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At least 937 records · Page 52Linked to original sources
PubMed2001

Expression of the human insulin gene in the gastric G cells of transgenic mice.

The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.

Animals↗
PubMed2002

Conserved regulation of the lymphocyte-specific expression of lck in the Fugu and mammals.

The lck gene encodes a lymphocyte-specific protein-tyrosine kinase that is implicated in T cell maturation and signaling. In mammals, the transcription of the lck gene is regulated by two independent promoters, the proximal promoter, which is active in thymocytes, and the distal promoter, which dominates in mature T cells. In the human and mouse lck gene loci, the two promoter elements are separated by at least 40 kb and 10 kb, respectively. In this study, we have cloned and sequenced 60 kb from the pufferfish (Fugu rubripes) lck locus. The promoter region of the Fugu lck spans only 4.2 kb and contains a proximal and a distal promoter in the 2.3-kb region adjacent to the coding sequence. By generating transgenic mice, we have demonstrated that the compact promoter of the Fugu lck contains regulatory elements that direct expression to lymphoid organs of mice. We were able to localize the regulatory elements to a short region of 830 bp without losing specificity to cultured human T cell line. These results show that the basic mechanisms that mediate lymphocyte-specific expression are conserved between teleosts and mammals. The short promoter of the Fugu lck isolated by us offers a powerful tool for labeling T cells, targeting expression, and manipulating T cell activity in fishes as well as in mammals.

Amino Acid Sequence↗
PubMed2002

Trans-activated interferon-alpha2 delivered to T cells by SV40 inhibits early stages in the HIV-1 replicative cycle.

Several lines of evidence suggest a potential major role for interferon (IFN) in controlling HIV-1 replication. However, this inhibition is moderate and is reversible upon IFN removal. To achieve prolonged high concentrations of IFN at the site of infection, we devised an SV40-based vector, SV[HIVLTR]IFN, to direct the synthesis of human IFN-alpha2, by employing a virus-trans-activated human IFN-alpha2 gene to be transcribed in response to HIV-1 infection. Expression of IFN-alpha2 was confirmed by Northern and Western blotting, in SV[HIVLTR]IFN-transduced, HIV-1-challenged human lymphocyte lines and primary human lymphocytes. SV[HIVLTR]IFN-transduced cells showed no evidence of HIV-1-related cytophatic effects when challenged with high doses of HIV-1(NL4-3). As measured by supernatant HIV-1 p24 antigen concentration, IFN-alpha2-expressing cell lines and peripheral blood lymphocytes (PBL) were protected from high-dose challenges of HIV-1. rSV40-delivered IFN-alpha2 inhibited gp120 protein synthesis and expression of HIV-1 mRNAs. Finally, Southern analysis revealed that levels of proviral DNA were markedly reduced in SV[HIVLTR]IFN-transduced cells compared to control cultures. IFN-alpha2 expression driven by HIVLTR delivered by an rSV40 vector thus strongly inhibits HIV-1 replication, probably by blocking a preintegration step in HIV-1 infection. Targeted expression of IFN-alpha2 delivered by SV40 can thus repress HIV-1 replication, and may be a useful approach to HIV-1 treatment.

Animals↗
PubMed2005

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry.

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Animals↗
PubMed2004

Multiple promoters direct expression of three AKAP12 isoforms with distinct subcellular and tissue distribution profiles.

A Kinase Anchoring Protein 12 (AKAP12; also known as src-suppressed C kinase substrate (SSeCKS) and Gravin) is a multivalent anchoring protein with tumor suppressor activity. Although expression of AKAP12 has been examined in a number of contexts, its expression control remains to be elucidated. Herein, we characterize the genomic organization of the AKAP12 locus, its regulatory regions, and the spatial distribution of the proteins encoded by the AKAP12 gene. Using comparative genomics and various wet-lab assays, we show that the AKAP12 locus is organized as three separate transcription units that are governed by non-redundant promoters coordinating distinct tissue expression profiles. The proteins encoded by the three AKAP12 isoforms (designated alpha, beta, and gamma) share >95% amino acid sequence identity but differ at their N termini. Analysis of the targeting of each isoform reveals distinct spatial distribution profiles. An N-terminal myristoylation motif present in AKAP12alpha is shown to be necessary and sufficient for targeted expression of this AKAP12 isoform to the endoplasmic reticulum, a novel subcellular compartment for AKAP12. Our results demonstrate heretofore unrecognized complexity within the AKAP12 locus and suggest a mechanism for genetic control of signaling specificity through distinct regulation of alternately targeted anchoring protein isoforms.

