Tim Townes - Academia.edu (original) (raw)

Papers by Tim Townes

Antioxidants & Redox Signaling, Feb 1, 2015

Aims: Transfusion with stored red blood cells (RBCs) is associated with increased morbidity and m... more Aims: Transfusion with stored red blood cells (RBCs) is associated with increased morbidity and mortality. Peroxiredoxin-2 (Prx-2) is a primary RBC antioxidant that limits hydrogen peroxide (H 2 O 2)-mediated toxicity. Whether Prx-2 activity is altered during RBC storage is not known. Results: Basal and H 2 O 2-induced Prx-2 activity was measured in RBCs (stored for 7-35 days). Basal Prx-2 thiol oxidation increased with RBC age, whereas H 2 O 2-dependent formation of dimeric Prx-2 was similar. However, reduction of Prx-2 dimers to monomers became progressively slower with RBC storage, which was associated with increased H 2 O 2-induced hemolysis. Surprisingly, no change in the NADPH-dependent thioredoxin (Trx)/Trx-reductase system, which recycles dimeric Prx-2, was observed in stored RBCs. Using mouse RBCs expressing human wild type (b93Cys) or hemoglobin (Hb) in which the conserved b93Cys residue is replaced by Ala (b93Ala), a role for this thiol in modulating Prx-2 reduction was demonstrated. Specifically, Prx-2 recycling was blunted in b93Ala RBC, which was reversed by carbon monoxide-treatment, suggesting that heme autoxidation-derived H 2 O 2 maintains Prx-2 in the oxidized form in these cells. Moreover, assessment of the oxidative state of the b93Cys in RBCs during storage showed that while it remained reduced on intraerythrocytic Hb in stored RBC, it was oxidized to dehydroalanine on hemolyzed or extracellular Hb. Innovation: A novel mechanism for regulated Prx-2 activity in RBC via the b93Cys residue is suggested. Conclusion: These data highlight the potential for slower Prx-2 recycling and b93Cys oxidation in modulating storage-dependent damage of RBCs and in mediating post-transfusion toxicity. Antioxid. Redox Signal. 22, 294-307.

Blood, May 31, 2012

fective erythropoiesis during iron deficiency with inhibition of differentiation at the basophili... more fective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri Ϫ/Ϫ fetal liver erythroid progenitors. Importantly, the Hri-eIF2␣P-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2␣P-Atf4 signaling with chemical compounds as pharmaceutical therapies for ␤-thalassemia. (Blood. 2012; 119(22):5276-5284)

Nature Communications, Mar 7, 2016

It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as ob... more It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, it is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming and show that mitosis may be a driving force of reprogramming.

Stem Cells, Feb 12, 2009

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts usin... more We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3′ long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic “hit and run” vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells. Disclosure of potential conflicts of interest is found at the end of this article.

Diabetes, Aug 18, 2009

OBJECTIVE-We evaluate a potential role of activating transcription factor 4 (Atf4) in invertebrat... more OBJECTIVE-We evaluate a potential role of activating transcription factor 4 (Atf4) in invertebrate and mammalian metabolism. RESEARCH DESIGN AND METHODS-With two parallel approaches-a fat body-specific green fluorescent protein enhancer trap screen in D. melanogaster and expression profiling of developing murine fat tissues-we identified Atf4 as expressed in invertebrate and vertebrate metabolic tissues. We assessed the functional relevance of the evolutionarily conserved expression by analyzing Atf4 mutant flies and Atf4 mutant mice for possible metabolic phenotypes. RESULTS-Flies with insertions at the Atf4 locus have reduced fat content, increased starvation sensitivity, and lower levels of circulating carbohydrate. Atf4 null mice are also lean, and they resist age-related and diet-induced obesity. Atf4 null mice have increased energy expenditure potentially accounting for the lean phenotype. Atf4 null mice are hypoglycemic, even before substantial changes in fat content, indicating that Atf4 regulates mammalian carbohydrate metabolism. In addition, the Atf4 mutation blunts diet-induced diabetes as well as hyperlipidemia and hepatosteatosis. Several aspects of the Atf4 mutant phenotype resemble mice with mutations in components of the target of rapamycin (TOR) pathway. Consistent with the phenotypic similarities, Atf4 null mice have reduced expression of genes that regulate intracellular amino acid concentrations and lower intracellular concentration of amino acids, a key TOR input. Further, Atf4 mutants have reduced S6K activity in liver and adipose tissues. CONCLUSIONS-Atf4 regulates age-related and diet-induced obesity as well as glucose homeostasis in mammals and has conserved metabolic functions in flies.

Blood, May 23, 2019

In this issue of Blood, Xu et al describe a novel CRISPR/Cas approach for correcting b-thalassemi... more In this issue of Blood, Xu et al describe a novel CRISPR/Cas approach for correcting b-thalassemias that result from aberrant donor or acceptor splice sites. 1 The authors designed guide RNA/Cas complexes that can be introduced efficiently into hematopoietic stem and progenitor cells (HSPCs) ex vivo without viral vectors and can create double-stranded DNA cuts at specific genomic sites. The cells rapidly repair these double-stranded breaks by nonhomologous endjoining, which typically results in small deletions around the cut site. If these deletions encompass the aberrant splice sites and avoid additional sequences necessary for efficient splicing (see figure), normal splicing can be restored.

