Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed αv integrin expression, about 10–1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (αvβ3 and αvβ5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.     

Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization

Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus- mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.     

Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies.

A major hurdle to adenovirus (Ad)-mediated gene transfer is that the target issue lacks sufficient levels of receptors to mediate vector attachment via its fiber coat protein. Endothelial and smooth muscle cells are primary targets in gene therapy approaches to prevent restenosis following angioplasty or to promote or inhibit angiogenesis. However, Ad poorly binds and transduces these cells because of their low or undetectable levels of functional Ad fiber receptor. The Ad-binding deficiency of these cells was overcome by targeting Ad binding to alpha v integrin receptors that are sufficiently expressed by these cells. In order to target alpha v integrins, a bispecific antibody (bsAb) that comprised a monoclonal Ab to the FLAG peptide epitope, DYKDDDDK, and a monoclonal Ab to alpha v integrins was constructed. In conjunction with the bsAb, a new vector, AdFLAG, which incorporated the FLAG peptide epitope into its penton base protein was constructed. Complexing AdFLAG with the bsAb increased the beta-glucuronidase transduction of human venule endothelial cells and human intestinal smooth muscle cells by seven- to ninefold compared with transduction by AdFLAG alone. The increased transduction efficiency was shown to occur through the specific interaction of the complex with alpha v integrins. These results demonstrate that bsAbs can be successfully used to target Ad to a specific cellular receptor and thereby increase the efficiency of gene transfer.     

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.     

Integrin alpha(v)beta1 is an adenovirus coreceptor

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.     

Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector

Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.     

Enhancement of adenovirus-mediated gene delivery to rheumatoid arthritis synoviocytes and synovium by fiber modifications: role of arginine-glycine-aspartic acid (RGD)- and non-RGD-binding integrins

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) do not express the coxsackie-adenovirus (Ad) receptor and are poorly permissive to Ad serotype 5 (Ad5). Genetically modified, coxsackie-Ad receptor-independent Ad5 vectors were studied for gene delivery in human RA FLS and synovium explants and murine collagen-induced arthritis. Short-fiber Ad5 vectors with seven fiber shaft repeats Ad5GFP-R7-knob, Ad5GFP-R7-arginine-glycine-aspartic acid (RGD) (RGD-liganded), and Ad5GFPDeltaknob (knob-deleted) were compared with Ad5GFP-FiWT, a conventional wild-type (WT) Ad5 vector. Gene transfer by Ad5GFP-R7-knob and Ad5GFP-R7-RGD was 40- to 50-fold and 25-fold higher, respectively, than Ad5GFP-FiWT in FLS. Ad5GFPDeltaknob was more efficacious than its knob-bearing version Ad5GFP-R7-knob in FLS transduction. Virus attachment and entry required RGD- and LDV-binding integrins including alpha(v), alpha(v)beta3, a(v)beta5, and beta1. Ad5GFP-R7-knob infection of FLS was partially neutralized by synovial fluid (SF), but remained 30- to 40-fold higher than Ad5GFP-FiWT in the presence of SF. Ad5GFPDeltaknob was partially neutralized by SF at low virus input, but escaped viral neutralization by SF at higher virus input. Gene transfer to human synovium ex vivo explants and murine collagen-induced arthritis in vivo was also more efficient with short fiber-modified vectors (with and without the knob domain) than Ad5GFPFiWT. Gene transfer by short fiber-modified vectors was enhanced by inflammatory cytokines in vitro and in the presence of inflammation in murine synovium in vivo. Our data indicated that the highly efficient gene delivery RA was mediated by RGD- and non-RGD-binding integrins and enhanced by inflammation. Short fiber modifications with knob ablation may be a strategy to enhance gene delivery, reducing vector dose and vector-induced inflammation and toxicity.     

Comparative analysis of adenovirus fiber-cell interaction: adenovirus type 2 (Ad2) and Ad9 utilize the same cellular fiber receptor but use different binding strategies for attachment.