3T3 Cells↗
PubMed2006

Evaluation of the HER2/neu-derived peptide GP2 for use in a peptide-based breast cancer vaccine trial.

BACKGROUND: E75 and GP2 are human leukocyte antigen (HLA)-A2-restricted immunogenic peptides derived from the HER2/neu protein. In a E75 peptide-based vaccine trial, preexisting immunity and epitope spreading to GP2 was detected. The purpose of this study was to further investigate GP2 for potential use in vaccination strategies. Importantly, a naturally occurring polymorphism (I-->V at position 2, 2VGP2) associated with increased breast cancer risk was addressed. METHODS: Prevaccination peripheral blood samples (PBMC) from HLA-A2 breast cancer patients and CD8+ T cells from HLA-A2 healthy donors were stimulated with autologous dendritic cells (DC) pulsed with GP2 and tested in standard cytotoxicity assays with HER2/neu+ tumor cells or GP2- or 2VGP2-loaded T2 targets. Additional cytotoxicity experiments used effectors stimulated with DC pulsed with E75, GP2, or the combination of E75+GP2. RESULTS: GP2-stimulated prevaccination PBMC from 28 patients demonstrated killing of MCF-7, SKOV3-A2, and the HLA-A2- control target SKOV3 of 28.8+/-3.7% (P<.01), 29.5+/-4.0% (P<.01), and 16.9+/-2.7%, respectively. When compared with E75, GP2-stimulated CD8+ T cells lysed HER2/neu+ targets at 43.8+/-5.2% versus 44.2+/-5.7% for E75 (P=.87). When combined, an additive effect was noted with 58.6+/-5.4% lysis (P=.05). GP2-stimulated CD8+ T cells specifically recognized both GP2-loaded (19.6+/-5.7%) and 2VGP2-loaded T2 targets (17.7+/-5.2%). CONCLUSIONS: GP2 is a clinically relevant HER2/neu-derived peptide with immunogenicity comparable to that of E75. Importantly, GP2-specific effectors recognize 2VGP2-expressing targets; therefore, a GP2 vaccine should be effective in patients carrying this polymorphism. GP2 may be most beneficial used in a multiepitope vaccine.

Breast Neoplasms↗
PubMed2025

Identification of a robust promoter in mouse and human hepatocytes by in vivo biopanning of a barcoded AAV library.

Recombinant adeno-associated viruses (AAVs) are leading vectors for in vivo human gene therapy. An integral vector element is promoters, which control transgene expression in either a ubiquitous or cell-type-selective manner. Identifying optimal capsid-promoter combinations is challenging, especially when considering on- versus off-target expression. Here, we report a pipeline for in vivo promoter biopanning in AAV building on our AAV capsid barcoding technology and illustrate its potential by screening 53 promoters in 16 murine tissues using an AAV9 vector. Surprisingly, the 2.2-kb human glial fibrillary acidic protein (GFAP) promoter was the top hit in the liver, where it outperformed robust benchmarks such as the human &#x3b1;-1-antitrypsin promoter or the clinically used liver-specific promoter 1 (LP1). Analysis of hepatic cell populations revealed preferred GFAP promoter activity in hepatocytes. Notably, the GFAP promoter also surpassed the LP1 and cytomegalovirus promoters in human hepatocytes engrafted in an immune-deficient mouse. These findings establish the GFAP promoter as an exciting alternative for research and clinical applications requiring efficient and specific transgene expression in hepatocytes. Our pipeline expands the arsenal of technologies for high-throughput in vivo screening of viral vector components and is compatible with capsid barcoding, facilitating the combinatorial interrogation of complex AAV libraries.

Dependovirus↗
PubMed2004

Expression of the NF-kappaB-responsive gene BTG2 is aberrantly regulated in breast cancer.

