- Allergy and Immunology
- Pediatric Allergy/Immun
Assistant Professor, Pediatrics Immunology & Allergy (2009 - Present)
Honors & Awards
Fellow, American Academy of Pediatrics (2008-)
Scholar, SPARK (2010-11,11-13,14-15)
Award, Bio-X Interdisciplinary Award (2010)
Boards, Advisory Committees, Professional Organizations
Defense Sciences Study Group, US Department of Defense (2014 - Present)
Education Committee, Clinical Immunology Society (2014 - Present)
Annual Retreat Co-Chair, Stanford Immunology (2012 - 2013)
Fellowship:Children's Hospital Boston (2006) MA
Residency:Children's Hospital of Philadelphia (2003) PA
Medical Education:Brown University - School of Medicine (1996) RI
Board Certification: Pediatrics, American Board of Pediatrics (2004)
Board Certification: Allergy and Immunology, American Board of Allergy and Immunology (2005)
Ph.D., UCSF, Biophysics (2000)
M.D., Brown Univ. School of Medicine, Medicine (1996)
Sc.B., Brown University, Physics (1993)
Manish J Butte, Marc Amor Bruce, Jianwei Liu. "United States Patent US 13/307,882 Atomic force microscope manipulation of living cells", The Board Of Trustees Of The Leland Stanford Junior University, Nov 30, 2010
Manish J. BUTTE, John J. LeBlanc. "United States Patent US 13/317,180 Microfluidic Waveguide Detector", The Board Of Trustees Of The Leland Stanford Junior University, Oct 12, 2010
Arlene H. Sharpe, Manish J. Butte, Shinji Oyama. "United States Patent PCT/US2011/020046 Modulators of Immunoinhibitory Receptor PD-1, and Methods of Use Thereof", President And Fellows Of Harvard College, Jan 4, 2010
Meital Reches, Michael D. Dickey, Manish J. Butte, George M. Whitesides. "United States Patent PCT/US2009/038702 Cotton thread as a low-cost multi-assay diagnostic platform", President And Fellows Of Harvard College, Mar 27, 2008
George M. Whitesides, Scott T. PHILLIPS, Andreas W. Martinez, Manish J. BUTTE, Amy Wong, Samuel W. Thomas, Hayat Sindi, Sarah J. VELLA, Emanuel CARRILHO, Katherine A. MIRICA, Yanyan LIU,. "United States Patent US 13/730,028 Lateral flow and flow-through bioassay devices based on patterned porous media, methods of making same, and methods of using same", President And Fellows Of Harvard College, Oct 18, 2006
Current Research and Scholarly Interests
Our laboratory's goal is to address fundamental and therapeutic questions in immunology using innovative nanotechnological and biophysical approaches to visualize and manipulate cells. Our primary focus is on understanding the molecular controls that balance T cell activation versus tolerance. The ultimate aim of our work is to manipulate T cell signaling pathways to control immunologically-mediated diseases.
Projects in the lab include:
1) Atomic force microscopy of T cells
2) Mechanobiology of biomaterials, cardiac cells, endothelial cells, and cancers
3) Discovery and characterization of new inhibitory pathways of T cells
4) High resolution visualization of receptor and ligands in the T cell immunological synapse
5) Manipulation of single-receptors on T cells to control activation versus tolerance
6) Development of diagnostic tools for immune monitoring and autoimmune diseases (such as Type 1 diabetes and multiple sclerosis)
7) Characterization of self-like antigens and their roles in autoimmunity (in multiple sclerosis and Type 1 diabetes)
Independent Studies (14)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr)
- Directed Reading in Pediatrics
PEDS 299 (Aut, Win, Spr)
- Early Clinical Experience
PEDS 280 (Aut, Win, Spr)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr)
- Graduate Research
PEDS 399 (Aut, Win, Spr)
- Medical Scholars Research
PEDS 370 (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr)
- Ph.D. Research
MATSCI 300 (Aut, Win, Spr, Sum)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr)
- Undergraduate Directed Reading/Research
PEDS 199 (Aut, Win, Spr)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr)
- Directed Reading in Biophysics
- Prior Year Courses
Compound Heterozygous Mutation of Rag1 Leading to Omenn Syndrome
2015; 10 (4)
Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in "non-core" regions of the Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a "core" region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination.