We have analyzed the binding of adenovirus (Ad) serotypes from subgroups B, C, and D through fiber-virus and fiber-fiber cross-competition experiments. Since viruses in these distinct subgroups display markedly different tropisms, it was unexpected that the subgroup C viruses Ad2 and 5 and the subgroup D virus Ad9 cross-competed for the same cellular fiber receptor. The subgroup B serotype Ad3 recognized a receptor distinct from the Ad2, 5, and 9 fiber receptor. However, despite sharing the same fiber receptor, Ad2 and Ad9 displayed markedly different binding characteristics that appeared to result from direct Ad9 binding to cells via alpha(v)-integrins. Unlike Ad2, Ad9 binding to many cell lines was not abrogated by competition with the fiber 9 knob (F9K). Ad9 binding to fiber receptor-deficient cells was blocked by a monoclonal antibody to alpha(v)-integrins. In contrast, Ad9 binding to alpha(v)-deficient cells that express fiber receptor was blocked by F9K. Transfection of an alpha(v)-integrin-deficient cell line with a plasmid that expresses alpha(v)beta5 resulted in Ad9 binding that was not significantly blocked by F9K but was blocked with a combination of F9K and penton base. These results imply that the shorter length of fiber 9 (11 nm) relative to fiber 2 (37 nm) permits fiber-independent binding of Ad9 penton base to alpha(v)-integrins. The difference in fiber length may explain the different binding characteristics and tissue tropisms of each virus despite both utilizing the same fiber and penton base receptors.     

Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2.

The penton base gene from adenovirus type 12 (Ad12) was sequenced and encodes a 497-residue polypeptide, 74 residues shorter than the penton base from Ad2. The Ad2 and Ad12 proteins are highly conserved at the amino- and carboxy-terminal ends but diverge radically in the central region, where 63 residues are missing from the Ad12 sequence. Conserved within this variable region is the sequence Arg-Gly-Asp (RGD), which, in the Ad2 penton base, binds to integrins in the target cell membrane, enhancing the rate or the efficiency of infection. The Ad12 penton base was expressed in Escherichia coli, and the purified refolded protein assembled in vitro with Ad2 fibers. In contrast to the Ad2 penton base, the Ad12 protein failed to cause the rounding of adherent cells or to promote attachment of HeLa S3 suspension cells; however, A549 cells did attach to surfaces coated with either protein and pretreatment of the cells with an integrin alpha v beta 5 monoclonal antibody reduced attachment to background levels. Treatment of HeLa and A549 cells with integrin alpha v beta 3 or alpha v beta 5 monoclonal antibodies or with an RGD-containing fragment of the Ad2 penton base protein inhibited infection by Ad12 but had no effect on and in some cases enhanced infection by Ad2. Purified Ad2 fiber protein reduced the binding of radiolabeled Ad2 and Ad12 virions to HeLa and A549 cells nearly to background levels, but the concentrations of fiber that strongly inhibited infection by Ad2 only weakly inhibited Ad12 infection. These data suggest that alpha v-containing integrins alone may be sufficient to support infection by Ad12 and that this pathway is not efficiently used by Ad2.     

Pulmonary immunity to viral infection: adenovirus infection of lung dendritic cells renders T cells nonresponsive to interleukin-2

Adenovirus (Ad) infection has been identified as predisposing hosts to the development of pulmonary disease through unknown mechanisms. Lung dendritic cells (DCs) are vital for initiating pulmonary immune responses; however, the effects of Ad infection on primary lung DC have not been studied. In contrast to the effects on bone marrow- and monocyte-derived DCs, the current study shows that Ad infection of murine BALB/c lung DCs in vitro and in vivo suppresses DC-induced T-cell proliferation. The effect of Ad on DCs was not due to a downregulation of major histocompatibility complex or costimulatory molecules. Analysis of the production of interleukin-12 (IL-12), alpha interferon (IFN-alpha), and IFN-gamma by the Ad-infected DCs shows no significant differences over noninfected control lung DCs. Ad-induced suppression was not due to a deficiency of IL-2 or other DC-secreted factors and was dependent on viral protein synthesis, as UV irradiation of Ad abrogated the suppressive effect. Results suggest that Ad-infected DCs induce T cells to be nonresponsive to IL-2 during primary coculture, as the addition of IL-2 in secondary cultures recovered T-cell proliferation. In vivo studies supported in vitro results showing that Ad infection resulted in lung T cells with decreased proliferative ability. This study demonstrates that Ad infection induces local immunoincompetence by altering DC-T-cell interactions.     