BTG2, a p53-inducible antiproliferative gene, is stimulated in breast cancer cells by activation of nuclear factor kappa B (NF-kappaB). In rat mammary glands, BTG2 is expressed in epithelial cells and levels decreased during pregnancy and lactation but recovered during involution. Estrogen and progestin suppress BTG2 expression, suggesting that these steroids, which stimulate proliferation and lobuloalveolar development of mammary epithelial cells, may downregulate BTG2 in the mammary gland during pregnancy. Consistent with the report that BTG2 inhibits cyclin D1 expression, suppression of BTG2 mRNA in the mammary gland during gestation, and by estrogen and progestin, correlated with stimulation of cyclin D1. Ectopic expression of BTG2 inhibited breast cancer cell growth by arresting cells in the G1 phase, an effect reversed by cyclin D1. BTG2 expression was very low or undetectable in human breast cancer cell lines compared with nontumorigenic mammary epithelial cells, and nuclear expression of BTG2 was absent in 65% of human breast tumors compared with adjacent matched normal glands. Spontaneous mammary tumors arising in a mouse model with targeted expression of the early region of the SV40 large tumor Ag demonstrated loss of BTG2 protein very early during the tumorigenic process. Thus deregulation of BTG2 may be an important step in the development of mammary tumors.

Animals↗
PubMed2006

A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster.

Drosophila melanogaster shows sexually dimorphic cuticular hydrocarbons, with monoenes produced in males and dienes produced in females. Here we describe a female-specific desaturase gene, desatF. RNAi knock-down led to a dramatic decrease in female dienes and increase in monoenes paralleled with an increase in copulation latency and a decrease in courtship index and copulation attempts by the males. The desatF gene was also expressed in females from D. sechellia, rich in dienes, but not D. simulans, which produce only monoenes. When hydrocarbons were feminized in D. melanogaster males by targeted expression of the transformer gene, the expression of desatF occurred. These results strongly suggest that desatF is a crucial enzyme for female pheromone biosynthesis and courtship behaviour in D. melanogaster.

Amino Acid Sequence↗
PubMed2000

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction.

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.

Adrenergic beta-Agonists↗
PubMed1998

Molecular aspects of vasopressin receptor function.

The molecular mechanisms governing the G protein coupling selectivity of different members of the vasopressin receptor family were studied by using a combined molecular genetic/biochemical approach. While the V1a and V1b vasopressin receptors are selectively linked to G proteins of the Gq/11 class, the V2 vasopressin receptor is preferentially coupled to Gs. Systematic functional analysis of V1a/V2 hybrid receptors showed that the second intracellular loop of the V1a receptor is required and sufficient for efficient coupling to Gq/11, whereas the third intracellular loop of the V2 receptor is required and sufficient for coupling to Gs. By using a strategy involving the coexpression of the wild type V1a receptor with chimeric G protein alpha s/alpha q subunits, two C-terminal alpha q/11 residues were identified that are critical for proper receptor recognition. We previously demonstrated -in transiently transfected COS-7 cells- that selected mutant V2 vasopressin receptors (all of which have been identified in X-linked nephrogenic diabetes insipidus patients) containing inactivating mutations in the C-terminal third of the receptor protein (including missense, frameshift, or nonsense mutations) can be functionally rescued by coexpression with a C-terminal V2 receptor fragment (V2-tail) spanning the region where the various mutations occur. Co-immunoprecipitation experiments and a newly developed sandwich ELISA revealed that the V2-tail polypeptide directly interacts with the mutant V2 receptors thus creating a functional receptor protein. To study the potential therapeutic usefulness of these findings, CHO cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors (E242stop, Y280C, and W284stop) were created and infected with a recombinant adenovirus coding for the V2-tail polypeptide. Following adenovirus infection, arginine vasopressin (AVP) gained the ability to stimulate cAMP formation in all CHO cell clones studied. Adenovirus-mediated gene transfer also proved to be a highly efficient method to achieve expression of the V2-tail fragment (as well as of the wild type V2 vasopressin receptor) in MDCK renal tubular cells. We therefore speculate that the targeted expression of receptor fragments in vivo may represent a novel strategy in the treatment of human diseases caused by inactivating mutations in distinct G protein-coupled receptors.

Amino Acid Sequence↗
PubMed2002

Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides.

Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially unaffected. Indeed, a PS-FANA oligomer containing a single deoxyribonucleotide residue core retained significant antisense activity. These findings correlated exactly with the ability of the various chimeric antisense molecules to elicit RNase H degradation of the target RNA in vitro, and suggest that this mode of inhibition is likely the most important determinant for potent antisense activity.

