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R C Hoeben

Publications and source records attributed to R C Hoeben.

At least 19 recordsLinked to original sources
PubMed2003

Association of the -2849 interleukin-10 promoter polymorphism with autoantibody production and joint destruction in rheumatoid arthritis.

OBJECTIVE: To analyze the -2849 A/G interleukin-10 (IL-10) promoter polymorphism, which is associated with high (AG/GG) and low (AA) IL-10 production, in a cohort of rheumatoid arthritis (RA) patients and controls in order to gain a better understanding of its role in the incidence and progression of RA. METHODS: Allele frequencies of the promoter polymorphism -2849 A/G and carriage rates were compared in 283 RA patients, 413 patients with other rheumatic diseases, and 1,220 healthy controls. The rate of joint damage and baseline levels of IgG and IgM rheumatoid factors and anti-citrullinated peptide antibodies were measured and were correlated with the IL-10 gene polymorphism. Furthermore, the correlation between the invasiveness of fibroblast-like synoviocytes (FLS) and the -2849 IL-10 genotype was tested. RESULTS: The IL-10 genotype was not associated with the incidence of RA, but instead, correlated with disease progression, as determined by the extent of joint destruction. A higher rate of joint destruction was observed in patients with the genotype associated with high IL-10 production. Since FLS are thought to be involved in joint destruction, we analyzed IL-10 genotypes in conjunction with FLS invasiveness. Although adenoviral gene transfer of IL-10 to FLS inhibited their invasiveness, no differences were observed in vitro in the FLS from RA patients who were -2849 non-G carriers compared with those who were G carriers. Instead, patients with the -2849 AG/GG genotype, which is associated with high IL-10 production, had higher autoantibody titers at baseline. CONCLUSION: The -2849 IL-10 promoter polymorphism is associated with autoantibody production and subsequent joint damage in RA.

Adult↗
PubMed2003

The protective effect of cardiac gene transfer of CuZn-sod in comparison with the cardioprotector monohydroxyethylrutoside against doxorubicin-induced cardiotoxicity in cultured cells.

Doxorubicin-induced cardiotoxicity is related to its production of free radicals that specifically affect heart tissue because of its low antioxidant status. Monohydroxyethylrutoside (monoHER), a potent antioxidant flavonoid, is under development as a protector against doxorubicin-induced cardiotoxicity. The overexpression of high levels of superoxide dismutase (sod) protects against free radical damage in transgenic mice. Seeking alternatives besides the few cardioprotectors that are presently under investigation, the aim of the present study was to investigate the protective effect of cardiac gene transfer of CuZn-sod compared with that of the presently most promising cardioprotector monoHER against doxorubicin-induced cardiotoxic effects on neonatal rat cardiac myocytes (NeRCaMs) in vitro. NeRCaMs were infected with different multiplicity of infections (MOIs) of adenovirus encoding CuZn-sod (AdCuZn-sod). A control infection with an adenovirus vector encoding a nonrelated protein was included. The overexpression of CuZn-sod was characterized within 3 days postinfection. For doxorubicin treatment, NeRCaMs were divided into three groups. The first group was infected with AdCuZn-sod before treatment with doxorubicin (0-50 microM). The second and third groups were treated with doxorubicin (0-50 microM) alone and with 1 mM monoHER, respectively. The LDH release and survival of treated cells were measured 24 and 48 hours after doxorubicin treatment. The beating rate was followed during the 3 days after doxorubicin (0-100 microM) treatment. At the third day after infection with an MOI of 25 plaque-forming unit (PFU) of AdCuZn-sod/cell, the activity of CuZn-sod significantly increased (five-fold, P=.029). Higher MOI produced cytopathic effects (CPEs). Doxorubicin alone produced significant concentration- and time-dependent reduction in NeRCaMs beating rate and survival (P < .0005). Doxorubicin (> or =50 microM)-treated cells ceased to beat after 24 hours. This cytotoxicity was associated with an increase in the LDH release from the treated cells (P <.0005). The five-fold increase in the activity of CuZn-sod did not protect against any of the cytotoxic effects of doxorubicin on NeRCaMs. In contrast, monoHER (1 mM) protected against the lethal effects of doxorubicin on the survival, LDH release and the beating rate of NeRCaMs (P <.004) during 48 hours after doxorubicin treatment. Doxorubicin-treated (< or =100 microM) cells continued beating for >72 hours in the presence of monoHER. The present study showed the lack of adenoviral CuZn-sod gene-transfer to protect myocardiocytes against doxorubicin-induced toxicity and confirms the efficacy of monoHER cardioprotection. Thus, a gene-therapy strategy involving overexpression of CuZn-sod to protect against doxorubicin-induced cardiotoxicity is not feasible with the currently available adenovirus vectors.

