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Biomedical subjects

M Nerenberg

Publications and source records attributed to M Nerenberg.

33 records · Page 2Linked to original sources
PubMed1995

Application of antisense technology to therapeutics.

Antisense oligonucleotides inhibit gene expression by binding in a sequence-specific manner to an RNA target. Modern nucleotide chemistry has enabled the synthesis of chemically modified oligonucleotides that are highly resistant to nuclease degradation. Among other applications, these agents are currently being evaluated as potential antiviral and anticancer drugs. However, several unsolved problems remain. Poor efficiency of delivery to cells, tissue toxicity and antisense-independent biological effects of oligonucleotides currently limit the widespread application of antisense oligonucleotides to human disease. This article reviews some of the applications of antisense oligonucleotides and discusses problems associated with these applications.

Animals↗
PubMed1994

Thymic selection and adaptability of cytotoxic T lymphocyte responses in transgenic mice expressing a viral protein in the thymus.

Upon primary challenge with lymphocytic choriomeningitis virus (LCMV), H-2d (BALB/cByJ) mice mount a cytotoxic T lymphocyte (CTL) response to a single immunodominant domain of the viral nucleoprotein (NP) but no detectable response to the viral glycoprotein (GP). To manipulate this CTL response, the viral NP gene was expressed in the thymus and peripheral T lymphocytes using the murine Thy1.2 promoter. As a result, such Thy1.2-NP (H-2d) transgenic (tg) mice deleted their high-affinity anti-LCMV-NP CTL, but generated equal numbers of lower-affinity NP CTL. Further, they made an alternative anti-LCMV-GP CTL response that is not normally found in non-tg mice indicating a hierarchial control of the CTL response. Unlike the H-2d mice, H-2b (C57Bl/6J) mice normally mount a CTL response to both LCMV-GP and -NP. When the LCMV-NP was expressed using the Thy1.2 promoter in these H-2b mice, the LCMV-NP-specific CTL response was completely aborted and no CTL to new, alternative viral epitopes were generated. Dilutions of H-2b or H-2d NP peptides indicated that 3-4 logs less H-2b NP peptide was required to sensitize syngeneic target cells for CTL-specific lysis, suggesting that the differing affinities of H-2b and H-2d major histocompatibility complex molecules for their peptides likely account for the total removal of NP CTL in the H-2b mice but only partial removal in H-2d mice made to express thymic NP. Thymic grafting experiments done with thymi from newborn Thy1.2-NP tg mice show that selection processes studied in this model are of central (thymic) origin and are not caused by Thy1.2-positive LCMV-NP-expressing T lymphocytes in the periphery.

Adaptation, Physiological↗
PubMed1994

A transgenic mouse model to assess the interaction of cytotoxic T lymphocytes with virally infected, class I MHC-expressing astrocytes.

Astrocytes provide crucial support for neurons and their impairment by viruses or their interactions with anti-viral or autoimmune responses could contribute to neurological disease. We have developed a transgenic mouse model to assess lymphocyte-astrocyte interactions. The major histocompatibility complex (MHC) class I molecule, Db, was expressed in astrocytes under the transcriptional control of regulatory sequences from the glial fibrillary acidic protein (GFAP) gene. Baseline cerebral MHC class I mRNA levels from transgenic mice were elevated over those of non-transgenic controls, and a prominent increase in cerebral MHC class I expression occurred following focal, injury-induced astroglial activation within transgenic brains but not in non-transgenic controls. FACS analysis of explant astrocyte cultures from established transgenic lines demonstrated astroglial expression of the GFAP-Db fusion gene at the protein level. Functional antigen-presenting capacity was conferred by the Db transgene, as virus-infected primary astrocytes obtained from transgenic BALB/c mice (KdIdDdLd) expressing the Db molecule were lysed by Db-restricted anti-viral CTL.

Animals↗
PubMed1992

Human T-cell leukemia virus type I tax transformation is associated with increased uptake of oligodeoxynucleotides in vitro and in vivo.