Arabinose↗
PubMed2006

Interferon-gamma induces regression of epithelial cell carcinoma: critical roles of IRF-1 and ICSBP transcription factors.

We have developed an epithelial cell carcinoma model for studying efficacy of IFNgamma gene therapy and have identified components of IFNgamma-signaling pathway responsible for its direct anti-tumor actions. The tumor results from ectopic expression of SV40 Large T-Antigen (SV40 T-Ag) oncogene in lens of transgenic mouse (alphaT3) and complete regression of the tumor is induced by targeting expression of IFNgamma into malignant lens cells. Inflammatory cells are absent in lens of alphaT3 or DT (co-expressing IFNgamma and SV40-T-Antigen) mice and the transformed lens cells are non-immunogenic, suggesting non-involvement of immunologic cells. We show that IFNgamma has direct growth-inhibitory effects on tumor cells, induces death of tumor cells by apoptosis and that these effects are mediated by two transcription factors, IRF-1 (interferon-regulatory factor-1) and ICSBP (interferon-consensus sequence-binding protein) induced by IFNgamma. Furthermore, stable transfection with ICSBP or IRF-1 construct inhibits lens carcinoma cell growth by upregulating Caspase-1, p21(WAF1) and p27 expression. In contrast, tumor progression in alphaT3 lens correlates with inhibition of IRF-1 and ICSBP expression. Our results suggest that IFNgamma gene therapy maybe effective in malignant diseases for which DNA tumor viruses are etiologic agents and that antitumor actions of IRF-1/ICSBP can be exploited therapeutically to circumvent adverse clinical effects associated with IFN therapy.

Animals↗
PubMed2006

Minimally invasive pharmacokinetic and pharmacodynamic technologies in hypothesis-testing clinical trials of innovative therapies.

Clinical trials of new cancer drugs should ideally include measurements of parameters such as molecular target expression, pharmacokinetic (PK) behavior, and pharmacodynamic (PD) endpoints that can be linked to measures of clinical effect. Appropriate PK/PD biomarkers facilitate proof-of-concept demonstrations for target modulation; enhance the rational selection of an optimal drug dose and schedule; aid decision-making, such as whether to continue or close a drug development project; and may explain or predict clinical outcomes. In addition, measurement of PK/PD biomarkers can minimize uncertainty associated with predicting drug safety and efficacy, reduce the high levels of drug attrition during development, accelerate drug approval, and decrease the overall costs of drug development. However, there are many challenges in the development and implementation of biomarkers that probably explain their disappointingly low implementation in phase I trials. The Pharmacodynamic/Pharmacokinetic Technologies Advisory committee of Cancer Research UK has found that submissions for phase I trials of new cancer drugs in the United Kingdom often lack detailed information about PK and/or PD endpoints, which leads to suboptimal information being obtained in those trials or to delays in starting the trials while PK/PD methods are developed and validated. Minimally invasive PK/PD technologies have logistic and ethical advantages over more invasive technologies. Here we review these technologies, emphasizing magnetic resonance spectroscopy and positron emission tomography, which provide detailed functional and metabolic information. Assays that measure effects of drugs on important biologic pathways and processes are likely to be more cost-effective than those that measure specific molecular targets. Development, validation, and implementation of minimally invasive PK/PD methods are encouraged.

Antineoplastic Agents↗
PubMed1989

GP120 specific cellular cytotoxicity in HIV-1 seropositive individuals. Evidence for circulating CD16+ effector cells armed in vivo with cytophilic antibody.