View details for DOI 10.1371/journal.pone.0121489
View details for Web of Science ID 000352477800062
View details for PubMedID 25849362
Taking T cell priming down a Notch: signaling through Notch receptors enhances T cell sensitivity to antigen.
2015; 42 (1): 6-8
Notch receptors are widely expressed and have recognized functions in thymocytes and mature T cells. In this issue, Laky et al. (2015) show that Notch interactions with Delta-like ligand 4 (DLL4) amplify priming of naive T cells.
View details for DOI 10.1016/j.immuni.2014.12.026
View details for PubMedID 25607451
- Customized atomic force microscopy probe by focused-ion-beam-assisted tip transfer APPLIED PHYSICS LETTERS 2014; 105 (5)
Use of bio-mimetic three-dimensional technology in therapeutics for heart disease.
2014; 5 (3): 193-197
Due to the limited self-renewal capacity of cardiomyocytes, the mammalian heart exhibits impaired regeneration and insufficient ability to restore heart function after injury. Cardiovascular tissue engineering is currently considered as a promising alternative therapy to restore the structure and function of the failing heart. Recent evidences suggest that the epicardium may play critical roles in regulation of myocardial development and regeneration. One of the mechanisms has been proposed for the restorative effect of the epicardium is the specific physiomechanical cues that this layer provides to the cardiac cells. In this article we explore whether a new generation of epicardium-mimicking, acellular matrices can be utilized to enhance cardiac healing after injury. The matrix consists of a dense collagen scaffold, with optimized biomechanical properties approaching those of embryonic epicardium. Grafting the epicardial patch onto the ischemic myocardium, promptly post the incidence of infarct, resulted in preserved contractility, attenuated ventricular remodeling, diminished fibrosis, and vascularization within the injured tissue in the adult murine heart.
View details for DOI 10.4161/bioe.27751
View details for PubMedID 24637710
Spatiotemporally and mechanically controlled triggering of mast cells using atomic force microscopy
2014; 58 (2-3): 211-217
Mast cells are thought to be sensitive to mechanical forces, for example, coughing in asthma or pressure in "physical urticarias." Conversion of mechanical forces to biochemical signals could potentially augment antigenic signaling. Studying the combined effects of mechanical and antigenic cues on mast cells and other hematopoietic cells has proven difficult. Here, we present an approach using a modified atomic force microscope cantilever to deliver antigenic signals to mast cells while simultaneously applying mechanical forces. We developed a strategy to concurrently record degranulation events by fluorescence microscopy during antigenic triggering. Finally, we also measured the mechanical forces generated by mast cells while antigen receptors are ligated. We showed that mast cells respond to antigen delivered by the atomic force microscopy cantilever with prompt degranulation and the generation of strong pushing and pulling forces. We did not discern any relationship between applied mechanical forces and the kinetics of degranulation. These experiments present a new method for dissecting the interactions of mechanical and biochemical cues in the signaling responses of immune cells.
View details for DOI 10.1007/s12026-014-8510-7
View details for Web of Science ID 000336333700006
Real-time GPU-based 3D Deconvolution
2013; 21 (4): 4766-4773
Confocal microscopy is an oft-used technique in biology. Deconvolution of 3D images reduces blurring from out-of-focus light and enables quantitative analyses, but existing software for deconvolution is slow and expensive. We present a parallelized software method that runs within ImageJ and deconvolves 3D images ~100 times faster than conventional software (few seconds per image) by running on a low-cost graphics processor board (GPU). We demonstrate the utility of this software by analyzing microclusters of T cell receptors in the immunological synapse of a CD4 + T cell and dendritic cell. This software provides a low-cost and rapid way to improve the accuracy of 3D microscopic images obtained by any method.