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha‐amylase: a comparative study on bacterial,fungal, and mammalian enzymes

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha‐amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha‐amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha‐amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha‐amylase surface in domain B. This domain shows differences in various alpha‐amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.     

RNA-containing adenovirus/polyethylenimine transfer complexes effectively transduce dendritic cells and induce antigen-specific T cell responses

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells in initiating primary immune responses. Given the unique properties of DCs, gene-modified DCs represent a particularly attractive approach for immunotherapy of diseases such as cancer. METHODS: Gene-modified DCs were obtained by a receptor-mediated gene delivery system using adenovirus (Ad) particles as ligand and RNA or DNA condensed by polyethylenimine (PEI). In vitro transcribed polyadenylated or non-polyadenylated RNA was used. RNA-transduced DCs were generated expressing chicken ovalbumin (OVA) or chimeric constructs thereof, and compared with DNA-transduced DCs. RESULTS: Ad/PEI transfection complexes efficiently delivered RNA into DCs. Such RNA-transduced DCs induced OVA-specific T cell responses more effectively than DNA-transduced DCs. Furthermore, DCs transduced with polyadenylated RNA were more potent in stimulating CD4(+) and CD8(+) T cell responses than DCs transduced with non-polyadenylated RNA and this was particularly important for CD4(+) T cell responses. CONCLUSIONS: Ad/PEI/RNA transfection is an efficient means for generating RNA-transduced DCs and for stimulating antigen-specific T cell responses. Polyadenylation of RNA enhances CD8(+) T cell responses and is essential for CD4(+) T cell responses.     

Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.     

Incorporation of Porcine Adenovirus 4 Fiber Protein Enhances Infectivity of Adenovirus Vector on Dendritic Cells: Implications for Immune-Mediated Cancer Therapy

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.     

Fiber-knob modifications enhance adenoviral tropism and gene transfer in malignant glioma

BACKGROUND: Malignant gliomas remain refractory to Ad5-mediated gene therapy due to deficiency of the coxsackie adenovirus receptor on tumor cells. The purpose of this study was to evaluate whether changes in adenoviral tropism can enhance gene transfer in the context of malignant glioma. METHODS: We have identified several receptors that are over-expressed on tumor cells and created a series of pseudotyped Ad5 vectors that recognize these receptors: Ad5-RGD which binds alpha(v)beta3/alpha(v)beta5 integrins; Ad5/3 which contains adenovirus serotype 3 knob and binds to CD46; Ad5-Sigma which incorporates the reovirus sigma knob and binds to junctional adhesion molecule-1; and Ad5-pk7 which contains the polylysine motif and binds heparan sulfate proteoglycans. We also investigated the Ad5-CAV1 vector, which contains the knob of canine adenovirus type 1, a virus previously shown to infect glioma via an unknown mechanism. In this study, we compared these modified vectors for their ability to promote the expression of luciferase transgene both in vitro and in vivo. RESULTS: Our results indicate that all five modified vectors attained higher mean luciferase activity vs. control. Among them, Ad5-CAV1 and Ad5-pk7 attained the highest transduction efficiency independent of different tumor lines or infection time. Ad5-Sigma and Ad5-pk7 also demonstrated the least nonspecific infection in normal human astrocytes. Most importantly, Ad5-pk7 achieved 1000-fold increased transgene expression in human glioma xenografts in vivo. CONCLUSIONS: These results indicate that modifications of adenoviral tropism can enhance gene transfer in tumors that are poorly susceptible to adenoviral vectors and warrant further development of Ad5-pk7 for glioma gene therapy.     

A system for the propagation of adenoviral vectors with genetically modified receptor specificities.

The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalization mediated by alpha v integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing alpha v integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.     

