Eric Lechman - Academia.edu (original) (raw)

Papers by Eric Lechman

Research paper thumbnail of Ectopic miR-125a Expression Induces Long-Term Repopulating Stem Cell Capacity in Mouse and Human Hematopoietic Progenitors

Cell Stem Cell, 2016

Highlights d Enforced expression of miR-125 increases hematopoietic stem frequency in vivo d miR-... more Highlights d Enforced expression of miR-125 increases hematopoietic stem frequency in vivo d miR-125 induces stem cell potential in murine and human progenitor cells d miR-125 represses, among others, targets of the MAP kinase signaling pathway d miR-125 function and targets are conserved in human and mouse

Research paper thumbnail of Contralateral Control Knees Disease in Both Injected and Experimental Arthritis Ameliorates Viral IL10 to Rabbit Knees with Direct Adenoviral Gene Transfer of

Research paper thumbnail of Injected and Uninjected Paws Collagen-Induced Arthritis in Both Suppresses Development of Gene Periarticularly to Mouse Paws Adenoviral Transfer of the Viral IL10

Research paper thumbnail of Abstract 2292: Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells

Research paper thumbnail of Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry

The American journal of physiology, 1996

In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimu... more In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimulation of apical entry enhances the ability of renal epithelial cells to reabsorb Na+, an important feature of the load-dependent regulation of renal tubular Na+ transport. To model this load dependency, apical Na+ entry was inhibited or stimulated for 18 h in A6 cells and vectorial transport was measured as short-circuit current (Isc) across monolayers on filter-bottom structures. Basal amiloride-sensitive Isc represents the activity of apical Na+ channels, whereas Isc after permeabilization of the apical membrane to cations with nystatin represents maximal activity of the basolateral Na(+)-K(+)-ATPase. Chronic inhibition of apical Na+ entry by 18-h apical exposure to amiloride or replacement of apical Na+ with tetramethylammonium (TMA+), followed by washing and restoration of normal apical medium, revealed a persistent decrease in Isc that remained despite exposure to nystatin. Both ba...

Research paper thumbnail of Human, viral or mutant human IL-10 expressed after local adenovirus-mediated gene transfer are equally effective in ameliorating disease pathology in a rabbit knee model of antigen-induced arthritis

Arthritis research & therapy, 2006

IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral r... more IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral responses. Human IL-10 (hIL-10) has anti-inflammatory, immunosuppressive as well as immunostimulatory characteristics, whereas viral IL-10 (vIL-10), a homologue of hIL-10 encoded by Epstein Barr virus (EBV), lacks several immunostimulatory functions. The immunostimulatory characteristic of hIL-10 has been attributed to a single amino acid, isoleucine at position 87, which in vIL-10 is alanine. A mutant hIL-10 in which isoleucine has been substituted (mut.hIL-10) is biologically active with only immunosuppressive, but not immunostimulatory, functions, making it a potentially superior therapeutic for inflammatory diseases. To compare the efficacy of mut.hIL-10 with hIL-10 and vIL-10 in blocking the progression of rheumatoid arthritis, we used replication defective adenoviral vectors to deliver intra-articularly the gene encoding hIL-10, vIL-10 or mut.hIL-10 to antigen-induced arthritic (AIA...

Research paper thumbnail of Ex vivo gene delivery of IL-1Ra and soluble TNF receptor confers a distal synergistic therapeutic effect in antigen-induced arthritis

Molecular therapy : the journal of the American Society of Gene Therapy, 2002

Intra-articular expression of antagonists of interleukin-1beta (IL-1beta) and tumor necrosis fact... more Intra-articular expression of antagonists of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in arthritic rabbit knee and mouse ankle joints by direct adenoviral-mediated intraarticular delivery results in amelioration of disease pathology in both the treated and contralateral untreated joints. Previous experiments suggest that direct adenoviral infection of resident antigen-presenting cells (APCs) and subsequent traveling of these cells to other sites of inflammation and lymph nodes might be responsible for this "contralateral effect." To determine whether genetic modification of APCs is required for the contralateral effect, we have used an ex vivo approach utilizing genetically modified fibroblasts to express IL-1 receptor antagonist protein (IL-1Ra) and soluble TNF-alpha receptor (sTNFR) locally in arthritic joints. Retroviral vectors carrying IL-1Ra, sTNFR-Ig, or both genes together were used to stably infect autologous rabbit fibroblasts that...

