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A Panet

Publications and source records attributed to A Panet.

At least 19 recordsLinked to original sources
PubMed2006

A novel system to study adenovirus tropism to normal and malignant colon tissues.

We describe here a new organ culture system for the evaluation of viral tropism to colon carcinomas and normal colon tissues. Organ cultures of mouse and human colon retained viability for several days and thus facilitated studies of viral tropism. Two adenoviral vectors (AD) were compared in the study: AD5, that utilizes the CAR receptor, demonstrated poor infectivity to both normal and carcinoma tissues, while a capsid-modified-AD, recognizing haparan-sulfate receptor, demonstrated efficient infectivity of both tissues. Immunohistochemistry analysis demonstrated different viral tropism; while AD5 infected only the colon epithelia, the capsid-modified-adeno infected both the epithelia and mesothelial layers. To investigate other determinants in the tissue that influence viral tropism, human cancer tissues were pretreated with collagenase and infected with the AD viruses. Increased infectivity and altered tropism were noted in the treated tumor tissue. Taken together, this ex vivo system indicated that receptor utilization and extracellular-matrix components influence AD viral tropism in solid tissues.

Adenoviridae↗
PubMed2006

Relationship between retroviral vector membrane and vector stability.

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.

Animals↗
PubMed2005

Solid tissues can be manipulated ex vivo and used as vehicles for gene therapy.

BACKGROUND: Organ fragments can be cultured for weeks in vitro if they are prepared of microscopic thickness and if the basic organ structure is preserved. Such organ fragments, which we termed micro-organs (MOs), express in culture endogenous tissue-specific gene products. We have exploited this methodology to engineer MOs ex vivo by gene transfer. METHODS: MOs prepared from spleen, lung, colon and skin were infected using: herpes simplex type-1, adeno virus, vaccinia virus and murine leukemia virus (MuLV), carrying the reporter gene beta-galactosidase. RESULTS: All four viral vectors infected MOs in culture, with adeno infection giving significantly higher values. After optimization, high levels of expression (> 15% positive cells), comparable to those obtained with the adeno construct, were also obtained using the MuLV construct both in vitro and after implantation into syngeneic hosts. After implantation, the engineered tissue was found to remain localized, become vascularized, and to express the transduced gene for several months. CONCLUSIONS: The system can be used to study interactions between viruses and tissues both ex vivo and in vivo. Furthermore, the approach proposes a novel platform for ex vivo gene therapy. Such engineered structures could be used as autologous biological pumps for continuous secretion in vivo of gene products of clinical importance.

Animals↗
PubMed2005

Expression of an anti apoptotic recombinant short peptide in mammalian cells.

Understanding the mechanisms of the apoptotic and anti apoptotic processes may lead to a better way to control these cascades. Here we demonstrated for the first time the feasibility to express a short functional peptide in mammalian cells that abrogates the apoptosis cascade through interference with the proteolytic activity of the initiator caspase 9 and the executing caspase 3 enzymes. The expression of a short peptide that includes the pseudo-substrate motif of the apoptosis inhibitor protein P35 (Asp-Gln-Met-Asp) leads to the abrogation of cell death induced through either the mitochondrial or the death receptors pathways. Short open reading frames have been detected in several mammalian mRNAs, primarily upstream of the main long reading frame (uORFs), however, direct evidence for de-novo peptides translation has not been provided. Utilizing biochemical and imaging techniques we demonstrate here that the functional recombinant peptide was localized to the cytpoplasmic fraction of the cell. In conclusion, this work demonstrates that ribosomes recognize short ORFs to translate stable short recombinant peptides in mammalian cells. Expression of these intracellular peptides results in the knock down of apoptotic processes to generate apoptosis resistant stable cells.

Animals↗
PubMed2003

Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library.

Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbalpha binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies.

Amino Acid Sequence↗
PubMed2001

Two splicing variants of a new inhibitor of apoptosis gene with different biological properties and tissue distribution pattern.

