Steven A Porcelli
Albert Einstein Col Of Med Yeshiva Univ
Project start date: 2011-01-01
Project end date: 2012-12-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Steven A Porcelli
DIGITAL EXTRA PARAMETER (DXP8)
Steven A Porcelli
Albert Einstein Col Of Med Yeshiva Univ, 1300 Morris Park Avenue, Bronx, Ny 10461
Grant 1S10RR027864-01 from National Center For Research Resources
Abstract: We are requesting funds for a Cytek Development DxP 8-color upgrade for an existing BD Biosciences FACSCalibur analytical flow cytometer. The instrument to be upgraded is shared core equipment that was purchased by the Department of Microbiology and Immunology in 2005. It currently has the standard 2 laser and 4 color configurations, and thus allows up to 4 color fluorescence analyses with a relatively restricted range of fluorochromes and fluorescent proteins. This instrument is used extensively by many of the laboratories in the Department, and has been well maintained and is in excellent working order. However, because of the increasing complexity of FACS analyses done by many of the Department´s investigators, this relatively simple flow cytometer is no longer able to meet current needs. A cost-effective solution to this problem is the purchase and installation of the Cytek digital 8 color upgrade requested by this application. Cytek Development is a well established company that specializes in the repair, maintenance, refurbishing and upgrading of flow cytometers. We have extensive experience with this company going back more than 5 years, and our experience with their products and services makes us confident that the proposed 8 color upgrade will be a cost- effective solution to our current need for a more advanced flow cytometer. At least 13 NIH funded faculty in the Department of Microbiology and Immunology have been identified as users for this equipment, and it is likely that additional users will be identified from laboratories run by the Department´s 16 primary and 19 secondary faculty members. The use and maintenance of this equipment will be supervised by the PI of this application, Dr. Steven A. Porcelli, who is Professor in the Department of Microbiology and Immunology and overall Scientific Director of all of the shared flow cytometry resources on the Einstein campus. A technician with experience in maintaining and operating FACSCalibur flow cytometers and a number of expert consultants are available to ensure that the features of the upgraded FACSCalibur will be used to full advantage. The DxP8 8 color upgrade will provide a large group of NIH-funded microbiologists and immunologists with access to advanced flow cytometry techniques that are critically important to a wide range of basic and translational research projects
Keywords: Back; Color; Cytofluorometry, Flow; Development; Dorsum; Electromagnetic, Laser; Ensure; Equipment; Faculty; Flow Cytofluorometries; Flow Cytometry; Flow Cytometry Shared Resource; Flow Microfluorimetry; Fluorescence; Fluorochrome; Funding; Immunologist; Immunology; Immunology (Including BRMP); Immunology (NCI Program); Investigators; Laboratories; Lasers; Maintenance; Maintenances; Methods and Techniques; Methods, Other; Microbiology; Microfluorometry, Flow; NIH; NIH RFA; National Institutes of Health; National Institutes of Health (U.S.); Proteins; R01 Mechanism; R01 Program; RPG; Radiation, Laser; Request for Applications; Research Grants; Research Personnel; Research Project Grants; Research Projects; Research Projects, R-Series; Researchers; Running; Science of Microbiology; Services; Solutions; Techniques; Translational Research; Translational Research Enterprise; Translational Science; United States National Institutes of Health; Work; cost; cost effective; digital; experience; flow cytophotometry; gene product; instrument; meetings; member; professor; repair; repaired; translation research enterprise
Project start date: 2010-07-01
Project end date: 2011-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PAR-09-028
1S10RR027864-01 (2010): $119552
Steven A Porcelli, Professor
Microbiology And Immunologyyeshiva University
Grant 1S10RR025678-01 from National Center For Research Resources IRG: ZRG1
Abstract: We are requesting funds for a Compucyte iCys Laser Scanning Cytometer (LSC). The LSC is a relatively recently developed instrument that joins many of the advantages of traditional microscopy and flow cytometry, and significantly extends the limits of single cell analyses. Rather than randomly imaging an entire field like a microscope does, it selects, images and analyzes cells that meet certain adjustable criteria (such as size or fluorescence). Unlike conventional flow cytometry, the LSC is usually used to analyze cells fixed to horizontal surface, such as cultured cells on slides or in a variety of culture vessels. Live cells can be imaged and cytometrically analyzed for fluorescent signals, and a variety of types of fixed specimens can also be studied in this way. The instrument uses an automated nanostep X-Y stage to move samples through the objective lens, applying up to three different lasers to excite fluorescent emissions and quantitating these using an array of four