View details for DOI 10.1364/OE.21.004766
View details for Web of Science ID 000315992600083
CD28 Costimulation Regulates Genome-Wide Effects on Alternative Splicing
2012; 7 (6)
CD28 is the major costimulatory receptor required for activation of naïve T cells, yet CD28 costimulation affects the expression level of surprisingly few genes over those altered by TCR stimulation alone. Alternate splicing of genes adds diversity to the proteome and contributes to tissue-specific regulation of genes. Here we demonstrate that CD28 costimulation leads to major changes in alternative splicing during activation of naïve T cells, beyond the effects of TCR alone. CD28 costimulation affected many more genes through modulation of alternate splicing than by modulation of transcription. Different families of biological processes are over-represented among genes alternatively spliced in response to CD28 costimulation compared to those genes whose transcription is altered, suggesting that alternative splicing regulates distinct biological effects. Moreover, genes dependent upon hnRNPLL, a global regulator of splicing in activated T cells, were enriched in T cells activated through TCR plus CD28 as compared to TCR alone. We show that hnRNPLL expression is dependent on CD28 signaling, providing a mechanism by which CD28 can regulate splicing in T cells and insight into how hnRNPLL can influence signal-induced alternative splicing in T cells. The effects of CD28 on alternative splicing provide a newly appreciated means by which CD28 can regulate T cell responses.
View details for DOI 10.1371/journal.pone.0040032
View details for Web of Science ID 000305892100181
View details for PubMedID 22768209
Atomic Force Mechanobiology of Pluripotent Stem Cell-Derived Cardiomyocytes
2012; 7 (5)
We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent, stem cell-derived cardiomyocytes, including contraction force, rate, duration, and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy, and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive, inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents, or as a platform for studying cardiomyocyte biology.
View details for DOI 10.1371/journal.pone.0037559
View details for Web of Science ID 000305343500135
View details for PubMedID 22624048
Counting cells with a low-cost integrated microfluidics-waveguide sensor
2012; 6 (1)
The capability to count cells from biofluids at low cost has important diagnostic implications in resource-poor settings. Many approaches have been developed to address this important need, and while most envision a low per-test cost, the detector instrument can be quite expensive. In this report, we present a novel device that enables low-cost and rapid counting of cells from a drop of blood. We demonstrate a shallow, buried, planar waveguide fabricated by ion exchange in glass that underlies a microfluidic structure for capturing cells. Laser light transmitted through the waveguide was attenuated by the number of metal nanoparticles tagged to the cells because of the interaction of the metal particles with the evanescent field of the waveguide. Calibration of the sensor using bead-tagged lymphocytes captured from human blood showed that the sensor could semi-quantitatively count as few as 100 cells/µL of blood. This technology enables the enumeration of specifically captured cells, allowing for a point-of-care, hand-held device for fast and affordable cell counting in screening, remote, or resource-poor settings.
View details for DOI 10.1063/1.3689857
View details for Web of Science ID 000302301000039
View details for PubMedID 22454696
Single molecule labeling of an atomic force microscope cantilever tip.
Applied physics letters
2012; 101 (16): 163705
In this paper, we present a method to functionalize the very apex of an atomic force microscope cantilever with a single or a few molecules. In force spectroscopy or interaction mapping, the cantilever must be functionalized with only a few molecules to avoid noise or spurious measurements. Here, we covalently attached single molecules to the cantilever by touching it to a paper wetted with a solution of quantum dots. The paper competes with wicking up the hydrophilic surface of the tip. This method has broad applications in scanning probe microscopy where small numbers of molecules are needed on the tip.
View details for PubMedID 23152642
Optical planar waveguide for cell counting.
Applied physics letters
2012; 100 (4): 43701-437015
Low cost counting of cells has medical applications in screening, military medicine, disaster medicine, and rural healthcare. In this report, we present a shallow, buried, planar waveguide fabricated by potassium ion exchange in glass that enables low-cost and rapid counting of metal-tagged objects that lie in the evanescent field of the waveguide. Laser light transmitted through the waveguide was attenuated proportionately to the presence of metal-coated microstructures fabricated from photoresist. This technology enables the low-cost enumeration of cells from blood, urine, or other biofluids.
View details for PubMedID 22331960
Epicardial FSTL1 reconstitution regenerates the adult mammalian heart
2015; 525 (7570): 479-?