Research paper thumbnail of Lessons learned from gene transfer approaches

Arthritis research, 1999

Recent technological advances allow the transfer of genes to the synovial lining of joints. As we... more Recent technological advances allow the transfer of genes to the synovial lining of joints. As well as opening novel opportunities for therapy, these techniques provide valuable new tools for the study of synovitis and other aspects of the biology of joints in health and disease. This article reviews briefly the results of experiments in which selected genes have been transferred to the knee joints of healthy rabbits and rabbits with antigen-induced arthritis.

Research paper thumbnail of Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1999

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery ... more Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatme...

Research paper thumbnail of Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees

Arthritis research & therapy, 2003

To examine the effect of transforming growth factor (TGF)-beta1 on the regulation of cartilage sy... more To examine the effect of transforming growth factor (TGF)-beta1 on the regulation of cartilage synthesis and other articular pathologies, we used adenovirus-mediated intra-articular gene transfer of TGF-beta1 to both naïve and arthritic rabbit knee joints. Increasing doses of adenoviral vector expressing TGF-beta1 were injected into normal and antigen-induced arthritis rabbit knee joints through the patellar tendon, with the same doses of an adenoviral vector expressing luciferase injected into the contralateral knees as the control. Intra-articular injection of adenoviral vector expressing TGF-beta1 into the rabbit knee resulted in dose-dependent TGF-beta1 expression in the synovial fluid. Intra-articular TGF-beta1 expression in both naïve and arthritic rabbit knee joints resulted in significant pathological changes in the rabbit knee as well as in adjacent muscle tissue. The observed changes induced by elevated TGF-beta1 included inhibition of white blood cell infiltration, stimul...

Research paper thumbnail of Identification of Hematopoietic Stem Cell-Specific miRNAs Enables Gene Therapy of Globoid Cell Leukodystrophy

Science Translational Medicine, 2010

Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal ... more Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.

Research paper thumbnail of Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor   soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Proceedings of the National Academy of Sciences, 1998

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I rece... more Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor alpha (TNFalpha) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFalpha receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

Research paper thumbnail of Stem cell gene expression programs influence clinical outcome in human leukemia

Nature Medicine, 2011

Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarc... more Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.

Research paper thumbnail of Identification of a synovial fibroblast-specific protein transduction domain for delivery of apoptotic agents to hyperplastic synovium

Molecular Therapy, 2003

Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pat... more Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pathologies associated with rheumatoid arthritis. To develop an approach for efficient delivery of proteins or agents to synovium to induce targeted apoptosis of hyperplastic synovial tissue, we have screened an M13 peptide phage display library for synovial-specific transduction peptides. We identified a novel synovial-targeted transduction peptide, HAP-1, which is able to facilitate specific internalization of protein complexes into human and rabbit synovial cells in culture and rabbit synovial lining in vivo. HAP-1 and a non-tissue-specific cationic protein transduction domain, PTD-5, were fused to an antimicrobial peptide, (KLAK)(2), to generate two proapoptotic peptides termed DP2 and DP1, respectively. Administration of these peptides was able to induce apoptosis of rabbit and human synovial cells in culture, with DP2 inducing synovial cell-specific apoptosis. Intra-articular injection of DP1 and DP2 into arthritic rabbit joints with synovial hyperplasia induced extensive apoptosis of the hyperplastic synovium, while reducing the leukocytic infiltration and synovitis. These results suggest that proapoptotic peptides and, in particular, DP2 can be clinically useful for treatment of synovial hyperplasia, as well as inflammation. Moreover, the results demonstrate the feasibility of identifying tissue-specific transduction peptides capable of mediating efficient transduction in vivo.

Research paper thumbnail of Exosomes Derived from Genetically Modified DC Expressing FasL Are Anti-inflammatory and Immunosuppressive

