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Biomedical subjects

M Mehtali

Publications and source records attributed to M Mehtali.

At least 37 records · Page 2Linked to original sources
PubMed1999

Gene therapy with cytokine-transfected xenogenic cells (Vero-IL-2) in patients with metastatic solid tumors: mechanism(s) of elimination of the transgene-carrying cells.

Eleven patients with advanced cancer were treated in a clinical gene therapy trial by repeated intra- tumoral injections with different doses of xenogenic fibroblasts secreting high amounts of human interleukin-2 (Vero-IL2). Treatments in a total of 14 courses were well tolerated and resulted in clinical responses and measurable biological effects. Together with increases in serum interleukin-2 (IL-2), modifications of the V-beta T cell receptor repertoire and induction of intratumoral T-cell infiltration were observed. When the intratumoral expression of endogenous cytokine genes and the persistence of the IL-2 transgene at the application site and in peripheral blood were investigated, rapid disappearance of the transgene at the application site appeared to be the most prominent biological effect. Tests detecting a single Vero-IL2 cell against a background of 10(5) non-transfected cells were not able to demonstrate significant expression of exogenous IL-2 (i.e. the transgene or transgene-carrying cells) in tumor biopsies or blood at different times. Therefore, further studies were performed to evaluate the mechanism(s) involved in the rapid disappearance of xenogenic carrier cells in more detail. We show here that significant in vitro cytotoxicity against transgene-carrying Vero cells can be observed in peripheral blood of all the patients before treatment as well as in healthy controls. "Cold" target inhibition shows that significant killing of Vero-IL2 cells is mediated by natural killer (NK) cells. This was confirmed by showing that established CD3(-)/CD16(+)/CD56(+) peripheral blood NK cell clones kill both K562 and Vero-IL2 target cells. The failure of other mechanisms (complement, antibody-dependent cell cytotoxicity or cytotoxic T lymphocytes) to destroy xenogenic, histoincompatible Vero cells in vitro suggests that NK cells also might be responsible for the killing of Vero-IL2 in vivo and for the failure to detect the transgene at the application site. These results might also be of importance for some aspects of the current discussion of xenotransplantation.

Adult↗
PubMed1999

Molecular determinants of adenovirus serotype 5 fibre binding to its cellular receptor CAR.

Adenovirus (Ad) tropism is mediated in part through the fibre protein. The common coxsackie B virus and Ad receptor (CAR) was recently identified as the major receptor for subgroup C Ad serotype 5 (Ad5) and serotype 2 (Ad2) fibres. Effects of mutations in the Ad5 fibre gene were studied to assess domains of the fibre capsomer that could alter virus tropism without altering virus assembly and replication. All mutants that accumulated as fibre monomers failed to assemble with a penton base and proved lethal for Ad5 which suggests that the absence of infectious virions resulted in part from a defect in fibre penton base assembly. Cell binding capacity of all fibre mutants was investigated in cell binding competition experiments with adenovirions using CHO-CAR cells (CHO cells that have been transfected with CAR cDNA and express functional CAR). The results suggest that the R-sheet of the Ad5 fibre knob monomer contains binding motifs for CAR and that beta-strands E and F, or a region close to them, may also be involved in receptor recognition.

Adenoviruses, Human↗
PubMed1999

Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: requirement for E4 ORF3.

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022-2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

Adenoviridae↗
PubMed1999

Replication-defective bovine adenovirus type 3 as an expression vector.

Although recombinant human adenovirus (HAV)-based vectors offer several advantages for somatic gene therapy and vaccination over other viral vectors, it would be desirable to develop alternative vectors with prolonged expression and decreased toxicity. Toward this objective, a replication-defective bovine adenovirus type 3 (BAV-3) was developed as an expression vector. Bovine cell lines designated VIDO R2 (HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and 6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1 proteins) were developed and found to complement the E1A deletion in BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most of the E1A and E3 regions was constructed. This virus could be grown in VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results demonstrated that the E1 region of HAV-5 has the capacity to transform bovine retina cells and that the E1A region of HAV-5 can complement that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A similar replication-defective vector expressing the hemagglutinin-esterase gene of bovine coronavirus from the E3 region was isolated. Although these viruses grew less efficiently than the replication-competent recombinant BAV-3 (E3 deleted), they are suitable for detailed studies with animals to evaluate the safety, duration of foreign gene expression, and ability to induce immune responses. In addition, a replication-competent recombinant BAV-3 expressing green fluorescent protein was constructed and used to evaluate the host range of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of replication-defective BAV-3 vectors is a major technical advancement for defining the use of BAV-3 as vector for vaccination against diseases of cattle and somatic gene therapy in humans.