The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.
View details for DOI 10.1038/nature15372
View details for Web of Science ID 000361599900042
View details for PubMedID 26375005
- PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY 2015; 135 (6): 1578-U294
PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity.
journal of allergy and clinical immunology
2015; 135 (6): 1578-88 e5
PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance.We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients.Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency.We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells.Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.
View details for DOI 10.1016/j.jaci.2015.01.040
View details for PubMedID 25842288
Differential fates of biomolecules delivered to target cells via extracellular vesicles.
Proceedings of the National Academy of Sciences of the United States of America
2015; 112 (12): E1433-42
Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.
View details for DOI 10.1073/pnas.1418401112
View details for PubMedID 25713383
- Differential fates of biomolecules delivered to target cells via extracellular vesicles PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2015; 112 (12): E1433-E1442
- Use of bio-mimetic three-dimensional technology in therapeutics for heart disease BIOENGINEERED 2014; 5 (3)
Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes
AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH
2014; 6 (6): 724-735
Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.
View details for Web of Science ID 000348299700009
The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction
2013; 34 (36): 9048-9055
Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.
View details for DOI 10.1016/j.biomaterials.2013.08.017
View details for Web of Science ID 000326426500006
View details for PubMedID 23992980
Polyvinylpyrrolidone microneedles enable delivery of intact proteins for diagnostic and therapeutic applications
2013; 9 (8): 7767-7774
We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy.
View details for DOI 10.1016/j.actbio.2013.04.045
View details for Web of Science ID 000322207700017
- High-Bandwidth AFM Probes for Imaging in Air and Fluid JOURNAL OF MICROELECTROMECHANICAL SYSTEMS 2013; 22 (3): 603-612
- Measurement of elastic properties in fluid using high bandwidth atomic force microscope probes APPLIED PHYSICS LETTERS 2013; 102 (10)
- Single molecule labeling of an atomic force microscope cantilever tip APPLIED PHYSICS LETTERS 2012; 101 (16)
Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells.
Stem cells and development
2012; 21 (15): 2798-2808
Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs (MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study investigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially pluripotent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10 days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka factors. Immunohistology and reverse transcription-polymerase chain reaction assays revealed that the MiPSCs expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell population and enable allogeneic cardiac stem cell therapy.
View details for DOI 10.1089/scd.2011.0435
View details for PubMedID 22530853
Apelin Enhances Directed Cardiac Differentiation of Mouse and Human Embryonic Stem Cells
2012; 7 (6)
Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors.
View details for DOI 10.1371/journal.pone.0038328
View details for Web of Science ID 000305339900024
View details for PubMedID 22675543
Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy
SCIENCE TRANSLATIONAL MEDICINE
2012; 4 (130)
Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric ?-actinin. When stimulated with a ?-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric ?-actinin distribution. Treatment with ?-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.
View details for DOI 10.1126/scitranslmed.3003552
View details for Web of Science ID 000303045900004
View details for PubMedID 22517884
- Optical planar waveguide for cell counting APPLIED PHYSICS LETTERS 2012; 100 (4)
Dependence of Avidity on Linker Length for a Bivalent Ligand-Bivalent Receptor Model System
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2012; 134 (1): 333-345
This paper describes a synthetic dimer of carbonic anhydrase, and a series of bivalent sulfonamide ligands with different lengths (25 to 69 Å between the ends of the fully extended ligands), as a model system to use in examining the binding of bivalent antibodies to antigens. Assays based on analytical ultracentrifugation and fluorescence binding indicate that this system forms cyclic, noncovalent complexes with a stoichiometry of one bivalent ligand to one dimer. This dimer binds the series of bivalent ligands with low picomolar avidities (K(d)(avidity) = 3-40 pM). A structurally analogous monovalent ligand binds to one active site of the dimer with K(d)(mono) = 16 nM. The bivalent association is thus significantly stronger (K(d)(mono)/K(d)(avidity) ranging from ~500 to 5000 unitless) than the monovalent association. We infer from these results, and by comparison of these results to previous studies, that bivalency in antibodies can lead to associations much tighter than monovalent associations (although the observed bivalent association is much weaker than predicted from the simplest level of theory: predicted K(d)(avidity) of ~0.002 pM and K(d)(mono)/K(d)(avidity) ~ 8 × 10(6) unitless).