Molecular Therapy, 2006

We previously have demonstrated the ability of primary murine bone marrow-derived DC (BM-DC), gen... more We previously have demonstrated the ability of primary murine bone marrow-derived DC (BM-DC), genetically modified by adenoviral infection to express FasL, to inhibit progression of established collagen-induced arthritis (CIA) following systemic delivery. Here we demonstrate that exosomes derived from genetically modified BM-DC expressing FasL are able to inhibit inflammation in a murine footpad model of delayed-type hypersensitivity (DTH). Local administration of exosomes derived from DC expressing FasL (Exo/FasL) as well as the parental DC/FasL resulted in a significant reduction in swelling in both the treated and the untreated distal paw. However, both the DC/FasL and the Exo/FasL were unable to suppress the DTH response in lpr (Fas-deficient) mice. Gene transfer of FasL to BM-DC from gld (FasL-deficient) mice resulted in restoration of the ability of DC as well as DC-derived exosomes to suppress DTH. The ability of DCderived exosomes and DC to suppress DTH responses was antigen specific and MHC class II dependent, but class I independent. The injected exosomes were found to be internalized into CD11c + cells at the site of injection and in the draining popliteal lymph node. Systemic injection of exosome/FasL into mice with established CIA resulted in significant disease amelioration. These results demonstrate that both systemic and local administration of exosomes derived from FasLexpressing DC are able to suppress antigen-specific immune responses through an MHC class IIdependent pathway, resulting in effective and sustained treatment of established collageninduced arthritis and suppression of the DTH inflammatory response. These results suggest that DC/FasL-derived exosomes could be used clinically for the treatment of inflammatory and autoimmune diseases.

Research paper thumbnail of Gene Delivery of IL-1Ra and Soluble TNF Receptor Confers a Distal Synergistic Therapeutic Effect in Antigen-Induced Arthritis

Research paper thumbnail of Zebrafish microRNA-126 determines hematopoietic cell fate through c-Myb

Leukemia, 2011

Precise regulatory mechanisms are required to appropriately modulate the cellular levels of trans... more Precise regulatory mechanisms are required to appropriately modulate the cellular levels of transcription factors controlling cell fate decisions during blood cell development. Here, we show that miR-126 is a novel physiological regulator of the proto-oncogene c-myb during definitive hematopoiesis. We show that knockdown of miR-126 results in increased c-Myb levels and promotes erythropoiesis at the expense of thrombopoiesis in vivo. We further provide evidence that specification of thrombocyte versus erythrocyte cell lineages is altered by the concerted activities of the miRNAs miR-126 and miR-150. Both microRNAs are required but not sufficient individually to precisely regulate the cell fate decision between erythroid and megakaryocytic lineages during definitive hematopoiesis in vivo. These results support the notion that microRNAs not only act to provide precision to developmental programs but also are essential determinants in the control of variable potential functions of a single gene during hematopoiesis.

Research paper thumbnail of Exosomes Derived from IL-10-Treated Dendritic Cells Can Suppress Inflammation and Collagen-Induced Arthritis

The Journal of Immunology, 2005

Research paper thumbnail of Element-tagged immunoassay with ICP-MS detection: Evaluation and comparison to conventional immunoassays

Journal of Immunological Methods, 2008

We have investigated the possibility of using element-tagged antibodies for protein detection and... more We have investigated the possibility of using element-tagged antibodies for protein detection and quantification in microplate format using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and compared the results to conventional immunoassays, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. The technique was further employed to detect low levels and measure DNA-binding activity of transcription factor p53 in leukemia cell lysates through its interaction with immobilized oligonucleotides and recognition by element-tagged antibodies. The advantages of ICP-MS detection for routine performance of immunoassays include increased sensitivity, wide dynamic range, minimal interference from complex matrices, and high throughput. Our approach advances the ICP-MS technology and demonstrates its applicability to proteomic studies through the use of antibodies directly labeled with polymer tags bearing multiple atoms of lanthanides. Development of this novel methodology will enable fast and quantitative identification of multiple analytes in a single well.

Research paper thumbnail of Fine specificity of equine infectious anaemia virus gp90-specific antibodies associated with protective and enhancing immune responses in experimentally infected and immunized ponies

Journal of General Virology, 1996

Equine infectious anaemia virus (EIAV) provides a model for examining the natural immunological c... more Equine infectious anaemia virus (EIAV) provides a model for examining the natural immunological control of a persistent lentivirus infection and for evaluating the efficacy of various vaccine strategies. As an initial characterization of antibody responses associated with protective or enhancing immune responses elicited by experimental infections or vaccinations, we have utilized synthetic peptide ELISA to characterize the fine specificity of antibodies to linear determinants of the EIAV surface glycoprotein, gp90. The data indicated that serum antibodies associated with protective or enhancing immune responses differed quantitatively and qualitatively in their pattern of reactivity to gp90 peptides. Protective and enhancing EIAV vaccines could also be distinguished by their ability to evoke anamnestic antibody responses to gp90 peptides. These studies demonstrate for the first time definitive differences in the specificity of protective and enhancing antibody responses to EIAV and emphasize the importance of using native viral glycoprotein immunogens in lentivirus vaccines.

