer of hLMP-3 could be developed as a bone-inductive therapeutic agent for clinical applications.

Human adenovirus type 35: nucleotide sequence and vector development.

In this report, we describe the complete 34,794 base pair genomic sequence of the human adenovirus serotype 35 (Ad35) Holden strain. The viral genome exhibits a compact organization similar to other adenoviral serotypes, with overlapping genes on both strands. In all, 47 open reading frames (ORFs) were identified, including early (E1, 2, 3, 4) and late (L1, 2, 3, 4, 5) regions conserved among the adenoviridae family. In addition, 14 ORFs were identified that do not encode known adenoviral genes. Comparison of the predicted translational products of the conserved genes with those of other adenoviruses revealed that Ad35 has high homology to Ad7, Ad3, Ad21, Ad17, and simian Ads25. Based on the complete Ad35 DNA sequence, E3-, E1-, and E1/E3-deleted Ad35-based vector systems were developed. An HEK293-derived cell line was established for the propagation of the E1-deleted Ad35 vector, avoiding the emergence of replication-competent adenovirus. Moreover, production of the E1-deleted recombinant Ad35 vector was achieved by transient transduction of a plasmid encoding the Ad35 E1B gene in HEK293 cells. Testing showed that the Ad35-based vector efficiently infects both human and rhesus macaque dendritic cells. Our novel Ad35-based vectors and their corresponding packaging cell lines will provide a versatile and powerful system for DNA-based vaccine development and gene therapy applications.

Intra-articular adenoviral-mediated gene transfer of trail induces apoptosis of arthritic rabbit synovium.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that primarily affects joints. In rheumatoid joints there is extensive synovial proliferation with diseased synovium becoming highly aggressive, attaching to the articular cartilage and bone to form what is termed a pannus. The formation of active pannus is central to erosive disease and resulting joint destruction. In this study, we examined the ability to eliminate the hyperplastic synovium by adenoviral-mediated gene transfer of human TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family that is able to induce apoptosis through interaction with receptors containing death domains, DR4 and DR5. Infection of synovial cells derived from RA patients with Ad.TRAIL resulted in significant apoptosis in three out of five lines. Moreover, primary rabbit synovial fibroblasts were also sensitive to Ad.TRAIL-mediated gene transfer. In a rabbit model of arthritis, intra-articular gene transfer of TRAIL induced apoptosis in cells within the synovial lining, reduced leukocytic infiltration and stimulated new matrix synthesis by cartilage. These results demonstrate that TRAIL can affect the viability of the cells populating the activated synovium in arthritic joints and suggest that the delivery of TRAIL to arthritic joints may represent a non-invasive mechanism for inducing pannus regression.

Genetic immunization with a melanocytic self-antigen linked to foreign helper sequences breaks tolerance and induces autoimmunity and tumor immunity.

Mechanisms maintaining peripheral tolerance to self-antigens present a major obstacle for the development of antigen-specific melanoma vaccines, presumably because self-antigens are not able to stimulate a CD4 T-helper response. Using the melanosomal enzyme tyrosinase-related protein 2 (TRP2) expressed by melanocytes and most melanoma cells as a model self-antigen, we investigated whether linkage with a foreign immunogenic protein providing strong CD4 helper sequences would be able to circumvent tolerance and enhance the induction of antigen-specific tumor immunity. We found that genetic immunization of mice with cDNA encoding a fusion protein between enhanced green fluorescent protein (EGFP) from jellyfish and autologous murine TRP2 (EGFP.mTRP2) resulted in the stimulation of TRP2-reactive T cells in vivo. Importantly, immunization with EGFP.mTRP2 effectively protected mice against metastatic growth of B16 melanoma in the lungs and was associated with fur depigmentation as a sign of autoimmune-mediated destruction of melanocytes. Our results show that tumor vaccines consisting of self-antigens linked to immunogenic helper sequences can be successfully applied to the immunotherapy of melanoma and provide a scientific basis for the translation of this strategy in future clinical investigations.

Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.

Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.

We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.

A role for mitochondrial Bak in apoptotic response to anticancer drugs.

In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells.

Local high-capacity adenovirus-mediated mCTLA4Ig and mCD40Ig expression prolongs recombinant gene expression in skeletal muscle.