View details for DOI 10.1021/ja2073033
View details for Web of Science ID 000301084200068
View details for PubMedID 22088143
Patient-Specific Induced Pluripotent Stem Cell as a Model for Familial Dilated Cardiomyopathy
LIPPINCOTT WILLIAMS & WILKINS. 2011
View details for Web of Science ID 000299738705089
Thread as a Matrix for Biomedical Assays
ACS APPLIED MATERIALS & INTERFACES
2010; 2 (6): 1722-1728
This paper describes the use of thread as a matrix for the fabrication of diagnostic assay systems. The kinds of thread used for this study are inexpensive, broadly available, and lightweight; some of them are already familiar materials in healthcare. Fluids wick along these threads by capillary action; no external power source is necessary for pumping. This paper demonstrates three designs for diagnostic assays that use different characteristics of the thread. The first two designs-the "woven array" and the "branching design"-take advantage of the ease with which thread can be woven on a loom to generate fluidic pathways that enable multiple assays to be performed in parallel. The third design-the "sewn array"-takes advantage of the ease with which thread can be sewn through a hydrophobic polymer sheet to incorporate assays into bandages, diapers and similar systems. These designs lead to microfluidic devices that may be useful in performing simple colorimetric assays that require qualitative results. We demonstrate the function of thread-based microfluidic devices in the context of five different colorimetric assays: detection of ketones, nitrite, protein, and glucose in artificial urine, and detection of alkaline phosphatase in artificial plasma.
View details for DOI 10.1021/am1002266
View details for Web of Science ID 000278963600027
View details for PubMedID 20496913
Interaction of human PD-L1 and B7-1
2008; 45 (13): 3567-3572
Numerous studies have pointed to the role of programmed death-1 ligand 1 (PD-L1) in regulating tolerance, chronic infection, and tumor immunity. Recently, we have identified murine B7-1 as a new binding partner for murine PD-L1. Human and mouse B7-1 share only 46% identity, leading us to question whether human B7-1 and PD-L1 can participate in a similar interaction. Here we show that human B7-1 can interact with human PD-L1 with affinity greater than that of B7-1 with CD28, but less than that of B7-1 with CTLA-4 or of PD-L1 with PD-1. We characterize a series of anti-human PD-L1 monoclonal antibodies and identify antibodies that can block interactions of PD-L1 with B7-1, PD-1, or both. Since PD-L1 and CD28 on T cells may compete for B7-1 as a binding partner and CD8 T cells may express high or low levels of CD28, we examined when PD-L1 and CD28 are co-expressed on CD8 T cells. We compared the time-course and extent of PD-L1 induction on CD8 CD28high versus CD28low T cells following stimulation with anti-CD3. We show that PD-L1 is induced to a higher level on CD28high T cells than on CD28low T cells upon activation. These results suggest that PD-L1 may play an important and undervalued role on human T cells.
View details for DOI 10.1016/j.molimm.2008.05.014
View details for Web of Science ID 000259473500008
View details for PubMedID 18585785
Cell Encapsulation in Sub-mm Sized Gel Modules Using Replica Molding
2008; 3 (5)
For many types of cells, behavior in two-dimensional (2D) culture differs from that in three-dimensional (3D) culture. Among biologists, 2D culture on treated plastic surfaces is currently the most popular method for cell culture. In 3D, no analogous standard method--one that is similarly convenient, flexible, and reproducible--exists. This paper describes a soft-lithographic method to encapsulate cells in 3D gel objects (modules) in a variety of simple shapes (cylinders, crosses, rectangular prisms) with lateral dimensions between 40 and 1000 microm, cell densities of 10(5)-10(8) cells/cm(3), and total volumes between 1x10(-7) and 8x10(-4) cm(3). By varying (i) the initial density of cells at seeding, and (ii) the dimensions of the modules, the number of cells per module ranged from 1 to 2500 cells. Modules were formed from a range of standard biopolymers, including collagen, Matrigel, and agarose, without the complex equipment often used in encapsulation. The small dimensions of the modules allowed rapid transport of nutrients by diffusion to cells at any location in the module, and therefore allowed generation of modules with cell densities near to those of dense tissues (10(8)-10(9) cells/cm(3)). This modular method is based on soft lithography and requires little special equipment; the method is therefore accessible, flexible, and well suited to (i) understanding the behavior of cells in 3D environments at high densities of cells, as in dense tissues, and (ii) developing applications in tissue engineering.