Adenoviridae↗
PubMed2001

Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells.

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.

Adenoviridae↗
PubMed2001

Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer.

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.

Adenoviridae↗
PubMed2000

Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells.

Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (Hyg(R)). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in Hyg(R) colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.

Bacteriophage mu↗
PubMed2000

Adenovirus E1A does not induce the Ewing tumor-associated gene fusion EWS-FLI1.

Rearrangement of the EWS gene with FLI1 is thought to occur early in the pathogenesis of Ewing's sarcoma family tumors (EFTs) because the chromosomal aberration is pathognomonic for this disease. Recently, adenovirus (Ad) 5 E1A protein has been reported to induce this gene rearrangement in a variety of cell types. This finding, if generally substantiated, not only suggests an etiological role for viral agents in the generation of oncogenic chromosomal aberrations but would also significantly impact the use of adenoviral vectors for gene therapy. In contrast, we now report on the absence of EWS-FLI1 chimeric products from short- and long-term cultures of stably Ad-transformed cells lines and from transiently E1A-expressing cell lines. In addition, we demonstrate the absence of E1A from EFTs. We conclude that there is no role for Ads in EFT pathogenesis. Consequently, evidence for a viral genesis of tumor-specific gene rearrangements is not available.

Adenovirus E1A Proteins↗
PubMed2000

Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells.

The host-immune response against adenoviruses forms a major obstacle for their use as gene therapy vectors for treatment of genetic defects. None the less, they are the preferred vectors for in vivo gene transfer in experimental gene therapy protocols for cancer. In this article we demonstrate the antitumor efficacy of adenovirus-mediated transfer of human interleukin-2 cDNA in the rat-CC531 model for hepatic metastases of colorectal cancer: intratumoral administration of 10 plaque-forming units of the hlL-2-expressing adenoviral vector, AdCAIL-2, resulted in a cessation of tumor growth in 80% of the injected tumors. In control groups receiving AdCnull, a vector with the same viral backbone, but lacking transgene expression, none of the tumors responded. However, intratumoral treatment with this vector significantly enhanced tumor regression induced by systemic IL-2 protein treatment, which was used as a positive control. In addition we show, by performing delayed-type of hypersensitivity assays, that AdCnull when injected intratumorally enhances recognition of tumor antigens by T lymphocytes to the same extent as intratumoral treatment with the IL-2-expressing vector. The replication-deficient adenoviruses appear to have a therapeutic advantage in cytokine-mediated immunotherapy: even adenovirus vectors that do not express a transgene, show adjuvant activity and stimulate an antitumor immune response.

Adenoviridae↗
PubMed2000

Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield.

In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 +/- 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4 precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.

Adenoviridae↗
PubMed2000

Apolipoprotein E2 (Lys146-->Gln) causes hypertriglyceridemia due to an apolipoprotein E variant-specific inhibition of lipolysis of very low density lipoproteins-triglycerides.