Fiber-modified adenovirus vectors decrease liver toxicity through reduced IL-6 production

Adenovirus (Ad) vectors are one of the most commonly used viral vectors in gene therapy clinical trials. However, they elicit a robust innate immune response and inflammatory responses. Improvement of the therapeutic index of Ad vector gene therapy requires elucidation of the mechanism of Ad vector-induced inflammation and cytokine/chemokine production as well as development of the safer vector. In the present study, we found that the fiber-modified Ad vector containing poly-lysine peptides in the fiber knob showed much lower serum IL-6 and aspartate aminotransferase levels (as a maker of liver toxicity) than the conventional Ad vector after i.v. administration, although the modified Ad vector showed higher transgene production in the liver than the conventional Ad vector. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine, chemokine, and IFN expression. Splenic CD11c(+) cells were found to secret cytokines. The tissue distribution of Ad vector DNA showed that spleen distribution was much reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. Liver toxicity by the conventional Ad vector was reduced by anti-IL-6R Ab, suggesting that IL-6 signaling is involved in liver toxicity and that decreased liver toxicity of the modified Ad vector was due in part to the reduced IL-6 production. This study contributes to an understanding of the biological mechanism in innate immune host responses and liver toxicity toward systemically administered Ad vectors and will help in designing safer gene therapy methods that can reduce robust innate immunity and inflammatory responses.     

Combination of two fiber-mutant adenovirus vectors, one encoding the chemokine FKN and another encoding cytokine interleukin 12, elicits notably enhanced anti-tumor responses

For achieving optimal cancer immunotherapy, it is anticipated that both the activation and infiltration of immune cells into tumor are indispensable. In the present study, fiber-mutant adenovirus vectors (Ad) encoding chemokine FKN, (AdRGD-FKN), and cytokine interleukin 12, (AdRGD-IL-12), were constructed. The in vivo gene expression of AdRGD was confirmed and the combination of both FKN and IL-12 encoding Ad elicited synergistic anti-tumor activity in ovarian carcinoma, which induced tumor regression in all tumor-bearing mice, while using FKN alone did not show notable tumor-suppressive effect. The treatment with both IL-12 and FKN induced long-term specific immunity against OV-HM tumors in tumor-rejected mice. The results of immunohistochemical staining for CD3(+ )and perforin-positive cells suggested that the failure of using FKN alone was because of the inactivation of infiltrated immune cells. In contrast, cotransduction with IL-12 and FKN could induce more activated tumor-infiltrating immune cells than that transducted with FKN or IL-12 alone. The results indicated that using both chemokine and cytokine might be a powerful tool and a promising way for effective cancer immunotherapy.     

TNF receptor-associated factor-2 is involved in both IL-1 beta and TNF-alpha signaling cascades leading to NF-kappa B activation and IL-8 expression in human intestinal epithelial cells

Cytokine signaling involves the participation of many adaptor proteins, including the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF-alpha signal through both the I kappa B/NF-kappa B and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (SAPK) pathways. The physiological role of TRAF proteins in cytokine signaling in intestinal epithelial cells (IEC) is unknown. We characterized the effect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnTRAF-2) on the cytokine signaling cascade in several IEC and also investigated whether inhibiting the TRAF-2-transmitting signal blocked TNF-alpha-induced NF-kappa B and IL-8 gene expression. A high efficacy and level of Ad5dnTRAF-2 gene transfer were obtained in IEC using a multiplicity of infection of 50. Ad5dnTRAF-2 expression prevented TNF-alpha-induced, but not IL-1 beta-induced, I kappa B alpha degradation and NF-kappa B activation in NIH-3T3 and IEC-6 cells. TNF-alpha-induced JNK activation was also inhibited in Ad5dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF-alpha was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in control Ad5LacZ-infected, cells. Surprisingly, IL-1 beta-mediated IL-8 gene expression was also inhibited in HT-29 cells as measured by Northern blot and ELISA. We concluded that TRAF-2 is partially involved in TNF-alpha-mediated signaling through I kappa B/NF-kappa B in IEC. In addition, our data suggest that TRAF-2 is involved in IL-1 beta signaling in HT-29 cells. Manipulation of cytokine signaling pathways represents a new approach for inhibiting proinflammatory gene expression in IEC.     

Expression of alpha-gal epitopes on HeLa cells transduced with adenovirus containing alpha1,3galactosyltransferase cDNA

Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.