View details for DOI 10.1371/journal.pone.0002258
View details for Web of Science ID 000262258700058
View details for PubMedID 18493609
PD-1 and its ligands in tolerance and immunity
ANNUAL REVIEW OF IMMUNOLOGY
2008; 26: 677-704
Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, deliver inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. Immune responses to foreign and self-antigens require specific and balanced responses to clear pathogens and tumors and yet maintain tolerance. Induction and maintenance of T cell tolerance requires PD-1, and its ligand PD-L1 on nonhematopoietic cells can limit effector T cell responses and protect tissues from immune-mediated tissue damage. The PD-1:PD-L pathway also has been usurped by microorganisms and tumors to attenuate antimicrobial or tumor immunity and facilitate chronic infection and tumor survival. The identification of B7-1 as an additional binding partner for PD-L1, together with the discovery of an inhibitory bidirectional interaction between PD-L1 and B7-1, reveals new ways the B7:CD28 family regulates T cell activation and tolerance. In this review, we discuss current understanding of the immunoregulatory functions of PD-1 and its ligands and their therapeutic potential.
View details for DOI 10.1146/annurev.immunol.26.021607.090331
View details for Web of Science ID 000255349300022
View details for PubMedID 18173375
TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells
2007; 27 (6): 927-940
The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Here we showed that TIM-4 is expressed on human and mouse macrophages and dendritic cells, and both TIM-4 and TIM-1 specifically bound phosphatidylserine (PS) on the surface of apoptotic cells but not any other phospholipid tested. TIM-4(+) peritoneal macrophages, TIM-1(+) kidney cells, and TIM-4- or TIM-1-transfected cells efficiently phagocytosed apoptotic cells, and phagocytosis could be blocked by TIM-4 or TIM-1 monoclonal antibodies. Mutations in the unique cavity of TIM-4 eliminated PS binding and phagocytosis. TIM-4 mAbs that blocked PS binding and phagocytosis mapped to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors whose recognition of PS is critical for the efficient clearance of apoptotic cells and prevention of autoimmunity.
View details for DOI 10.1016/j.immuni.2007.11.011
View details for Web of Science ID 000251907300012
View details for PubMedID 18082433
IL-7 receptor deficient SCID with a unique intronic mutation and post-transplant autoimmunity due to chronic GVHD
2007; 125 (2): 159-164
Severe combined immunodeficiency (SCID) may result from a variety of genetic defects that impair the development of T cells. Signaling mediated by the cytokine interleukin-7 is essential for the differentiation of T cells from lymphoid progenitors, and mutations of either the interleukin-7 receptor alpha chain (IL-7Ralpha) or its associated cytokine receptor chain, the common gamma chain (gammac), result in SCID. Here we report a case of SCID due to heterozygous mutations of the IL7R gene encoding IL-7Ralpha. A previously unrecognized mutation found within intron 3 created a new exon between exons 3 and 4 in the mRNA transcribed from this allele, producing a truncated, unstable mRNA. This mutation illustrates the necessity of evaluating both coding and non-coding regions of genes when searching for pathogenic mutations. Following hematopoietic stem cell transplantation of our patient, immune reconstitution was accompanied by two unusual complications, immune-mediated myositis and myasthenia gravis.