The apolipoprotein E2 (Lys146-->Gln) variant is associated with a dominant form of familial dysbetalipoproteinemia. Heterozygous carriers of this variant have elevated levels of plasma triglycerides, cholesterol, and apolipoprotein E (apoE). It was hypothesized that the high amounts of triglycerides in the very low density lipoprotein (VLDL) fraction are due to a disturbed lipolysis of VLDL. To test this hypothesis, apoE knockout mice were injected with an adenovirus containing the human APOE*2 (Lys146-->Gln) gene, Ad-E2(146), under the control of the cytomegalovirus promoter. ApoE knockout mice injected with an adenovirus vector encoding human apoE3 (Ad-E3) were used as controls. Five days after adenovirus injection, plasma cholesterol levels of mice injected with a high dose of Ad-E2(146) (2x10(9) plaque-forming units) were not changed compared with preinjection levels, whereas in the group who received a low dose of Ad-E2(146) (5x10(8) plaque-forming units) and in the groups injected with a low or a high dose of Ad-E3, plasma cholesterol levels were decreased 5-, 6-, and 12-fold, respectively. Plasma triglycerides were not affected in mice injected with Ad-E3. In contrast, a 7-fold increase in plasma triglycerides was observed in mice injected with the low dose of Ad-E2(146) compared with mice injected with Ad-E3. Injection with the high dose of Ad-E2(146) resulted in a dramatic increase of plasma triglycerides (50-fold compared with Ad-E3 injection). In vitro lipolysis experiments showed that the lipolysis rate of VLDLs containing normal amounts of apoE2 (Lys146-->Gln) was decreased by 54% compared with that of VLDLs containing comparable amounts of apoE3. The in vivo VLDL-triglyceride production rate of Ad-E2(146)-injected mice was not significantly different from that of Ad-E3-injected mice. These results demonstrate that expression of apoE2 (Lys146-->Gln) causes hypertriglyceridemia due to an apoE variant-specific inhibition of the hydrolysis of VLDL-triglycerides.

Adenoviridae↗
PubMed1999

Specific tumor-cell killing with adenovirus vectors containing the apoptin gene.

Specificity is an essential prerequisite for cancer gene therapy. Recently we described that apoptin, a protein of 121 amino acids which is derived from the chicken anemia virus, induces programmed cell death or apoptosis in transformed and malignant cells, but not in normal, diploid cells (Danen-van Oorschot AAAM et al, Proc Natl Acad Sci USA 1997; 94: 5843-5847). This protein has an intrinsic specificity that allows it to selectively kill tumor cells, irrespective of the p53 or Bcl-2 status of these cells. Hence, it is attractive to explore the use of the apoptin gene for therapeutic applications, viz cancer gene therapy. In this paper, we describe the generation and characterization of an adenovirus vector, AdMLPvp3, for the expression of apoptin. Despite the fact that apoptin ultimately induces apoptosis in the helper cells, which are transformed by the adenovirus type 5 early region 1 (E1), the propagation kinetics and yields of AdMLPvp3 are similar to those of control vectors. Infection with AdMLPvp3 of normal rat hepatocytes in cell culture did not increase the frequency of apoptosis. In contrast, in the hepatoma cell lines HepG2 and Hep3b, infection with AdMLPvp3, but not with control vectors, led to a rapid induction of programmed cell death. Experiments in rats demonstrated that AdMLPvp3 could be safely administered by intraperitoneal, subcutaneous or intravenous injection. Repeated intravenous doses of AdMLPvp3 were also well tolerated, indicating that the apoptin-expressing virus can be administered without severe adverse effects. In a preliminary experiment, a single intratumoral injection of AdMLPvp3 into a xenogeneic tumor (HepG2 cells in Balb/Cnu/nu mice) resulted in a significant reduction of tumor growth. Taken together, our data demonstrate that adenovirus vectors for the expression of the apoptin gene may constitute a powerful tool for the treatment of solid tumors.

Adenoviridae↗
PubMed1999

Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation.

Recombinant adenovirus vectors are popular tools for gene transfer and gene therapy. However biosafety constraints require that all handling of the vectors and vector-containing samples is restricted to dedicated containment laboratories, unless they had undergone a validated virus-inactivation procedure, which decontaminates the samples from any active virus. In this study we evaluated the feasibility of photodynamic treatment (PDT) with visible light to inactivate recombinant adenovirus vectors in biological samples, with minimum associated effects on other biological activities. Several photosensitizers were tested for their capacity to inactivate a model human adenovirus vector, AdCMVLuc, upon illumination. Four photosensitizers (methylene blue (MB), rose bengal (RB), uroporphyrin (UP) and aluminum phthalocynine tetrasulphonate (AIPcS4)) could inactivate the adenovirus, as measured by expression of the luciferase reporter gene and by plaque assay. Of these, MB demonstrated to be the most effective sensitizer in phosphate-buffered saline (PBS), giving > 7 log10 inactivation of the adenovirus. DNA isolated from MB- and light-treated virions was inefficient as a template for transcription. Furthermore, Southern blot analysis revealed fragmentation of the viral DNA. Based on its preference for DNA, MB is suited for adenovirus inactivation in blood plasma. Spiking experiments in which AdCMVLuc was added to plasma samples demonstrated a reduction (> 4 log10-fold) of reporter gene expression to almost background levels. In contrast to MB, photodynamic treatment with RB, UP or AIPcS4 did not lead to DNA damage. Although alterations of the viral capsid could not be detected, the binding pattern of the particles to target cells was significantly changed. Taken together, our data demonstrate that PDT is an efficient, convenient and useful method for the inactivation of adenovirus vectors in biological samples.