Using homology searches, we identified a novel human inhibitor of apoptosis (IAP) gene. This gene has two splicing variants that contain open reading frames of 298 and 280 amino acids and both contained a single copy of baculovirus IAP repeat (BIR) and RING domain. We refer here to the longer and shorter variants as Livin alpha and beta, respectively. Semiquantitative reverse transcriptase-polymerase chain reaction demonstrated a tissue-specific and non-correlated expression pattern in both adult and fetal tissues. Both mRNA variants were detected in various transformed cell lines. Despite their very close similarity, the two isoforms have different antiapoptotic properties. Both isoforms have a significant antiapoptotic activity in the Jurkat T cell line after triggering apoptosis via tumor necrosis factor and CD95 receptors. The Livin alpha but not beta protects cells from apoptosis induced by staurosporine, but in contrast, apoptosis initiated by etoposide was blocked only by the beta isoform. This difference in biological activities may indicate the presence of critical amino acids outside the BIR and RING domains. These functional and tissue distribution differences of Livin alpha and beta suggest that Livin may play a complex role in the regulation of apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Apolipoprotein E protects against bacterial lipopolysaccharide-induced lethality. A new therapeutic approach to treat gram-negative sepsis.

Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present. Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines. Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo. We now report that intravenously administered LPS strongly increases the serum levels of apoE. In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents. Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice. These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia.

Animals↗
PubMed2001

Imaging transgene expression in live animals.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.

Adenoviridae↗
PubMed2001

Gene transfer by viral vectors into blood vessels in a rat model of retinopathy of prematurity.

AIMS: To test the feasibility of gene transfer into hyaloid blood vessels and into preretinal neovascularisation in a rat model of retinopathy of prematurity (ROP), using different viral vectors. METHODS: Newborn rats were exposed to alternating hypoxic and hyperoxic conditions in order to induce ocular neovascularisation (ROP rats). Adenovirus, herpes simplex, vaccinia, and retroviral (MuLV based) vectors, all carrying the beta galactosidase (beta-gal) gene, were injected intravitreally on postnatal day 18 (P18). Two sets of controls were also examined: P18 ROP rats injected with saline and P18 rats that were raised in room air before the viral vectors or saline were injected. Two days after injection, the rats were killed, eyes enucleated, and beta-gal expression was examined by X-gal staining in whole mounts and in histological sections. RESULTS: Intravitreal injection of the adenovirus and vaccinia vectors yielded marked beta-gal expression in hyaloid blood vessels in the rat ROP model. Retinal expression of beta-gal with these vectors was limited almost exclusively to the vicinity of the injection site. Injection of herpes simplex yielded a punctuate pattern of beta-gal expression in the retina but not in blood vessels. No significant beta-gal expression occurred in rat eyes injected with the retroviral vector. CONCLUSIONS: Adenovirus is an efficient vector for gene transfer into blood vessels in an animal model of ROP. This may be a first step towards utilising gene transfer as a tool for modulating ocular neovascularisation for experimental and therapeutic purposes.

Animals↗
PubMed2001

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation.

A synthetic heparin-mimicking polyaromatic anionic compound RG-13577 (polymer of 4-hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr approximately 5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of (14)C-RG-13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor-related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG-13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild-type and heparan sulfate-deficient Chinese hamster ovary (CHO) cells, as well as normal- and LDL receptor negative- human skin fibroblasts bind RG-13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor-related protein (LRP) expressed a markedly reduced binding of RG-13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG-13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG-13577, suggesting that this inhibition is mediated by RG-13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and thereby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis.

Animals↗
PubMed2000

Avian hemangioma retrovirus induces cell proliferation via the envelope (env) gene.

Several years ago, a field strain retrovirus, avian hemangioma virus (AHV), was isolated from hemangioma tumors in layer hens. Sequence analysis indicated that the AHV genome contains the three prototypic retroviral genes, gag, pol, and env, and is devoid of an oncogene. In cultured endothelial cells, however, AHV induced a significant cytopathic effect through a typical apoptotic cascade. We now demonstrate that AHV also induces cell proliferation and anchorage-independent growth of BSC-1 epithelial cells and NIH-3T3 fibroblasts. This was shown by measurements of (1) cell viability, (2) DNA synthesis, (3) flow cytometry analysis of the cell DNA content, and (4) clonogenic efficiency of the infected cells. Anchorage-independent cell growth was demonstrated by colony formation in soft agar. Moreover, the AHV env gene was cloned into a MuLV-based retroviral vector, and infection of NIH-3T3 cells with this vector induced cell proliferation as well as clonogenic growth. These results suggest that AHV, which is devoid of an oncogene, is a pleiotropic activator capable of inducing either apoptosis or cellular proliferation, depending on the infected cell type.