We have utilized antisense oligodeoxynucleotides (ODNs) to modulate transcriptional activation by the human T-cell leukemia virus type I (HTLV-I) tax gene, the major transcriptional regulator of this virus. 3'-Terminal phosphorothioate-modified antisense ODNs were shown to efficiently inhibit Tax protein expression both in vitro and in vivo. Terminal substitution did not affect the affinity of ODNs for their target sequence but conferred a 9-fold increase in tax inhibition in vitro. When delivered into mice by intraperitoneal injection, ODNs inhibited tax expression in established tumors by 90%. Unmanipulated tax-transformed mouse fibroblasts, or HTLV-I-transformed human lymphocytes, showed at least 5-fold higher ODN binding and uptake over control cells. Balb/3T3 cell binding was induced to similar levels by cellular activators. This suggests that constitutive activation by tax transformation may increase susceptibility of HTLV-I-transformed cells to antisense therapy, providing a rationale for the use of antisense ODN therapeutics in HTLV-I-associated diseases.

3T3 Cells↗
PubMed1992

Ablation of transplanted HTLV-I Tax-transformed tumors in mice by antisense inhibition of NF-kappa B.

Mice transgenic for the human T cell leukemia virus (HTLV-I) Tax gene develop fibroblastic tumors that express NF-kappa B-inducible early genes. In vitro inhibition of NF-kappa B expression by antisense oligodeoxynucleotides (ODNs) inhibited growth of these culture-adapted Tax-transformed fibroblasts as well as an HTLV-I-transformed human lymphocyte line. In contrast, antisense inhibition of Tax itself had no apparent effect on cell growth. Mice treated with antisense to NF-kappa B ODNs showed rapid regression of transplanted fibrosarcomas. This suggests that NF-kappa B expression may be necessary for the maintenance of the malignant phenotype and provides a therapeutic approach for HTLV-I-associated disease.

3T3 Cells↗
PubMed1992

Failure to detect HTLV-I by in situ hybridization in the biopsied muscles of viral carriers with polymyositis.

Direct infection of muscle fibers by human T-lymphotropic virus type I (HTLV-I) has recently been reported in a patient with polymyositis infected with both HTLV-I and human immunodeficiency virus (HIV). Coinfections of these viruses are frequently found in the United States. In Kagoshima, Japan, patients with polymyositis have a significantly increased incidence of seropositivity to HTLV-I alone, when compared with the general population of Kagoshima. In this study, we examined muscle tissue from 6 HTLV-I-positive patients with polymyositis from Kagoshima. To detect HTLV-I products, sensitive immunohistochemistry and in situ hybridization analysis were performed. These were compared with muscle fibers from a well-characterized transgenic mouse model which expressed HTLV-I tax. No specific signals were detected in the biopsied muscles of patients with polymyositis infected with HTLV-I alone. HIV co-infection may, therefore, augment HTLV-I expression through either immunosuppression or direct viral interactions.

Adult↗
PubMed1991

Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response.

We investigated the potential association between viruses and insulin-dependent (type 1) diabetes (IDDM) by developing a transgenic mouse model. By inserting into these mice a unique viral protein that was then expressed as a self-antigen in the pancreatic islets of Langerhans, we could study the effect on that expressed antigen alone, or in concert with an induced antiviral (i.e., autoimmune) response manifested later in life in causing IDDM. Our results indicate that a viral gene introduced as early as an animal's egg stage, incorporated into the germline, and expressed in islet cells does not produce tolerance when the host is exposed to the same virus later in life. We observed that the induced anti-self (viral) CTL response leads to selective and progressive damage of beta cells, resulting in IDDM.

Animals↗
PubMed1991

Vacuolar myositis with expression of both MHC class I and class II antigens on skeletal muscle fibers.

We describe here a 10-year-old patient with high levels of serum IgE and inflammatory myopathy whose muscle fibers exhibit excessive autophagy. Previous studies have demonstrated surface expression of class I MHC antigens on muscle fibers from patients with inflammatory myopathy. The muscle fibers of this patients showed marked expression of both class I and class II MHC antigens. The reaction products were demonstrated not only on sarcolemma but also in and around some vacuoles. Both CD4-positive and CD8-positive T-lymphocytes were noted in inflammatory exudates surrounding these fibers but B-lymphocytes were rare. We hypothesize that myocyte expression of both class I and class II antigens may play a role in the pathogenesis of this new type of inflammatory myopathy.

Autoimmune Diseases↗
PubMed1990

Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control.

To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.

Animals↗
PubMed1987

The tat gene of human T-lymphotropic virus type 1 induces mesenchymal tumors in transgenic mice.