Adenoviridae↗
PubMed1999

Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset.

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.

Adenoviruses, Human↗
PubMed1998

New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses.

The presence of replication-competent adenoviruses (RCAs) in batches of replication-defective adenovirus (Ad) vectors is a major problem for the application of these vectors in gene therapy. RCAs are generated by recombination between sequences in the Ad vector and homologous Ad sequences in the helper cells, resulting in the acquisition by the vector of early region 1. To prevent the formation of RCAs, we have developed helper cell lines, which we named PER, and matched Ad vectors that do not have sequence overlap. PER cells contain the Ad serotype 5 (Ad5) E1A- and E1B-encoding sequences (Ad5 nucleotides 459-3510) under the control of the human phosphoglycerate kinase (PGK) promoter. We demonstrate that PER cells synthesize high levels of the Ad5 E1A and E1B proteins. The yields from PER cells of E1-deleted Ads are similar to those obtained from earlier helper cells, such as 911 and 293 cells. Propagation of matched Ad vectors, which lack Ad5 nucleotides 459-3510, in one of the PER clones, PER.C6, does not result in the generation of RCAs, in contrast to propagation in 293 cells. We conclude that the combination of PER.C6 cells and nonoverlapping E1-deleted adenoviral vectors eliminates the problem of RCA generation by homologous recombination, and allows cost-effective production of safe, clinical-grade batches of recombinant Ad vectors.

Adenovirus E1A Proteins↗
PubMed1998

[Gene therapy for hemophilia: feasible in principle but not yet clinically applicable].

The current treatment of haemophiliacs consists of injection of concentrates of blood clotting factors VIII (haemophilia A) and IX (haemophilia B). The inconvenience of the multiple injections needed, and the risk of transmission of infectious agents (HIV, hepatitis) prompted the development of alternative therapies. Gene therapy aims at introducing functional factor VIII and IX genes into the body cells of patients in order to make these cells produce the desired clotting factors. There are two strategies for gene therapy: (a) in the laboratory cells of the patient may be provided with the desired gene, followed by reintroduction of the cells that now produce factor VIII, into the patient (ex vivo strategy); (b) vectors with the desired genes may be injected into the patient in order to induce the modification (in vivo strategy) For both routes, the formal proof-of-principle has been acquired recently in animal experiments: cells modified by factor VIII or IX genes will produce adequate concentrations of the clotting products in plasma and will correct the bleeding tendency. Before the clinical evaluation and widespread application of the technology can be considered many technical problems have to be solved.

Factor IX↗
PubMed1998

Towards gene therapy for colorectal liver metastases.

Hepatic gene therapy may provide a new approach to treating hepatic malignancies. A promising strategy involves the infection of tumor and liver cells with replication-defective adenoviral vectors carrying suicide genes. The products of these genes convert prodrugs into cytotoxic derivates. In the transduced cells the subsequently administered prodrugs are thus activated and destroy replicating tumor cells, whereas the infected liver cells are thought to be unaffected because of their minimal proliferative activity. A major concern about this approach is that the suicide genes can theoretically enter the germline and other organs with high mitotic activity and as a consequence, cause their destruction. In vivo gene delivery should therefore be tissue-specific, and methods for targeted gene delivery are required. Targeting the colorectal liver metastases is pursued by combining the suicide gene principle and the surgical technique of isolated perfusion of the liver.

Adenoviridae↗