Blood, Nov 16, 2004

Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the tr... more Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the treatment of hemoglobinopathies such as β thalassemia and sickle cell disease. One genetic strategy for the correction of β thalassemia is to replace mutant or deleted β-globin alleles with a wild-type gene by homologous recombination in ES cells. Thalassemic mice that mimic the disorder have been generated by targeted gene deletion of the adult murine β-globin genes (PNAS 92: 9259–9263). We derived ES cells from our β-globin knockout mice and produced genetically identical mutant mice by injecting the ES cells into tetraploid embryos. These cloned β thalassemic mice have a severe microcytic anemia characterized by a marked reduction of the erythrocyte mean corpuscular volume (MCV), hemoglobin level (Hb), and hematocrit (Hct), and a marked increase in reticulocytes and red cell distribution width (RDW) compared to cloned wild-type control animals. In contrast to the normochromic, normocytic erythrocytes of wild-type clones, erythrocytes in peripheral blood smears of β thalassemic mice were hypochromic and exhibit extreme anisopoikilocytosis. A targeting construct containing 8.7 kb of mouse homology flanking a human γ- and β-globin gene cassette and a hygromycin marker gene was electroporated into the β thalassemic ES cells. After selection, DNA from 48 ES cell colonies was analyzed by PCR to identify homologous recombinants. Nineteen colonies (40%) had correctly integrated the human globin genes into the deleted mouse β-globin locus. Correctly targeted cells were injected into tetraploid blastocysts to produce mice that are derived solely from the corrected ES cells. These cloned mice synthesize high levels of human β-globin polypeptide that corrects the α- to β-globin chain imbalance, thereby eliminating the thalassemic erythrocyte morphology. The MCV, Hb, Hct, RDW, and reticulocyte levels in the blood of these mice are normal. These results demonstrate that a severe hemoglobinopathy can be cured after targeted gene replacement of a mutant gene(s) with a wild-type allele by homologous recombination in ES cells.

JCI insight, Feb 19, 2019

PubMed, 1985

Goats switch their hemoglobins during development in a manner similar to humans and thus provide ... more Goats switch their hemoglobins during development in a manner similar to humans and thus provide a useful model system for studying the control of hemoglobin synthesis. Initially, goats synthesize embryonic hemoglobin, zeta 2 epsilon 2, which is replaced by fetal hemoglobin, alpha 2 beta F 2, as erythropoiesis moves to the liver and bone marrow. At birth, the fetal hemoglobin is replaced by juvenile hemoglobin, alpha 2 beta C 2, which in turn is replaced by adult hemoglobin, alpha 2 beta A 2, during the first year of life. In order to understand these switches, we have cloned the alpha and beta globin loci of goats. The alpha globin locus is composed of three genes, an embryonic and two adult genes, zeta-I alpha-II alpha. The beta globin locus is composed of twelve genes arranged in the following order, epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F. Close inspection of the beta globin locus indicates that it has arisen from a triplication of a four-gene set, epsilon-epsilon-beta-beta. Interestingly, the fetal globin gene has originated from an adult beta globin gene rather than from a second position gene as it has in humans. The gene at the end of the first four gene set, beta C, is expressed during pre-adult life while the gene at the end of the second set is the adult beta A gene. The last gene of the third set, beta F, is expressed during fetal development. Because the beta C, beta A and beta F genes have arisen quite recently during evolution, they have very similar nucleotide sequences. It is reasonable to assume that the few differences which are seen are important in developmental control. As one approach to defining regions involved in the regulation of the beta A, beta C and beta F genes their chromatin structure at different times of development has been characterized. Both DNase I sensitivity and accessibility to restriction endonucleases have been employed. While the entire beta globin locus is more sensitive to DNase in erythroid than non-erythroid cells, specific regions such as the 5' end of the genes are more accessible in cells expressing that particular gene.