photomultiplier tube (PMT) detectors. Thus, the LSC is essentially a powerful hybrid technology that combines imaging and cytometric analysis in a single platform. Importantly, with the LSC image becomes a parameter, and detailed microscopic pictures are stored and referenced directly to cytometric data. A highly appealing feature of the instrument that we plan to use extensively is its ability to gather quantitative fluorescence data from microscopic areas of fluorescently labeled tissue sections, and to display and analyze such data in a manner that is comparable in many cases to true cytometric data such as one obtains with traditional flow cytometry. We plan to house and operate the iCys LSC in our well established central Flow Cytometry Core Laboratory, where there will be ample expertise and oversight to ensure that the instrument is used to its full capacity. The Core Laboratory has an experienced senior faculty member (Dr. Steven Porcelli) as its supervisor, and employs three full time faculty level associates to direct operations and provide technical supervision. There is also a range of internal consultants with expertise in areas of major relevance to the development of applications that will use the LSC. The Core Laboratory has a well developed and carefully monitored financial plan, and receives substantial institutional support from the institution and from our NCI-funded Cancer Center to ensure its efficient and stable operation. By incorporating the iCys LSC into this core laboratory, a large body of investigators pursuing many diverse NIH-funded basic and translational research projects will gain convenient and affordable access to this important new technology. This application requests funding that will allow our Flow Cytometry Core Laboratory to acquire a new analytical instrument called the Compucyte iCys Laser Scanning Cytometer. This instrument will provide many researchers at our institution with a powerful new way to analyze cells and tissues. The studies that will be done with this instrument will advance our understanding of a wide spectrum of areas with high relevance to biomedical science, including cancer biology, immunology, autoimmune diseases and infectious diseases
Project start date: 2008-12-22
Project end date: 2009-12-21
MoFlow High Speed Fluorescence Activated Cell Sorter
Steven A Porcelli, Professor
Yeshiva University 500 W 185th St New York, Ny 10033
Grant 1S10RR020999-01A2 from National Center For Research Resources IRG: ZRG1
Abstract: This is a revised application for funding of a DakoCytomation MoFlo high speed fluorescence activated cell sorter. There is currently only one high speed cell sorter available to most investigators at the Albert Einstein College of Medicine (AECOM), which is inadequate to meet current needs. The requested funding will enable us to add a second high speed sorter to our main FACS Core Facility, which will double cell sorting capacity and extend the range of applications that can be supported. The Facility provides most FACS-based resources for approximately 275 users associated with the laboratories of more than 50 NIH- funded investigators at AECOM. The Facility has a highly evolved plan of operations, sound financial oversight, and a long history of strong institutional support. There are two full-time technical staff with extensive background in operation of the MoFlo sorter, and a third person with comparable skills will soon be hired. There is a faculty supervisor (Dr. Steven A. Porcelli, PI of this SIG application) who is a Professor of Microbiology and Immunology and an internationally known research immunologist. A steering committee composed of seven distinguished AECOM faculty meets regularly to set policy for current operations and future growth, and provide quality assurance. The instrument for which we have requested funding will have three lasers to enable excitation of a broad range of different fluorescent probes. Cells and subcellular structures in suspension can be characterized and sorted at speeds exceeding 25,000 events per second using simultaneous measurement of two light scatter parameters and up to seven channels of fluorescence. This instrument permits the isolation of specific cell types from among complex mixed cell suspensions, and is the most powerful and precise method available for purification of cells and certain subcellular structures based on the binding of specific fluorescent probes or antibodies. The requested instrument will be housed in biosafety level-2 (BSL-2) laboratory space and will be equipped with an aerosol containment module, thus enabling us to perform high speed sorting of samples containing infectious agents requiring BSL-2 precautions in accordance with our Institutional Biosafety Committee. The provision of funds to support the purchase of this instrument will accelerate research into the causes and treatment of major public health problems including cancer, diabetes, cardiovascular and infectious diseases. The acquisition of this instrument will also play a significant role in facilitating the continuing growth of AECOM as a major center for advanced biomedical research and education.