Research paper thumbnail of Ectopic miR-125a Expression Induces Long-Term Repopulating Stem Cell Capacity in Mouse and Human Hematopoietic Progenitors

Cell Stem Cell, 2016

Highlights d Enforced expression of miR-125 increases hematopoietic stem frequency in vivo d miR-... more Highlights d Enforced expression of miR-125 increases hematopoietic stem frequency in vivo d miR-125 induces stem cell potential in murine and human progenitor cells d miR-125 represses, among others, targets of the MAP kinase signaling pathway d miR-125 function and targets are conserved in human and mouse

Research paper thumbnail of Contralateral Control Knees Disease in Both Injected and Experimental Arthritis Ameliorates Viral IL10 to Rabbit Knees with Direct Adenoviral Gene Transfer of

Research paper thumbnail of Injected and Uninjected Paws Collagen-Induced Arthritis in Both Suppresses Development of Gene Periarticularly to Mouse Paws Adenoviral Transfer of the Viral IL10

Research paper thumbnail of Abstract 2292: Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells

Research paper thumbnail of Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry

The American journal of physiology, 1996

In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimu... more In several settings in vivo, prolonged inhibition of apical Na+ entry reduces and prolonged stimulation of apical entry enhances the ability of renal epithelial cells to reabsorb Na+, an important feature of the load-dependent regulation of renal tubular Na+ transport. To model this load dependency, apical Na+ entry was inhibited or stimulated for 18 h in A6 cells and vectorial transport was measured as short-circuit current (Isc) across monolayers on filter-bottom structures. Basal amiloride-sensitive Isc represents the activity of apical Na+ channels, whereas Isc after permeabilization of the apical membrane to cations with nystatin represents maximal activity of the basolateral Na(+)-K(+)-ATPase. Chronic inhibition of apical Na+ entry by 18-h apical exposure to amiloride or replacement of apical Na+ with tetramethylammonium (TMA+), followed by washing and restoration of normal apical medium, revealed a persistent decrease in Isc that remained despite exposure to nystatin. Both ba...

Research paper thumbnail of Human, viral or mutant human IL-10 expressed after local adenovirus-mediated gene transfer are equally effective in ameliorating disease pathology in a rabbit knee model of antigen-induced arthritis

Arthritis research & therapy, 2006

IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral r... more IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral responses. Human IL-10 (hIL-10) has anti-inflammatory, immunosuppressive as well as immunostimulatory characteristics, whereas viral IL-10 (vIL-10), a homologue of hIL-10 encoded by Epstein Barr virus (EBV), lacks several immunostimulatory functions. The immunostimulatory characteristic of hIL-10 has been attributed to a single amino acid, isoleucine at position 87, which in vIL-10 is alanine. A mutant hIL-10 in which isoleucine has been substituted (mut.hIL-10) is biologically active with only immunosuppressive, but not immunostimulatory, functions, making it a potentially superior therapeutic for inflammatory diseases. To compare the efficacy of mut.hIL-10 with hIL-10 and vIL-10 in blocking the progression of rheumatoid arthritis, we used replication defective adenoviral vectors to deliver intra-articularly the gene encoding hIL-10, vIL-10 or mut.hIL-10 to antigen-induced arthritic (AIA...

Research paper thumbnail of Ex vivo gene delivery of IL-1Ra and soluble TNF receptor confers a distal synergistic therapeutic effect in antigen-induced arthritis

Molecular therapy : the journal of the American Society of Gene Therapy, 2002

Intra-articular expression of antagonists of interleukin-1beta (IL-1beta) and tumor necrosis fact... more Intra-articular expression of antagonists of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in arthritic rabbit knee and mouse ankle joints by direct adenoviral-mediated intraarticular delivery results in amelioration of disease pathology in both the treated and contralateral untreated joints. Previous experiments suggest that direct adenoviral infection of resident antigen-presenting cells (APCs) and subsequent traveling of these cells to other sites of inflammation and lymph nodes might be responsible for this "contralateral effect." To determine whether genetic modification of APCs is required for the contralateral effect, we have used an ex vivo approach utilizing genetically modified fibroblasts to express IL-1 receptor antagonist protein (IL-1Ra) and soluble TNF-alpha receptor (sTNFR) locally in arthritic joints. Retroviral vectors carrying IL-1Ra, sTNFR-Ig, or both genes together were used to stably infect autologous rabbit fibroblasts that...