Fresh circulating PBMC from HIV-1 seropositive individuals have been found to mediate specific, non-MHC restricted lysis of targets expressing the major envelope glycoprotein of HIV-1, gp120, in 6-h 51Cr release assays. This gp120 specific cell-mediated cytotoxicity (CMC) is broadly reactive against target cells infected with a wide range of viral isolates, is IL-2 augmentable, and is mediated by a CD16+, Leu-7+, CD15-, CD3- population of NK/K cells. The presence of FcR (CD16) on these cells suggested that the lytic specificity for gp120 might be directed by cytophilic antibody bound to the cell surface. Affinity purified F(ab')2 antibody fragments specific for the Fc and F(ab')2 portions of human IgG were used in attempts to block gp120 specific lysis. A 1/50 dilution of these antibodies inhibited gp120 specific cytolytic activity by more than 90% while exhibiting a minimal effect on NK/K cell lysis of K562 targets. The blocking activity of these fragments demonstrates the direct involvement of cytophilic antibody in CMC. In attempts to isolate this cytophilic anti-HIV-1 antibody, short 56 degrees C incubations were used to dissociate antibodies from the surface of PBMC of seropositive individuals. The supernatants generated in this manner exhibited specific gp120 activity in antibody-dependent cellular cytotoxicity assays. The ability of Staphylococcal protein A to remove this activity confirms the presence of cytophilic antibody on freshly isolated PBMC. Selective enrichment of specific cell subpopulations revealed the origin of the cytophilic antibody to be CD16+ NK/K cells and not B cells, T cells, or monocytes/macrophages. These studies show that the gp120-specific CMC seen in HIV-1 seropositive individuals is directed by cytophilic antibody bound to circulating CD16+ NK/K cells and represents a form of direct antibody-dependent cellular cytotoxicity which may provide a primary cytotoxic host defense.

Antibody Specificity↗
PubMedDate not supplied

Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen.

Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.

Animals↗
PubMed2004

High-level expression of Dok-1 in neurons of the primate prefrontal cortex and hippocampus.

The docking protein p62Dok-1 (Dok-1) has a central role in cell signaling mediated by a wide range of protein tyrosine kinases, including intrinsic membrane kinases, such as the insulin-like growth factor-1 (IGF-1) receptor. To elucidate potential IGF signaling mechanisms, we used DNA array technology to investigate novel kinase targets expressed in the primate dorsolateral prefrontal cortex (DLPFC). Dok-1 transcripts were among the most abundant found in this structure. Because Dok-1 expression has not been characterized in brain, we evaluated its expression pattern using immunoblotting, in situ hybridization, and immunohistochemistry in the rhesus monkey prefrontal cortex and hippocampal formation. Dok-1 antibodies identified a 62-kDa band in lysates from the DLPFC, consistent with the known size for Dok-1. In situ hybridization showed that Dok-1 mRNA was expressed in all layers of the DLPFC and in all neuronal subregions of the hippocampal formation. Immunohistochemical analysis showed Dok-1 immunoreactivity concentrated in pyramidal neurons of cortical layers IV-V and throughout Ammon's horn and in granule neurons of the dentate gyrus. Dok-1 expression was also identified in endothelial cells of cerebral blood vessels. These expression patterns are very similar to those of the IGF-1 receptor and suggest that Dok-1 could be among the downstream targets of IGF signaling in areas of the primate brain involved in learning and memory.

Animals↗
PubMed1997

Fas-mediated cytotoxicity by intestinal intraepithelial lymphocytes during acute graft-versus-host disease in mice.

BACKGROUND & AIMS: Host-derived intestinal intraepithelial lymphocytes (IELs) increase in number during acute graft-versus-host disease (GVHD) in mice. In the present study, we examined Fas-mediated cytotoxicity by host-derived IELs against Fas-expressing target cells to see whether Fas/Fas ligand (Fas-L) interaction is involved in the pathogenesis of enteropathy during acute GVHD. METHODS: Acute GVHD was induced by injection of parental spleen cells into nonirradiated F1 mice. The expression of Fas antigen on the intestinal epithelial cells (IECs) was examined by flow cytometry, and the expression of messenger RNA (mRNA) for Fas-L, interleukin 2, and interferon gamma in host-derived IELs was assessed by reverse-transcription polymerase chain reaction. Fas-mediated cytotoxicity by host-derived IELs was assessed using Fas-transfected cells, IECs, and Fas immunoglobulin Fc fusion protein (Fas Fc). RESULTS: Fas antigen was constitutively expressed on the cell surface of IECs before and after GVHD induction. Although Fas-L mRNA was not detected or detected scarcely in either alphabeta or gammadelta IELs before GVHD induction, both IELs expressed high levels of mRNA for Fas-L and interferon gamma after GVHD induction. Host-derived IELs during acute GVHD showed cytotoxicity against Fas-transfected target cells and IECs, which was partly blocked by addition of Fas Fc. CONCLUSIONS: Fas/Fas-L-mediated cytotoxicity by host-derived IELs may be partly responsible for the enteropathy during acute GVHD.

Animals↗