Keywords: cell, flow cytometry, aerosol, antibody, base, cell sorting, cell type, communicable disease, education, fluorescence, fluorescent dye /probe, housing, immunology, laser, light scattering, measurement, medicine, microbiology, neoplasm /cancer, play, public health, role, sound, suspension, teacher, university
Project start date: 2007-06-01
Project end date: 2008-05-31
1S10RR020999-01A2 (2007): $431552
FLOW CYTOMETRY SHARED RESOURCE
Steven A Porcelli
Albert Einstein Col Of Med Yeshiva Univ, 1300 Morris Park Avenue, Bronx, Ny 10461
Abstract: The Flow Cytometry Shared Resource provides AECC investigators with convenient and affordable access to a broad spectrum of important technologies in flow cytometry, and supports numerous research studies that rely on high speed fluorescence activated cell sorting (FACS) and advanced multiparameter fluorescence analysis. In addition to providing advanced flow cytometry equipment, the shared resource provides technical expertise and training to guide AECC investigators and their staff in the application of basic and advanced flow cytometry techniques. The facility also organizes and supports a program of seminars on new technologies that inform AECC investigators about newly emerging applications of FACS that can enhance their research. The major usage of this shared resource is for multiparameter analysis and high speed sorting of cell populations based on labeling with fluorochrome-conjugated antibodies or other fluorescent protein or chemical dye reagents. Training of many investigators in the use of the facility´s analytical instruments has led to a very large and well-trained user base. During 2005-2006, the facility provided services to more than 275 registered users, consisting of faculty, postdoctoral fellows, students and technicians from 78 different laboratories. AECC members were principal investigators of 45 of these laboratories, and two thirds of total hours of usage of all facility services in 2005-2006 was attributable to the laboratories of AECC investigators. These investigators have access around the clock and seven days per week to the facility´s analyzers, which include Becton-Dickinson FACScan (one laser, 3-color), FACSCalibur (two laser, 4-color), and LSR-II (4 laser, 12 color) instruments. Standard fluorescent microscopes and computer work stations with an extensive software library for processing of FACS data are also routinely available. High speed cell sorting with the facility´s 3-laser, 8-color Dako-Cytomation MoFlo sorter is performed exclusively by the trained facility staff during regularly scheduled weekday hours. This shared resource has numerous value-added features, and receives substantial institutional support that has greatly assisted the acquisition of state-of-the-art instrumentation and a superb staff of flow cytometry experts
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
5P30CA013330-38_9008 (2010): $172321
3P30CA013330-37S3_9008 (2009): $2193
3P30CA013330-37S4_9008 (2009): $14564
5P30CA013330-37_9008 (2009): $194129
Binding And Presentation Of Lipid Antigens By CD1
Steven A Porcelli, Professor
Microbiology And Immunologyyeshiva University
500 W 185th St
new York, Ny 10033
Grant 5R01AI045889-11 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: The CD1 system of MHC class I-like proteins is firmly established as a group of antigen presenting molecules that activates T cells specific for lipids and glycolipids. A population of T lymphocytes known as NK T cells recognizes specific lipid ligands presented by the CD1d protein, and this component of the CD1-dependent immune response is highly conserved between humans and mice. Many detailed studies in mouse models have shown that CD1d-restricted NK T cells contribute to immune responses against pathogens, the elimination of malignant tumors, and the prevention of autoimmune diseases. A synthetic alpha-galactosyl ceramide known as KRN7000 has been identified as a glycolipid ligand that binds to CD1d and strongly activates multiple effector functions of NK T cells. Through collaborative efforts with established experts in the area of synthetic organic chemistry, has developed novel approaches to the creation of alpha-galactosyl ceramides with a range of structural alterations. Many of these compounds preserve the ability to activate CD1d-restricted NK T cells, and preliminary studies indicate that the have immunomodulatory activities that are significantly different from those of KRN7000. The current proposal will investigate the immunomodulatory activities of a large panel of novel alpha-galactosyl ceramides. Properties of these compounds to be investigated include induction of Th1 (inflammatory) versus Th2 (anti-inflammatory) cytokine production, specific targeting of NK T cell activation by certain types of antigen presenting cells or in certain tissues, and their ability to cause expansion as opposed to apoptosis of NK T cells. The proposed experiments will include in vitro cell culture studies of both human and murine NK T cells, and in vivo studies using mice. There is a high expectation that the proposed studies will contribute directly to the development of clinically useful immunomodulatory agents that act on NK T cells