Adenovirus E1A Proteins↗
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↗
PubMed1999

Fiberless recombinant adenoviruses: virus maturation and infectivity in the absence of fiber.

In vivo targeting of therapeutic genes to specific tissues has become a major issue in gene therapy, in particular when recombinant adenovirus vectors are used. Restriction of the viral tropism to selected cell types requires the abrogation of the interaction between the viral fiber and its natural cellular receptors and the introduction of a new binding specificity into the virion. In this context, fiberless adenoviruses are attractive vectors, since they may be used as substrates for the insertion of a new ligand in other capsid proteins. In this study, we confirm by using cloned full-length adenovirus genomes with the fiber gene deleted that efficient virus particle formation can occur in the absence of fiber. As expected, the infectivity of such fiberless viruses was severely reduced, but it could be only partially restored when the viruses were produced in cells stably providing the fiber in trans. Although incorporation of penton base into the fiberless particles was normal and binding of the particles to the cellular integrins was functional, several pieces of experimental evidence suggest that later steps in the cell entry process are impaired in correlation with an incorrect maturation of several structural proteins of the fiberless particles. These observations support the hypothesis that the fiber protein may have additional biological functions besides its role in cell binding. Together with the fiber complementation cells, such fiberless vectors constitute unique tools to investigate the role of the fiber in virus assembly, maturation, and cell entry and to explore the possibility of deriving gene transfer vectors with novel target specificities.

Adenoviruses, Human↗
PubMed1999

Deletion of the fiber gene induces the storage of hexon and penton base proteins in PML/Sp100-containing inclusions during adenovirus infection.

The present study has documented changes in the in situ distribution of viral DNA and capsid proteins in 293 cells infected with fiber gene-deleted adenoviruses. It shows that infection results in the intense production of progeny viruses which appear morphologically intact although they are devoid of fiber-coding sequence in their genome and hence of fiber protein in their capsid. The data confirm, therefore, that fiber protein is not essential for the assembly of progeny viruses. The main contribution of our observations concerns specific intranuclear structures induced by infection with either wild-type or fiber gene-deleted viruses. These clear amorphous inclusions contain two cellular proteins, PML and Sp100, which in non-infected cells co-localize to a special type of nuclear bodies. PML and Sp100 nuclear bodies appear to directly modulate or to be altered in a wide variety of situations including viral infections, cell death and transformation. In cells infected with fiber gene-deleted viruses, the clear amorphous inclusions now accumulate non-used hexon and penton base proteins, whereas the absence of fiber protein prevents the assembly of capsid proteins in crystallin arrays. Taken together, the data suggest that the clear amorphous inclusions may correspond to storage sites of structural and regulatory proteins. Consequently, these virus-induced structures may promote the productive cycle of adenoviruses by regulating the amount of over-produced viral proteins and the shutoff of the host cell metabolism.

Adenoviridae↗
PubMed1998

Construction and characterization of E3-deleted bovine adenovirus type 3 expressing full-length and truncated form of bovine herpesvirus type 1 glycoprotein gD.

Using the homologous recombination machinery of E. coli, a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid. Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3. E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replication in vitro. Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3. E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals.

Adenovirus E3 Proteins↗
PubMed1998

Gene therapy with cytokine-transfected xenogeneic cells in metastatic tumors.

On the basis of compelling preclinical data in cats and dogs we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human IL-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to CT- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. Endpoints of the study were feasibility, toxicity, and clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological and molecular analyses were performed on biopsy specimens of tumors and blood samples from before, during and after treatment. Low levels of serum antibodies to Vero cells developed in 2/9 patients. Analysis of tumor biopsies showed increased expression of CD3 mRNA and enhanced tumor infiltration with varying lymphocyte subpopulations after treatment. In addition, monoclonal alterations of the TCR repertoire of blood and tumor lymphocytes were observed. Treatment was well tolerated and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft tissue sarcoma showed a more than 90% and more than 50% reduction of the volume of two distant, non-injected metastases, respectively, lasting for 22+ months. Four other patients showed stabilization of their disease for three to nine months, among whom was a patient with melanoma who developed marked vitiligo. We conclude that repeated injection of up to 5 x 10(7) Vero-IL-2 cells was safe and showed biological and clinical activity in heavily pretreated patients with advanced solid tumors. Further evaluation of intratumoral application of Vero-IL-2 seems warranted.