Research paper thumbnail of Lessons learned from gene transfer approaches

Arthritis research, 1999

Recent technological advances allow the transfer of genes to the synovial lining of joints. As we... more Recent technological advances allow the transfer of genes to the synovial lining of joints. As well as opening novel opportunities for therapy, these techniques provide valuable new tools for the study of synovitis and other aspects of the biology of joints in health and disease. This article reviews briefly the results of experiments in which selected genes have been transferred to the knee joints of healthy rabbits and rabbits with antigen-induced arthritis.

Research paper thumbnail of Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1999

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery ... more Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatme...

Research paper thumbnail of Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees

Arthritis research & therapy, 2003

To examine the effect of transforming growth factor (TGF)-beta1 on the regulation of cartilage sy... more To examine the effect of transforming growth factor (TGF)-beta1 on the regulation of cartilage synthesis and other articular pathologies, we used adenovirus-mediated intra-articular gene transfer of TGF-beta1 to both naïve and arthritic rabbit knee joints. Increasing doses of adenoviral vector expressing TGF-beta1 were injected into normal and antigen-induced arthritis rabbit knee joints through the patellar tendon, with the same doses of an adenoviral vector expressing luciferase injected into the contralateral knees as the control. Intra-articular injection of adenoviral vector expressing TGF-beta1 into the rabbit knee resulted in dose-dependent TGF-beta1 expression in the synovial fluid. Intra-articular TGF-beta1 expression in both naïve and arthritic rabbit knee joints resulted in significant pathological changes in the rabbit knee as well as in adjacent muscle tissue. The observed changes induced by elevated TGF-beta1 included inhibition of white blood cell infiltration, stimul...

Research paper thumbnail of Identification of Hematopoietic Stem Cell-Specific miRNAs Enables Gene Therapy of Globoid Cell Leukodystrophy

Science Translational Medicine, 2010

Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal ... more Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.

Research paper thumbnail of Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor   soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Proceedings of the National Academy of Sciences, 1998

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I rece... more Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor alpha (TNFalpha) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFalpha receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

Research paper thumbnail of Stem cell gene expression programs influence clinical outcome in human leukemia

Nature Medicine, 2011

Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarc... more Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.

Research paper thumbnail of Identification of a synovial fibroblast-specific protein transduction domain for delivery of apoptotic agents to hyperplastic synovium

Molecular Therapy, 2003

Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pat... more Synovial hyperplasia, resulting in erosion of cartilage and bone, represents one of the major pathologies associated with rheumatoid arthritis. To develop an approach for efficient delivery of proteins or agents to synovium to induce targeted apoptosis of hyperplastic synovial tissue, we have screened an M13 peptide phage display library for synovial-specific transduction peptides. We identified a novel synovial-targeted transduction peptide, HAP-1, which is able to facilitate specific internalization of protein complexes into human and rabbit synovial cells in culture and rabbit synovial lining in vivo. HAP-1 and a non-tissue-specific cationic protein transduction domain, PTD-5, were fused to an antimicrobial peptide, (KLAK)(2), to generate two proapoptotic peptides termed DP2 and DP1, respectively. Administration of these peptides was able to induce apoptosis of rabbit and human synovial cells in culture, with DP2 inducing synovial cell-specific apoptosis. Intra-articular injection of DP1 and DP2 into arthritic rabbit joints with synovial hyperplasia induced extensive apoptosis of the hyperplastic synovium, while reducing the leukocytic infiltration and synovitis. These results suggest that proapoptotic peptides and, in particular, DP2 can be clinically useful for treatment of synovial hyperplasia, as well as inflammation. Moreover, the results demonstrate the feasibility of identifying tissue-specific transduction peptides capable of mediating efficient transduction in vivo.

Research paper thumbnail of Exosomes Derived from Genetically Modified DC Expressing FasL Are Anti-inflammatory and Immunosuppressive