Keywords: CD1 molecule, analog, antigen presentation, cerebroside, chemical binding, immune response, immunoregulation, leukocyte activation /transformation, natural killer cell B lymphocyte, Schwann cell, T cell receptor, cell free system, combinatorial chemistry, dendritic cell, gastrointestinal epithelium, intracellular transport, keratinocyte, macrophage, recombinant protein cell line, clinical research, enzyme linked immunosorbent assay, genetically modified animal, human subject, hybridoma, laboratory mouse, microarray technology
Project start date: 1999-07-01
Project end date: 2010-06-30
5R01AI045889-11 (2008): $336311
5R01AI045889-10 (2007): $335044
5R01AI045889-09 (2006): $337268
Sponsored Links Excellgen http://Excellgen.com
5R01AI045889-08 (2005): $336488
2R01AI045889-07 (2004): $327721
5R01AI045889-13 (2010): $410850
2R01AI045889-12 (2009): $415000
BINDING AND PRESENTATION OF LIPID ANTIGENS BY CD1B
Steven A Porcelli, Professor
Microbiology And Immunologyyeshiva University
500 W 185th St
new York, Ny 10033
Grant 5R01AI045889-04 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: Recent studies have identified the CD1 family of cell surface glycoproteins as novel antigen presenting molecules encoded by genes located outside of the major histocompatibility complex. Identification of naturally occurring antigens presented by CD1 has revealed the surprising finding that these prominently include a range of foreign lipids and glycolipids, such as several that are known to exist in the cell walls and membranes of pathogenic mycobacteria. Among the currently known CD1-presented antigens, the best structurally characterized are the mycolic acids and their monoglucosylated analogue, glucose monomycolate (GMM). These compounds have a lipid structure that is unlike any found in mammalian tissues, and occur abundantly as a major outer cell membrane component in a variety of pathogenic bacteria, including Mycobacterium tuberculosis. Recognition of these lipids appears to be a frequent feature of CD1b-restricted M. tuberculosis-specific T cells in humans, suggesting that T cell responses to mycolic acids and GMM may play a significant role in the protective host response to this important pathogen. Studies in the current proposal will assess whether mycolic acids or GMM are among the immunodominant T cell antigens of M. tuberculosis in humans infected with this bacterium, and will seek to clarify the molecular basis for the presentation and recognition of these novel T cell antigens. A combination of preparative and synthetic chemistry will be used to generate a range of structural analogues of mycolic acids and GMM. These will be studied for their ability to be recognized by CD1b-restricted T cells, and for their ability to bind directly to CD1b proteins. In addition, the structural basis for the binding of these antigens to the CD1b protein will be analyzed by carrying out site directed mutagenesis of residues forming the predicted ligand binding groove of CD1b. These studies will lead to a detailed understanding of the fundamental rules governing the interaction of a newly recognized class of T cell antigens with their antigen presenting molecule. The identification and detailed analysis of this newly recognized pathway for T cell antigen recognition is likely to be important for understanding the human immune response to M. tuberculosis and related pathogens, and has potential implications for future vaccine development efforts
Keywords: CD1 molecule, Mycobacterium tuberculosis, antigen presentation, bacterial antigen, chemical binding T lymphocyte, binding site, chemical structure, hydroxy fatty acid, microorganism immunology chemical synthesis, clinical research, human subject, site directed mutagenesis
Project start date: 1999-07-01
Project end date: 2004-06-30
5R01AI045889-04 (2001): $256900
5R01AI045889-03 (2000): $249413
1R01AI045889-01 (1999): $252981
Prevention Of Diabetes With Lipid Immunomodulators
Steven A Porcelli, Professor
Yeshiva University 500 W 185th St New York, Ny 10033
Grant 5R01AI064424-02 from National Institute Of Allergy And Infectious Diseases IRG: ZDK1