View details for DOI 10.1016/j.clim.2007.06.007
View details for Web of Science ID 000250466900007
View details for PubMedID 17827065
Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8(+) T-cell-mediated injury in the heart
2007; 116 (18): 2062-2071
PD-L1 and PD-L2 are ligands for the inhibitory receptor programmed death-1 (PD-1), which is an important regulator of immune responses. PD-L1 is induced on cardiac endothelial cells under inflammatory conditions, but little is known about its role in regulating immune injury in the heart.Cytotoxic T-lymphocyte-mediated myocarditis was induced in mice, and the influence of PD-L1 signaling was studied with PD-L1/L2-deficient mice and blocking antibodies. During cytotoxic T-lymphocyte-induced myocarditis, the upregulation of PD-L1 on cardiac endothelia was dependent on T-cell-derived interferon-gamma, and blocking of interferon-gamma signaling worsened disease. Genetic deletion of both PD-1 ligands [PD-L1/2(-/-)], as well as treatment with PD-L1 blocking antibody, transformed transient myocarditis to lethal disease, in association with widespread polymorphonuclear leukocyte-rich microabscesses but without change in cytotoxic T-lymphocyte recruitment. PD-L1/2(-/-) mice reconstituted with bone marrow from wild-type mice remained susceptible to severe disease, which demonstrates that PD-L1 on non-bone marrow-derived cells confers the protective effect. Finally, depletion of polymorphonuclear leukocytes reversed the enhanced susceptibility to lethal myocarditis attributable to PD-L1 deficiency.Myocardial PD-L1, mainly localized on endothelium, is critical for control of immune-mediated cardiac injury and polymorphonuclear leukocyte inflammation.
View details for DOI 10.1161/CIRCULATIONAHA.107.709360
View details for Web of Science ID 000250518000010
View details for PubMedID 17938288
Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses
2007; 27 (1): 111-122
Pathways in the B7:CD28 family of costimulatory molecules regulate T cell activation and tolerance. B7-dependent responses in Cd28(-/-)Ctla4(-/-) T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 molecules (PD-L1 or PD-L2) have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine production were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient versus PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient versus CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. Our findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an additional dimension to immunoregulatory functions of the B7:CD28 family.
View details for DOI 10.1016/j.immuni.2007.05.016
View details for Web of Science ID 000248398100013
View details for PubMedID 17629517
- Patterned paper as a platform for inexpensive, low-volume, portable bioassays ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 2007; 46 (8): 1318-1320
Pentobarbital desensitization in a 3-month-old child
ALLERGY AND ASTHMA PROCEEDINGS
2004; 25 (4): 225-227
Barbiturates, a class of medications commonly used as antiepileptics and sedatives, are known to cause adverse reaction, with the most commonly reported immune-mediated reactions being anticonvulsant hypersensitivity syndrome. Other types of allergic reactions such as immediate hypersensitivity reactions also can occur. We present a 3-month-old child with refractory generalized convulsive status epilepticus who required pentobarbital therapy in the context of phenobarbital sensitivity because of progressive generalized seizures unresponsive to other aggressive therapies. Skin tests to pentobarbital and phenobarbital were negative. In the intensive care unit setting, intravenous pentobarbital desensitization was performed without reaction. To our knowledge, this is the first reported protocol for pentobarbital desensitization.
View details for Web of Science ID 000223899700002
View details for PubMedID 15510580
Neurotrophic factor structures reveal clues to evolution, binding, specificity, and receptor activation
CELLULAR AND MOLECULAR LIFE SCIENCES
2001; 58 (8): 1003-1013
The neutrophin family, the glial-derived neurotrophic factor family, and the ciliary neurotrophic factor are the best described growth factors specific for developing neurons and neutral crest cells. As might be expected for regulatory molecules of the complex central and peripheral nervous system, these factors show considerable receptor specificity and cross-talk. Thanks to a decade of intense research by numerous laboratories, the structures of many of these factors are now available. This review discusses the structural bases of receptor binding, specificity, and activation in each of these systems. Using structure-based sequence alignments, the evolutionary implications of these molecules and their receptors are discussed, followed by suggestions for further directions for research on the structure and function of these neurotrophic factors.