PubMed, 2013

Two of the proposed mechanisms by which red blood cells (RBC) mediate hypoxic vasorelaxation by c... more Two of the proposed mechanisms by which red blood cells (RBC) mediate hypoxic vasorelaxation by coupling hemoglobin deoxygenation to the activation of nitric oxide signaling involve ATP-release from RBC and S-nitrosohemoglobin (b93C(SNO)Hb) dependent bioactivity. However, different studies have reached opposite conclusions regarding the aforementioned mechanisms. Using isolated vessels, hypoxic vasorelaxation induced by human, C57BL/6 or mouse RBC which exclusively express either native human hemoglobin (HbC93) or human hemoglobin in which the conserved b93cys was replaced with Ala (HbC93A) were compared. All RBCs stimulated hypoxic vasodilation to similar extents suggesting the b93cys is not required for this RBC-mediated function. Hypoxic vasorelaxation was inhibited by co-incubation of ATP-pathway blockers including L-NAME (eNOS inhibitor) and Apyrase. Moreover, we tested if modulation of adenosine-dependent signaling affected RBC-dependent vasorelaxation using pan- or subtype specific adenosine receptor blockers, or adenosine deaminase (ADA). Interestingly, ADA and adenosine A2 receptor blockade, but not A1 receptor blockade, inhibited HbC93, HbC93A dependent hypoxic vasorelaxation. Equivalent results were obtained with human RBC. These data suggest that using isolated vessels, RBC do not require the presence of the b93cys to elicit hypoxic vasorelaxation and mediate this response via ATP- and a novel adenosine-dependent mechanism.

Stem Cells Translational Medicine, Feb 24, 2017

Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated b... more Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) CC chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat-induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS-ECs overexpressing IL8RA/B (RiPS-IL8RA/B-ECs), CCR2/5 (RiPS-CCR2/5-ECs), or both receptors (RiPS-IL8RA/B1CCR2/5-ECs) will inhibit inflammatory responses and neointima formation in balloon-injured rat carotid artery. Twelve-week-old male Sprague-Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS-Null-ECs (ECs transduced with empty virus), (c) RiPS-IL8RA/B-ECs, (d) RiPS-CCR2/5-ECs, or (e) RiPS-IL8RA/B1CCR2/5-ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS-ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injuryinduced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS-ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS-ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases.

Hematology, 2008

Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse model... more Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing anti-sickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (β β β β β S) with a normal copy of the gene (β β β β β A). We recently derived embryonic stem (ES) cells from a novel knock-in mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. Animals derived after gene replacement produced high levels of normal human hemoglobin (HbA), and the pathology associated with SCD was corrected. These experiments provided a foundation for similar studies in which our group collaborated with Rudolf Jaenisch's laboratory to correct SCD by gene replacement in iPS (induced pluripotent stem) cells derived by direct reprogramming of sickle skin fibroblasts. Corrected iPS cells were differentiated into hematopoeitic progenitors that were transplanted into irradiated sickle recipients. The transplanted animals produced high levels of normal human HbA, and the pathology of SCD was corrected. These proof-of-principle studies provide a foundation for the development of gene replacement therapy for human patients with SCD and other blood disorders.

PLOS ONE, May 14, 2014

Human induced pluripotent stem cells (hiPSCs) have enormous potential for the treatment of inheri... more Human induced pluripotent stem cells (hiPSCs) have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR) diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The cd TCR locus is rearranged first and is followed by rearrangement of the ab locus. Both cd and ab T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-a, IFN-c, IL-2) and cytolytic proteins (Perforin and Granzyme-B). These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy.

Journal of The American Society of Nephrology, Mar 27, 2017

Sickle cell disease (SCD)-associated nephropathy is a major source of morbidity and mortality in ... more Sickle cell disease (SCD)-associated nephropathy is a major source of morbidity and mortality in patients because of the lack of efficacious treatments targeting renal manifestations of the disease. Here, we describe a long-term treatment strategy with the selective endothelin-A receptor (ET A) antagonist, ambrisentan, designed to interfere with the development of nephropathy in a humanized mouse model of SCD. Ambrisentan preserved GFR at the level of nondisease controls and prevented the development of proteinuria, albuminuria, and nephrinuria. Microscopy studies demonstrated prevention of podocyte loss and structural alterations, the absence of vascular congestion, and attenuation of glomerulosclerosis in treated mice. Studies in isolated glomeruli showed that treatment reduced inflammation and oxidative stress. At the level of renal tubules, ambrisentan treatment prevented the increased excretion of urinary tubular injury biomarkers. Additionally, the treatment strategy prevented tubular brush border loss, diminished tubular iron deposition, blocked the development of interstitial fibrosis, and prevented immune cell infiltration. Furthermore, the prevention of albuminuria in treated mice was associated with preservation of cortical megalin expression. In a separate series of identical experiments, combined ET A and ET B receptor antagonism provided only some of the protection observed with ambrisentan, highlighting the importance of exclusively targeting the ET A receptor in SCD. Our results demonstrate that ambrisentan treatment provides robust protection from diverse renal pathologies in SCD mice, and suggest that longterm ET A receptor antagonism may provide a strategy for the prevention of renal complications of SCD.

Journal of Biological Chemistry, Apr 1, 2004

Trends in Genetics, 1990

A region of DNA located far upstream of the human beta-globin locus is critically involved in the... more A region of DNA located far upstream of the human beta-globin locus is critically involved in the regulation of the beta-globin gene family. Recent experiments in transgenic mice suggest that switching from fetal to adult globin gene expression during human development results from competition among individual globin gene family members for interaction with sequences in this region. The phenotypes of patients with defined hemoglobinopathies support this hypothesis.

Free Radical Biology and Medicine, Nov 1, 2014