Abstract: In recent years, CD1d-restricted natural killer T cells (NK T cells) have been identified as a critical population of regulatory T cells that function to control autoimmune responses. Defects in these cells have been implicated as contributing factors in the progression of autoimmune islet beta cell destruction in type 1 diabetes, both in humans and in nonobese diabetic (NOD) mice. A synthetic glycolipid known as KRN7000, which is a form of alpha-galactosyl ceramide (aGalCer), has the capacity to specifically activate NK T cells. Treatment of NOD mice with this compound can significantly delay or prevent diabetes. Available data indicate that KRN7000 exerts its beneficial effects by stimulating NK T cells to produce protective Th2-type cytokines such as IL-4. However, KRN7000 also triggers production of Th1-type cytokines by NK T cells and by secondary stimulation of dendritic cells and natural killer cells. This could limit its therapeutic efficacy in diseases associated with a harmful Th1 inflammatory response such as type 1 diabetes. Dr. Porcelli s laboratory has recently identified structural analogues of aGalCer that stimulate altered responses of NK T cells, such as the preferential secretion of Th2 cytokines without concurrent Th1 cytokines. Our central hypothesis is that analogues of aGalCer that have an enhanced capacity to stimulate the production of Th2 cytokines will have an even greater therapeutic efficacy in diabetes prevention than aGalCer, and that the beneficial effects of aGalCer and its analogues are due at least in part to the suppression of cytotoxic T lymphocytes (CTL) specific for islet beta-cell autoantigens. In this collaborative partnership, the Principal Investigators (Drs. Porcelli and DiLorenzo) will work together to identify glycolipid activators of NK T cells that efficiently block or reverse the autoimmune-mediated destruction of pancreatic islets in the NOD mouse model of type 1 diabetes. The mechanisms by which these glycolipids alter the repertoire and effector functions of autoreactive T cells will be analyzed, and treatment protocols that optimize the use of these potential therapeutic agents in vivo will be developed. The project will thus combine Dr. DiLorenzo s established expertise in analysis of T cell responses and disease progression in the NOD model with Dr. Porcelli s expertise in the areas of glycolipid chemistry, CD1, and NK T cell biology. The Specific Aims are (1) To test the ability of novel analogues of aGalCer that elicit altered response of NK T cells to prevent or reverse diabetes in NOD mice; (2) To assess the ability of these aGalCer analogues to promote syngeneic islet graft survival in diabetic NOD mice; (3) To determine the effects of in vivo analogue treatment on the numbers, distribution and functions of CD1d-restricted NK T cells; (4) To assess the effects of aGalCer analogues on the activation, expansion and distribution of diabetogenic beta-cell cytotoxic CD8+ T cells.
Keywords: cellular immunity, disease /disorder prevention /control, glycolipid, immunomodulator, insulin dependent diabetes mellitus, autoantigen, autoimmune disorder, cytotoxic T lymphocyte, dendritic cell, helper T lymphocyte, interleukin 4, leukocyte activation /transformation, natural killer cell, pancreatic islet, NOD mouse
Project start date: 2004-09-01
Project end date: 2007-08-31
5R01AI064424-02 (2005): $229600
1R01AI064424-01 (2004): $229600
ANTIGEN PRESENTATION BY CD I PROTEINS
Steven A Porcelli, Professor
Boston University Medical Campus 715 Albany St, 560 Boston, Ma 021182394
Grant 5P41RR010888-030046 from National Center For Research Resources
Abstract: Human CDI proteins are a family of norimajor histocompatibility complex (MHC) encoded transmembrane glycoproteins expressed in association with 02-microglobulin on the surface of antigen-presenting cells (APCs). Unlike the well known M[HC class I and class 11 proteins that present peptide antigens to T cells, the human group I CD1 proteins (CD1a, CD1b andCDlc) mediate specific T cell recognition of bacterial lipid and glycolipid antigens. Previous studies of mycobacteria specific T cells have identified two classes of CDI-restricted lipid antigens. These are the free mycolic acids, a family of cc-branched, 0-hydroxy long chain fatty acids, and the phosphatidylinositol-containing glycolipids including lipoarabinomannan (LAM) andthe phosphatidylinositol mannosides (PlMs). Mycohacterium leprae infection has recently revealed evidence for a third class of CDI restricted lipid antigens. The CDI proteins play a central role in the specific T cell recognition of foreign lipid antigens, but the molecular mechanisms underlying lipid antigen presentation are not known. During this past year, we have identified a novel CDI-restricted glycolipid antigen, glucose monomycolate (GM[M), which is allowing a systematic analysis of the structural features that determine its recognition by T cells. Analogues of GNM that differed substantially in their acyl chain lengths and other chemical features of the lipid moiety were recognized by T cells. In contrast, T cells demonstrated fine specificity for the carbohydrate portion ?of mycolyl glycolipids, even discriminating among carbohydrate isomers differing only in the orientation of a single hydroxyl group (e.g., only glucose and not mannose or galactose). In combination with recent studies of the crystal structure of CD1, these results provide strong support for a molecular model of antigen presentation in which the acyl chains of the antigen bind relatively nonspecifically within the deep, hydrophobic pocket of the CD1 protein, resulting in presentation of the hydrophilic elements of antigens for highly specific interactions with the T cell receptor.