3T3 Cells↗
PubMed1998

Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase.

The cellular localization and virion association of the human cytomegalovirus (HCMV) UL97 protein were studied. UL97 protein demonstrated early nuclear localization followed by late perinuclear accumulation. It was found to be a structural virion constituent detected in all three enveloped forms of extracellular viral particles and shown to be phosphorylated by the virion-associated protein kinase. UL97 protein immunoprecipitated from virions and from infected cells demonstrated protein kinase activity manifested by autophosphorylation. This activity was reduced in the presence of a ganciclovir-resistance mutation at residue 460, implicated in nucleotide binding. A mutant virus, from which the proposed UL97 kinase catalytic domain had been deleted, could not be propagated in the absence of a helper wild-type virus. The characterization of UL97 protein as a virion-associated protein kinase which appears essential for viral replication, provides further insight into HCMV replication and could identify a potential novel target for antiviral therapy.

Animals↗
PubMed1998

Herpes simplex virus type 1 latency-associated transcripts suppress viral replication and reduce immediate-early gene mRNA levels in a neuronal cell line.

During herpes simplex virus type 1 (HSV-1) latent infection in human dorsal root ganglia, limited viral transcription, which has been linked to HSV-1 reactivation ability, takes place. To study the involvement of this transcription in HSV-1 replication in neuronal cells and consequently in viral latency, we constructed stably transfected neuronal cell lines containing (i) the entire HSV-1 latency transcriptionally active DNA fragment, (ii) the same DNA sequence with deletions of the latency-associated transcript (LAT) promoters, or (iii) the DNA coding sequence of the LAT domain. Replication of HSV-1 or a LAT-negative mutant was markedly repressed in the LAT-expressing cells, a phenomenon mediated by the LATs. To study the mechanism responsible for this effect, we examined LAT influence upon expression of HSV-1 immediate-early (IE) genes ICP0, ICP4, and ICP27, by Northern blot analysis. Following infection of a LAT-expressing neuronal cell line with a LAT-negative mutant, the steady-state levels of all three IE mRNAs were reduced compared to those for control cells. Transient transfections into a neuronal cell line indicated that the LAT suppressive effect upon ICP0 mRNA was mediated directly and was not due to the LAT effect upon the ICP0 promoter. We therefore propose that the LATs may repress viral replication in neuronal cells by reducing IE gene mRNA levels and thus facilitate the establishment of HSV-1 latency in nervous tissue.

Animals↗
PubMed1998

Tissue specific distribution of the herpes simplex virus type 1 latency-associated transcripts on polyribosomes during latent infection.

Transcription of herpes simplex virus type 1 (HSV-1) during latency produces two abundant latency-associated transcripts (LATs). We have recently shown, that during HSV-1 latency in mice trigeminal ganglia (TG) LATs are bound to polyribosomes (J Virol, 1997, 71, 2897-2904). In order to study the possible role of this binding in the latency process, we now extend the polyribosomal analysis to brainstem tissues of latently infected mice, that unlike TG do not support viral reactivation. We report here that the relative amounts of the LATs associated with polyribosomes in the brainstems of mice are significantly lower than those present in TG. We therefore show that binding of the 1.5 and 2.0 kilobases LATs to polyribosomes is tissue specific and hypothesize that this association may have a role in the reactivation function of HSV-1.

Animals↗
PubMed1997

Variant mouse lymphoma cells with modified response to interferon demonstrate enhanced immunogenicity.