Adult↗
PubMed1998

In vitro and in vivo antitumor effects of retrovirus-mediated herpes simplex thymidine kinase gene-transfer in human medulloblastoma.

Herpes simplex virus thymidine kinase gene (HSVtk) transfer together with treatment with the prodrug ganciclovir (GCV) represents the most commonly used suicide gene approach for the gene therapy of human central nervous system malignancies. Despite encouraging results reported in clinical trials conducted in adults, very little is known about the feasibility of this approach for the treatment of CNS tumors of childhood. We studied the effects of the HSVtk/GCV system on human medulloblastoma cells in vitro and in vivo. The transfer of tk gene to medulloblastoma cells was capable of mediating cell suicide in vitro and in vivo upon treatment with GCV, but the overall effect in vivo appeared to be suboptimal. The relatively low sensitivity of the medulloblastoma cells to viral infection and a limited bystander effect, coupled with a low expression of connexin-43 protein, might partially explain these results. Whether this is a peculiarity of the cell line studied or a general characteristic of medulloblastoma remains to be determined. These findings should be taken into account for the future planning of gene therapy trials for human medulloblastoma.

Cerebellar Neoplasms↗
PubMed1998

In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted.

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Adenovirus E1 Proteins↗
PubMed1997

Protection of SIVmac-infected macaque monkeys against superinfection by a simian immunodeficiency virus expressing envelope glycoproteins of HIV type 1.

The infection of macaque monkeys by attenuated simian immunodeficiency virus can vaccinate against pathogenic molecular clones and isolates of the same virus. The correlates of this potent protective immunity are not fully understood but may be the key to an effective AIDS vaccine for humans. Aiming to determine whether host immune responses to envelope glycoprotein are an essential component of the immunity to primate lentiviruses, we have tried to superinfect SIVmac-infected macaque monkeys with SHIVsbg, a chimeric primate lentivirus constructed from the SIVmac239 genome with the env, rev, tat, and vpu genes from HIV-1 Lai. After inoculation of a large dose of SHIVsbg, the chimeric virus was isolated by coculture of mononuclear blood cells from four of five SIV-infected monkeys, but three animals were protected from extracellular SHIV viremia and did not seroconvert to HIV-1 glycoproteins. In the two SIV-infected monkeys that did develop SHIV viremia, cell-associated viral load was reduced at least 100-fold. These data indicate that an antiviral response capable of effectively controlling primate lentivirus replication might not necessarily involve the envelope glycoprotein.

AIDS Vaccines↗
PubMed1997

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response.

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Adenoviruses, Human↗
PubMed1997

[Genetic therapy for hemophiliacs--therapeutic potential and technological limits].

Defects in the genes encoding the human coagulation factor VIII (hFVIII) and IX (hFIX) result in life-threatening haemorrhages and severe arthropathies. While haemophiliacs are currently treated by blood-derived factors or recombinant hFVIII and hFIX, a number of recent technical advances make the prospect of using gene therapy to treat such genetic diseases a realistic goal. Several gene therapy strategies have therefore been developed and evaluated in recent years. Most of the initial protocols were ex vivo gene transfer approaches in which the target cells (fibroblasts, keratinocytes, myoblasts, ...) were expanded and genetically-engineered in the laboratory and then implanted in the host. However, the complexity of most ex vivo gene therapy strategies, together with the disappointing results obtained in various animal models stimulated the development of more direct in vivo gene therapy protocols. In aiming to establish such an in vivo gene transfer protocol for haemophilia B, we constructed and tested in vitro and in vivo various recombinant adenovirus vectors expressing human FIX. Intravenous administration of this vector into various strains of immunocompetent and immunodeficient mice led to an efficient hFIX gene transfer in liver and lung. As a consequence, the hFIX protein was correctly produced and secreted at high levels in the blood of the treated animals. However, expression was transient in all immunocompetent mice, except surprisingly in C57B1/6 animals. A systematic molecular and immunological analysis allowed us to identify the parameters that prevent the long-term in vivo expression of the human molecule and to improve the current adenovirus vectors.