Keywords: bacteria, biological product, biomaterial, biomedical resource, carbohydrate, immunology, infection, inflammation, lipid, lymphatic system, structural biology
Sponsored Links Excellgen http://Excellgen.com
ANTIGEN PRESENTATION BY CD1 PROTEINS
Steven A Porcelli, Professor
Boston University Medical Campus 715 Albany St, 560 Boston, Ma 021182394
Grant 3P41RR010888-05S10052 from National Center For Research Resources
Abstract: Glycosyl transferases are important components of the biosynthetic system that generates and modifies glycoproteins. Inherited disorders that affect the levels or structures of the transferases can result in degenerative diseases such as Carbohydrate Deficient Glycoprotein syndrome (CDGS). In order to facilitate investigation of these pathways, rabbit GnT I and human GnT II have been expressed in a transfer vector system that secretes the hybrid enzymes from the insect cells with an attached H]H[HHOHH sequence. The signal peptide is derived from mellittin and it is expected that the signal peptidase sequence .... YIYADPM... is cleaved at its Ala-Asp linkage. The molecular weights of the expressed proteins are being checked by MALDI and ESI MS to determine whether the correct proteins have been obtained and to look for post-translational modifications.
Keywords: biological product, biomedical resource, genetics, nervous system, orphan disease /drug, spectrometry, structural biology, technology /technique
Steven A Porcelli
Albert Einstein Col Of Med Yeshiva Univ, 1300 Morris Park Avenue, Bronx, Ny 10461
Abstract: Flow cytometry is one of the most widely used and indispensable techniques for the analysis and isolation of specific cell populations. The CFAR Flow Cytometry Core was established in 2003 with initial seed funds from the CFAR grant to provide CFAR investigators with improved access to advanced flow cytometry equipment and techniques. The CFAR Core is unique among the flow cytometry resources at Einstein in providing biohazard containment for carrying out analysis and sorting of samples requiring Biosafety Level-2 or higher precautions, and is thus a major resource for work involving HIV and a variety of AIDS-related opportunistic pathogens. The CFAR Flow Cytometry Core provides CFAR investigators with ready access to a variety of advanced instruments for analytical flow cytometry and cell sorting, and extensive technical expertise and assistance for performance of established flow cytometry techniques and implementation of new technologies in this area. In the four years since its inception, the Core has grown substantially with regard to space, equipment and personnel. This reflects the important role that this core facility has played in CFAR related research projects, and also the considerable institutional support that underscores the value added features of the Core. The activities of the Core are organized according to the following four specific aims Aim 1. To maintain the necessary research infrastructure needed for flow cytometry studies, with appropriate biosafety containment safeguards. Aim 2. To maintain cutting edge capabilities by regularly updating existing flow cytometry resources and introducing new equipment and techniques in a timely manner. Aim 3. To provide convenient and cost-effective access to equipment and technical expertise for the performance and interpretation of flow cytometry. Aim 4. To provide education and training to assist CFAR investigators in the implementation of new and established flow cytometry techniques. RELEVANCE Flow cytometry allows researchers to study many properties of cells or micro-organisms and rapidly gather information that is crucial to understanding disease processes in animal models and human subjects. The CFAR Flow Cytometry Core supports instruments and personnel that are required for this type of essential research, and organizes these resources in a centralized facilty that provides access for a large number of investigators working on a wide range of problems relevant to HIV infection and AIDS