Human T-lymphotropic virus type 1 (HTLV-1) is a suspected causative agent of adult T-cell leukemia. One of the viral genes encodes a protein (tat) that not only results in transactivation of viral gene expression but may also regulate the expression of certain cellular genes that are important for cell growth. Transgenic mice that expressed the authentic tat protein under the control of the HTLV-1 long terminal repeat were generated, and cell types that are permissive for the viral promoter and the effects of the tat gene on these cells were studied. Three of eight founder mice with high levels of expression of the transgene in muscle were bred and then analyzed. All developed soft tissue tumors at multiple sites between 13 to 17 weeks of age. This phenotype was transmitted to nine of nine offspring that inherited the tat gene and were available for analysis. The remaining five founders expressed the transgene in the thymus, as well as in muscle. This second group of mice all exhibited extensive thymic depletion and growth retardation; in all of these mice, death occurred between 3 to 6 weeks of age before tumors became macroscopically visible. The tat gene under the control of the HTLV-1 regulatory region showed tissue-specific expression and the tat protein efficiently induced mesenchymal tumors. The data establish tat as an oncogenic protein and HTLV-1 as a transforming virus.

Animals↗
PubMed1987

A transgenic mouse model for human neurofibromatosis.

Human T-lymphotropic virus type 1 (HTLV-1) has been associated with the neurologic disorder tropical spastic paraparesis and possibly with multiple sclerosis. The tat gene of HTLV-1 under control of its own long terminal repeat is capable of inducing tumors in transgenic mice. The morphologic and biologic properties of these tumors indicate their close resemblance to human neurofibromatosis (von Recklinghausen's disease), the most common single gene disorder to affect the nervous system. The high spontaneous incidence of this disease, together with the diverse clinical and pathologic features associated with it, suggests that environmental factors may account for some of the observed cases. Multiple tumors developed simultaneously in the transgenic tat mice at approximately 3 months of age, and the phenotype was successfully passed through three generations. The tumors arise from the nerve sheaths of peripheral nerves and are composed of perineural cells and fibroblasts. Tumor cells from these mice adapt easily to propagation in culture and continue to express the tat protein in significant amounts. When transplanted into nude mice, these cultured cells efficiently induce tumors. Evidence of HTLV-1 infection in patients with neural and other soft tissue tumors is needed in order to establish a link between infection by this human retrovirus and von Recklinghausen's disease and other nonlymphoid tumors.

Animals↗
PubMed1987

Abundant synthesis of functional human T-cell leukemia virus type I p40x protein in eucaryotic cells by using a baculovirus expression vector.

The human T-cell leukemia virus type I (HTLV-I) p40x protein is a 40-kilodalton polypeptide encoded in the 3'-terminal region of the virus. This protein is responsible for positive transcriptional trans-activation of promoter elements located within the HTLV-I long terminal repeat. We introduced the protein-coding region of HTLV-I p40x into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus. After infection of the insect Spodoptera frugiperda (SF9) cell line, this recombinant strain of baculovirus produced approximately 200 mg of intact p40x protein per 2.5 X 10(8) cells. The protein was biologically active in trans-activation of an HTLV-I long terminal repeat-human beta-globin construct. Biochemical analyses of the protein suggest that the p40x polypeptide underwent posttranslational modification in these eucaryotic SF9 cells.

Biological Assay↗
PubMed1986

Expression of the complete human T-cell leukemia virus type I pX coding sequence as a functional protein in Escherichia coli.

Human T-cell leukemia virus type I (HTLV-I), a virus associated with adult T-cell leukemia, contains a long open reading frame (LOR) in the 3' end of its genome between the env region and the 3' long terminal repeat (LTR). This open reading frame encodes a 40-kDa protein (designated p40x) that has been implicated as a positive control element for transcription from the HTLV-I LTR in a phenomenon known as trans-activation. We now report the expression of the complete p40x coding sequence as a 40-kDa protein in Escherichia coli. The p40x protein produced in bacteria is shown, using the protoplast fusion technique, to possess biological activity by its ability to trans-activate a HTLV-I LTR-chloramphenicol acetyltransferase plasmid that is stably integrated into the genome of mouse L cells. This stimulatory activity could be detected within 2 hr after fusion, suggesting the possibility of a direct role for p40x in trans-activation of the HTLV-I LTR. The production of p40x in large quantities in E. coli, together with the rapid protoplast fusion assay for its biological activity, should facilitate the analysis of p40x mutants and the elucidation of the molecular mechanism of trans-activation.

Cloning, Molecular↗