Molecular Therapy, 2006

We previously have demonstrated the ability of primary murine bone marrow-derived DC (BM-DC), gen... more We previously have demonstrated the ability of primary murine bone marrow-derived DC (BM-DC), genetically modified by adenoviral infection to express FasL, to inhibit progression of established collagen-induced arthritis (CIA) following systemic delivery. Here we demonstrate that exosomes derived from genetically modified BM-DC expressing FasL are able to inhibit inflammation in a murine footpad model of delayed-type hypersensitivity (DTH). Local administration of exosomes derived from DC expressing FasL (Exo/FasL) as well as the parental DC/FasL resulted in a significant reduction in swelling in both the treated and the untreated distal paw. However, both the DC/FasL and the Exo/FasL were unable to suppress the DTH response in lpr (Fas-deficient) mice. Gene transfer of FasL to BM-DC from gld (FasL-deficient) mice resulted in restoration of the ability of DC as well as DC-derived exosomes to suppress DTH. The ability of DCderived exosomes and DC to suppress DTH responses was antigen specific and MHC class II dependent, but class I independent. The injected exosomes were found to be internalized into CD11c + cells at the site of injection and in the draining popliteal lymph node. Systemic injection of exosome/FasL into mice with established CIA resulted in significant disease amelioration. These results demonstrate that both systemic and local administration of exosomes derived from FasLexpressing DC are able to suppress antigen-specific immune responses through an MHC class IIdependent pathway, resulting in effective and sustained treatment of established collageninduced arthritis and suppression of the DTH inflammatory response. These results suggest that DC/FasL-derived exosomes could be used clinically for the treatment of inflammatory and autoimmune diseases.

Research paper thumbnail of Gene Delivery of IL-1Ra and Soluble TNF Receptor Confers a Distal Synergistic Therapeutic Effect in Antigen-Induced Arthritis

Research paper thumbnail of Zebrafish microRNA-126 determines hematopoietic cell fate through c-Myb

Leukemia, 2011

Precise regulatory mechanisms are required to appropriately modulate the cellular levels of trans... more Precise regulatory mechanisms are required to appropriately modulate the cellular levels of transcription factors controlling cell fate decisions during blood cell development. Here, we show that miR-126 is a novel physiological regulator of the proto-oncogene c-myb during definitive hematopoiesis. We show that knockdown of miR-126 results in increased c-Myb levels and promotes erythropoiesis at the expense of thrombopoiesis in vivo. We further provide evidence that specification of thrombocyte versus erythrocyte cell lineages is altered by the concerted activities of the miRNAs miR-126 and miR-150. Both microRNAs are required but not sufficient individually to precisely regulate the cell fate decision between erythroid and megakaryocytic lineages during definitive hematopoiesis in vivo. These results support the notion that microRNAs not only act to provide precision to developmental programs but also are essential determinants in the control of variable potential functions of a single gene during hematopoiesis.

Research paper thumbnail of Exosomes Derived from IL-10-Treated Dendritic Cells Can Suppress Inflammation and Collagen-Induced Arthritis

The Journal of Immunology, 2005

Research paper thumbnail of Element-tagged immunoassay with ICP-MS detection: Evaluation and comparison to conventional immunoassays

Journal of Immunological Methods, 2008

We have investigated the possibility of using element-tagged antibodies for protein detection and... more We have investigated the possibility of using element-tagged antibodies for protein detection and quantification in microplate format using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and compared the results to conventional immunoassays, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. The technique was further employed to detect low levels and measure DNA-binding activity of transcription factor p53 in leukemia cell lysates through its interaction with immobilized oligonucleotides and recognition by element-tagged antibodies. The advantages of ICP-MS detection for routine performance of immunoassays include increased sensitivity, wide dynamic range, minimal interference from complex matrices, and high throughput. Our approach advances the ICP-MS technology and demonstrates its applicability to proteomic studies through the use of antibodies directly labeled with polymer tags bearing multiple atoms of lanthanides. Development of this novel methodology will enable fast and quantitative identification of multiple analytes in a single well.

Research paper thumbnail of Fine specificity of equine infectious anaemia virus gp90-specific antibodies associated with protective and enhancing immune responses in experimentally infected and immunized ponies

Journal of General Virology, 1996

Equine infectious anaemia virus (EIAV) provides a model for examining the natural immunological c... more Equine infectious anaemia virus (EIAV) provides a model for examining the natural immunological control of a persistent lentivirus infection and for evaluating the efficacy of various vaccine strategies. As an initial characterization of antibody responses associated with protective or enhancing immune responses elicited by experimental infections or vaccinations, we have utilized synthetic peptide ELISA to characterize the fine specificity of antibodies to linear determinants of the EIAV surface glycoprotein, gp90. The data indicated that serum antibodies associated with protective or enhancing immune responses differed quantitatively and qualitatively in their pattern of reactivity to gp90 peptides. Protective and enhancing EIAV vaccines could also be distinguished by their ability to evoke anamnestic antibody responses to gp90 peptides. These studies demonstrate for the first time definitive differences in the specificity of protective and enhancing antibody responses to EIAV and emphasize the importance of using native viral glycoprotein immunogens in lentivirus vaccines.