We have previously developed an experimental model for the xenogenization of malignant lymphoma. From highly tumorigenic S49 mouse lymphoma cells that proliferate in suspension culture (designated T-25), we selected variant clones that grew as an adherent monolayer (designated T-25-Adh) and were non-tumorigenic in syngeneic mice. Furthermore, priming of syngeneic hosts with T-25-Adh cells protected them against subsequent challenges with the tumorigenic T-25 cells. Several lines of evidence have indicated that antigens of an endogenous mouse mammary tumor virus (MMTV) are involved in the immunogenicity of T-25-Adh cells. Since interferon (IFN) is known to affect retroviral assembly and maturation on the cell membrane, we have studied the effects of IFN on endogenous MMTV-related structures, as well as on the immunogenicity of T-25-Adh cells. We observed that mouse alpha and beta interferons affect the morphogenesis of intracellular MMTV-related precursors in the immunogenic T-25-Adh cells, but not in tumorigenic T-25 cells. From T-25-Adh cells we selected variants that were either high responders or low responders to the above-mentioned interferon effect. The high-response variants were significantly more protective against tumorigenic T-25 cells than the low-response variants. Involvement of MMTV-related antigens in the immune response of the host to T-25-Adh cells was further suggested by immunoelectron-microscopical analysis, demonstrating that antisera from mice, immunized with T-25-Adh cells, interacted specifically with cell-surface MMTV budding particles. These findings indicate a novel method for xenogenization of lymphoma cells by IFN. Since endogenous retroviruses are present in all tissues of the mouse, this approach might be applicable to a wide variety of tumors.

Animals↗
PubMed1997

The abundant latency-associated transcripts of herpes simplex virus type 1 are bound to polyribosomes in cultured neuronal cells and during latent infection in mouse trigeminal ganglia.

During herpes simplex virus type 1 (HSV-1) latency, limited viral transcription takes place. This transcription has been linked to the ability of the HSV-1 genome to reactivate and consists of abundant 2.0- and 1.5-kb collinear latency-associated transcripts (LATs), spanned by minor hybridizing RNA (mLAT). The 1.5-kb LAT is derived from the 2.0-kb LAT by splicing, and both transcripts contain two large overlapping open reading frames. The molecular action mechanisms of the latency-associated gene expression are unknown, and no HSV-1 latency-encoded proteins have been convincingly demonstrated. We have cloned the entire latency-associated transcriptionally active HSV-1 DNA fragment (10.4 kb) under control of a constitutive promoter and generated a neuronal cell line (NA4) stably transfected with the viral LAT's region. NA4 cells produced the 2.0- and the 1.5-kb LATs. Northern blotting and reverse transcription-PCR analysis of RNA from NA4 cells and from trigeminal ganglia of mice latently infected with HSV-1 revealed that the two abundant LAT species were present in the polyribosomal RNA fractions. After addition of EDTA, which causes dissociation of mRNA-ribosome complexes, both LATs could be detected only in subpolyribosomal, but not in polyribosomal fractions. These results show that (i) HSV-1 LATs are bound to polyribosomes during latency in vivo, as well as in neuronal cells in vitro, and therefore might be translated, and that (ii) the NA4 cell line is a suitable tool with which to look for HSV-1 latency-encoded gene products.

Animals↗
PubMed1997

Human T cells recovered from human/Balb radiation chimeras are hypersensitive to human immunodeficiency virus type 1 infection.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated by virus-encoded regulatory proteins, as well as by a variety of cellular factors. Productive infection of human T lymphocytes by HIV-1 is dependent upon the activation status of the target cells. In general, short-term mitogenic stimulation of CD4 T cells is used to enhance infection of peripheral blood mononuclear cells (PBMC) in vitro. Recently, we demonstrated that adoptive transfer of human PBMC into lethally irradiated BALB/c mice, radioprotected with severe combined immunodeficiency (SCID) mouse bone marrow, leads to marked T-cell activation and proliferation. In the present study, we investigated the effect of such xenoactivation of human T cells on their susceptibility to HIV-1 infection. Human cells that were recovered from human/Balb radiation chimeras supported efficient replication of laboratory strains of HIV-1, as well as of HIV-1 clinical isolates. The multiplicity of infection required to attain effective virus replication in the recovered xenoactivated human cells was 10- to 100-fold lower than that needed for infection of short- or long-term phytohemagglutinin (PHA)-stimulated blasts or of various T-cell lines. Analysis of human cell surface activation markers has indicated that xenoactivation in the mouse, in contrast to in vitro stimulation with PHA, is associated with a marked downregulation of CD25 (interleukin 2 receptor). Our results demonstrate that human cells recovered from human/Balb radiation chimeras, which are hypersensitive to HIV-1 infection, differ from in vitro-stimulated cells in their activation status. Therefore, this system could be used to study host factors that participate in HIV-1 infection and replication in vitro and in vivo.

Animals↗