Adenoviridae↗
PubMed1997

Adenovirus-mediated gene transfer: influence of transgene, mouse strain and type of immune response on persistence of transgene expression.

E1-deleted adenovirus (Ad) vectors expressing the human coagulation factor IX (hFIX) or the bacterial beta-galactosidase (lacZ) were injected intravenously into various strains of immunocompetent (C57BI/6, BALB/c, CD1, CBA/J, C3H) and immunodeficient (BALB/c-nu/nu, C57BI/6-nu/nu, SCID, NIH-bg-nu-xid) mice. Regular analysis of mouse sera and tissues showed a persistent expression of both transgenes in immunodeficient mice, while detection diminished very rapidly in immunocompetent mice. The mechanisms responsible for the transient detection of the two transgenes were however not identical. Rapid decline of lacZ expression was correlated with a rapid decrease of viral DNA sequences, and consequently to the induction of a cellular immune response to the lacZ antigen. In contrast, absence of detectable levels of serum hFIX in immunocompetent animals was not associated with a loss of viral DNA but was strictly correlated with the induction of anti-hFIX antibodies. Surprisingly, anti-hFIX antibodies were never detected in C57BI/6 mice, leading to prolonged detection of hFIX. These results suggest that cellular immunity to viral antigens plays a minor role in the early extinction of transgene expression and illustrate the influence of the cellular (eg lacZ) or humoral (eg hFIX) immunity to transgene-encoded products on the persistence of transgene expression.

Adenoviridae↗
PubMed1997

Experimental gene therapy: the transfer of Tat-inducible interferon genes protects human cells against HIV-1 challenge in vitro and in vivo in severe combined immunodeficient mice.

OBJECTIVES: To evaluate in vitro and in vivo a strategy for gene therapy for AIDS based on the transfer on interferon (IFN)-alpha, -beta and -gamma genes to human cells. DESIGN: Human U937 promonocytic cells were stably transfected with Tat-inducible IFN expression vectors conferring an antiviral state against infection with HIV. METHODS: Transfected cells were either infected by HIV-1 in vitro or transplanted into severe combined immunodeficient (SCID) mice for an HIV challenge in vivo. RESULTS: U937 cell lines stably carrying IFN transgenes under the positive control of the HIV-1 Tat protein were highly resistant to HIV-1 replication in vitro. This antiviral resistance was associated with a strong induction of IFN synthesis immediately following the viral infection. HIV-1 proteins were found to be specifically trapped within the genetically modified cells. In contrast, all IFN-U937 cells permitted full HIV-2 replication. Transfected cells injected into SCID mice and challenged against HIV-1 were strongly resistant to infection when cells were transduced with IFN-alpha of IFN-beta genes. However, IFN-gamma-transfected cells permitted HIV-1 infection in vivo despite the induction of a high level of IFN-gamma secretion. The quantity of proviral DNA was 10(5)-fold lower in IFN-alpha- or IFN-beta-transfected U937 cells collected from these SCID mice than that in non-transfected cells. CONCLUSIONS: Our results substantiated the validity of a strategy, bases on the transfer of HIV-1-inducible IFN-alpha or IFN-beta genes, to confer antiviral resistance to human cells.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Conditional repression of the E2 transcription unit in E1-E3-deleted adenovirus vectors is correlated with a strong reduction in viral DNA replication and late gene expression in vitro.

An E1-E3-deleted recombinant adenovirus vector expressing the hybrid protein TetR-KRAB has been produced. In this virus, AdTG9562, the E2 transcription is regulated by TetR-KRAB and tetO sequences inserted in cis. In the absence of tetracycline, a strong reduction in E2A gene expression, viral DNA replication, and late gene expression was observed in noncomplementing A549 cells, and a reduction in viral growth was seen in the E1-expressing 293 cells. In contrast, there was no repression in the presence of the regulator tetracycline. We propose that regulation by TetR-KRAB is a valuable tool with which to study the effects of viral gene expression in vitro.

Adenoviridae↗