View details for Web of Science ID 000170535200002
View details for PubMedID 11529493
Functional studies of the BTB domain in the Drosophila GAGA and Mod(mdg4) proteins
NUCLEIC ACIDS RESEARCH
2000; 28 (20): 3864-3870
The BTB/POZ (BTB) domain is an approximately 120 residue sequence that is conserved at the N-terminus of many proteins in both vertebrates and invertebrates. We found that the protein encoded by a lethal allele of the Drosophila modifier of mdg4 [mod(mdg4)] gene has two mutated residues in its BTB domain. The identities of the residues at the positions of these mutations are highly conserved in the BTB domain family of proteins, and when the corresponding mutations were engineered into the BTB domain-containing GAGA protein, the activity of GAGA as a transcription activator in a transient transfection assay was severely reduced. The functional equivalence of the BTB domains was established by showing that the BTB domain of the mod(mdg4) protein can effectively substitute for that of GAGA.
View details for Web of Science ID 000089954300004
View details for PubMedID 11024164
Pediatric myocardial infarction after racemic epinephrine administration
1999; 104 (1)
Myocardial infarction is a previously unreported complication of treatment with racemic epinephrine that is used commonly in the emergency department for severe respiratory distress in bronchiolitis or croup syndrome. We describe a pediatric patient who presented with the croup syndrome and severe respiratory distress that required multiple doses of nebulized racemic epinephrine in the emergency department. The patient developed ventricular tachycardia and mild chest discomfort during one treatment, which resolved spontaneously on discontinuation of the nebulization. Persistently abnormal electrocardiograms and elevated creatine phosphokinase MB isoenzyme (CPK-MB) levels suggested a myocardial infarction had occurred. Subsequent echocardiography, cardiac catheterization, and angiography revealed an anatomically normal heart with normal coronary circulation; however, a stress nuclear study showed a small myocardial infarct. The significance of this previously unreported complication of racemic epinephrine is discussed, along with recommendations for proper use in the emergency department.
View details for Web of Science ID 000081258700024
View details for PubMedID 10390295
Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors
1998; 37 (48): 16846-16852
Neurotrophin-3 (NT-3) is a cystine knot growth factor that promotes the survival, proliferation, and differentiation of developing neurons and is a potential therapeutic for neurodegenerative diseases. To clarify the structural basis of receptor specificity and the role of neurotrophin dimerization in receptor activation, the structure of the NT-3 homodimer was determined using X-ray crystallography. The orthorhombic crystals diffract to 2.4 A, with dimer symmetry occurring about a crystallographic 2-fold axis. The overall structure of NT-3 resembles that of the other neurotrophins, NGF and BDNF; each protomer forms a twisted four-stranded beta sheet, with three intertwined disulfide bonds. There are notable differences, however, between NT-3 and NGF in the surface loops and in three functionally important regions, shown in previous mutagenesis studies to be critical for binding. One such difference implies that NT-3's binding affinity and specificity depend on a novel hydrogen bond between Gln 83, a residue important for binding specificity with TrkC, and Arg 103, a residue crucial for binding affinity with TrkC. NT-3's extensive dimer interface buries much of the otherwise solvent-accessible hydrophobic surface area and suggests that the dimeric state is stabilized through the formation of this hydrophobic core. A comparison of the dimer interface between the NT-3 homodimer and the BDNF/NT-3 heterodimer reveals similar patterns of hydrogen bonds and nonpolar contacts, which reinforces the notion that the evolutionarily conserved neurotrophin interface resulted from the need for receptor dimerization in signal initiation.
View details for Web of Science ID 000077339600002
View details for PubMedID 9836577
TOPOLOGICAL AND PHENOMENOLOGICAL CLASSIFICATION OF BURSTING OSCILLATIONS
BULLETIN OF MATHEMATICAL BIOLOGY
1995; 57 (3): 413-439
We describe a classification scheme for bursting oscillations which encompasses many of those found in the literature on bursting in excitable media. This is an extension of the scheme of Rinzel (in Mathematical Topics in Population Biology, Springer, Berlin, 1987), put in the context of a sequence of horizontal cuts through a two-parameter bifurcation diagram. We use this to describe the phenomenological character of different types of bursting, addressing the issue of how well the bursting can be characterized given the limited amount of information often available in experimental settings.
View details for Web of Science ID A1995QM41500003
View details for PubMedID 7728115