Multiple forms of muscular dystrophy are due to the absence of cytoskeletal muscle proteins that normally protect the integrity of muscle cells. The lack of any adequate treatments for these devastating diseases propels research toward the development of strategies for gene delivery to skeletal muscle. High-capacity adenoviral vectors (HC-AdV) devoid of all viral coding sequences have been developed to avoid expression of viral proteins by the gene therapy vector. However, the capsid proteins that are an essential component of the input viral vector and any residual helper virus in the vector preparation could induce an immune response. Furthermore, the therapeutic protein provided by a gene transfer vector presents the potential to induce an immune response in a patient who does not express a normal cellular protein due to genetic mutation. Therefore, we hypothesize that some immune suppression will be required with therapeutic gene delivery designed for the treatment of patients with inherited muscle diseases. In this study, we constructed and rescued three HC-AdVs expressing murine CTLA4Ig, murine CD40Ig, or both. The backbone vector without a gene insert was rescued as a negative control vector. The production of relevant proteins from each vector was determined in vitro. In vivo function of each of the immunosuppressant vectors was assayed by co-injection with an enhanced green fluorescent protein (EGFP)-expressing first-generation adenoviral vector (AdEGFP) into the tibialis anterior muscle of C57BL/10 mice. Higher levels of muscle EGFP expression were observed in animals receiving an immunosuppressant vector. Furthermore, the production of total anti-AdV and anti-EGFP antibodies was reduced in mice treated with each of the three immunosuppressant vectors. A second intramuscular administration of AdEGFP alone 4 weeks after the initial co-injection was successful in all immunosuppressant vector-treated groups, but not in the negative control vector-treated group. All groups had a high antibody response to adenoviral proteins after the second injection of AdEGFP alone, indicating that the initial co-injection did not tolerize against vector capsid antigens.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

Dendritic cells acquire antigens from live cells for cross-presentation to CTL.

Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 microm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.

Adenovirus-mediated gene therapy to liver grafts: successful gene transfer by donor pretreatment.

BACKGROUND: We have previously shown excellent adenoviral (Ad) gene transfection to transplanted liver grafts with the clamp technique (CT) where viral vector was delivered ex vivo and trapped in cold preserved liver grafts. In this study, we adopted a new gene therapy approach to achieve early transgene expression by donor pretreatment with viral vector and compared the efficacy of these two methods by using Ad vector encoding enhanced green fluorescent protein (AdEGFP) marker gene. METHODS: AdEGFP (1 x 10(9)plaque forming units) was delivered to the liver grafts by: (1) single intravenous injection to donor Lewis rats 48 hours before harvesting, (2) ex vivo cold infusion into the harvested liver with CT, or (3) a combination of both methods. Liver grafts were stored in University of Wisconsin solution at 4 degrees C for 18 hours and then orthotopically transplanted into syngeneic recipients, and the expression of EGFP was studied. RESULTS: With intravenous pretreatment of donor liver grafts, EGFP-expressing cells were detected as early as 3 hours after transplant, and moderate expression was seen by 12 hours. In contrast, EGFP was not detected until 12 to 24 hours after transplant with CT. High levels of EGFP-producing cells were seen with each technique at 7 days ( approximately 30% transfection efficiency). A combination of both methods did not enhance infectivity. Liver preservation injury was comparable between groups. CONCLUSIONS: Gene transfer by donor pretreatment with AdEGFP induces early and efficient gene transduction to liver grafts compared with back-table delivery with CT. This method is simple and provides early transgene expression in liver grafts that potentially could be used to deliver genes to decrease preservation injury or rejection.

Transduction of dendritic cells with Bcl-xL increases their resistance to prostate cancer-induced apoptosis and antitumor effect in mice.

We have shown that prostate cancer (PCa) causes apoptosis of dendritic cells (DC), which might block the development of specific antitumor immune responses. Analysis of murine prostatic carcinoma tissues revealed the significant decrease in intratumoral DC number during tumor progression. We demonstrated that the cytokine-mediated increase in DC survival was accompanied by an elevated expression of the anti-apoptotic protein Bcl-xL. Next, we evaluated the resistance to tumor-induced apoptosis and the antitumor efficiency of genetically engineered DC overexpressing Bcl-xL. DC were transduced with an adenoviral vector encoding the murine Bcl-xL gene and injected intratumorally. Data analysis revealed that treatment of PCa-bearing mice with Bcl-xL-transduced DC resulted in significant inhibition of tumor growth compared with the administration of nontransduced DC. Thus, our data suggest that the protection of DC from PCa-induced apoptosis might significantly increase the efficacy of DC-based therapies in cancer even in the absence of available tumor-specific Ags.

Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I.

Interleukin-1beta is a potent pro-inflammatory cytokine that has been shown to inhibit islet beta cell function as well as to activate Fas-mediated apoptosis in a nitric oxide-dependent manner. Furthermore, this cytokine is effective in recruiting lymphocytes that mediate beta cell destruction in IDDM onset. The insulin-like growth factor I (IGF-I) has been shown to block IL-1beta actions in vitro. We hypothesized that gene transfer of the insulin-like growth factor I to intact human islets could prevent IL-1beta-induced beta cell dysfunction and sensitization to Fas-triggered apoptosis activation. Intact human islets were infected with adenoviral vectors encoding IGF-I as well as beta-galactosidase and enhanced green fluorescent protein as controls. Adenoviral gene transfer of human IGF-I prevented IL-1beta-mediated nitric oxide production from human islets in vitro as well as the suppression of beta cell function as determined by glucose-stimulated insulin production. Moreover, IGF-I gene transfer prevented IL-1beta-induced, Fas-mediated apoptosis. These results suggest that locally produced IGF-I from cultured islets may be beneficial in maintaining beta cell function and promoting islet survival before and following islet transplantation as a potential therapy for type I diabetes.