Keywords: AIDS; AIDS Virus; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immune Deficiency Syndrome Virus; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Acquired Immunodeficiency Syndrome Virus; Animal Model; Animal Models and Related Studies; Area; Cell Isolation; Cell Segregation; Cell Separation; Cell Separation Technology; Cells; Consultations; Containment; Containment of Biohazards; Core Facility; Cytofluorometry, Flow; Disease; Disorder; Environment; Equipment; Expertise, Technical; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Funding; Grant; HIV; HIV Infections; HTLV-III; HTLV-III Infections; HTLV-III-LAV Infections; Human Immunodeficiency Viruses; Human Resources; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Immunologic Deficiency Syndrome, Acquired; Improve Access; Infrastructure; Investigators; LAV-HTLV-III; Lymphadenopathy-Associated Virus; Manpower; Methods and Techniques; Methods, Other; Microfluorometry, Flow; Organism; Performance; Plant Embryos; Play; Population; Process; Property; Property, LOINC Axis 2; R01 Mechanism; R01 Program; RPG; Research; Research Grants; Research Infrastructure; Research Personnel; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Researchers; Resources; Role; Sampling; Seeds; Sorting - Cell Movement; T-Lymphotropic Virus Type III Infections, Human; Technical Expertise; Techniques; Training and Education; Update; Virus-HIV; Work; Zygotes, Plant; biohazard control; cell sorting; cost; disease/disorder; flow cytophotometry; human subject; information gathering; instrument; living system; model organism; new technology; pathogen; personnel; seed; social role; sorting
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
5P30AI051519-08_9009 (2010): $146111
5P30AI051519-07_9009 (2009): $164820
DETERMINANTS OF T CELL IMMUNITY TO TUBERCULOSIS VACCINES
Steven A Porcelli
Albert Einstein Col Of Med Yeshiva Univ, 1300 Morris Park Avenue, Bronx, Ny 10461
Grant 5P01AI063537-05_0002 from National Institute Of Allergy And Infectious Diseases
Abstract: Effective host immunity to Mycobacterium tuberculosis is dependent on T cell-mediated responses against specific antigens of the bacillus. Although more than 2 million people die from tuberculosis every year, approximately 90% of infected humans who mount a normal immune response to the bacterium never develop clinical disease. This demonstrates the importance of a potent immune response in limiting or Preventing disease caused by M. tuberculosis, and provides a rationale for the development of an effective vaccine to prevent disease. Recently, it has become clear that many virulence-promoting factors produced by M. tuberculosis are lipids, which can have profound effects on modulating the immune response of the host. Functional inactivation of two genetic loci involved in lipid biosynthesis in M. tuberculosis, pcaA and panCD, are associated with reduced virulence and enhanced clearance of the bacilli in mice. A third genetic locus called RD1 plays a major role in virulence of M. tuberculosis most likely by controlling the secretion of toxic proteins. We hypothesize that M. tuberculosis harboring functional deletions of these genes will generate enhanced T cell responses and more robust protective immunity in infected animals. In addition, we propose that the T cell response against M. tuberculosis-associated protein antigens can be further enhanced by using novel synthetic glycolipid adjuvants in the alpha-galacotosyl ceramide family that activate CD1-restricted NK T cells. The following three specific aims are proposed 1) To assess the impact of deletion of pcaA, panCD and RD1 on T cell responses to MHC class I and class II presented epitopes; 2) To analyze the basis for adaptive immunity to attenuated vaccine strains of M. tuberculosis in CD4-deficient mice; and 3) to determine the effect of synthetic immunomodulatory lipids in the alpha-galactosyl ceramide family on the T cell response and protective immunity induced by attenuated M. tuberculosis vaccine strains. This project will be enabled through an extensive network of collaborations with other members of this program project and the use of all of its core facilities. Combining these approaches for enhancing immunogenicity may lead to the development of highly effective attenuated live M. tuberculosis vaccines. This would be a major tool for enhancing the currently inadequate efforts to reduce the global burden of tuberculosis and prevent the emergence of multidrug resistant strains
Keywords: ATGN; Adjuvant; Animals; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Antigenic Determinants; Antigens; Attenuated; Attenuated Vaccines; Bacillus; Bacillus (bacterium); Bacteria; Binding Determinants; Body Tissues; Cells; Ceramide (lipids); Ceramides; Clinical; Collaborations; Core Facility; Development; Disease; Disorder; Drug Resistance, Multiple; Drug Resistant, Multiple; Epitopes; Family; Galactocerebrosides; Galactosyl Ceramides; Galactosylceramides; Gene Deletion; Genes, Class I; Genes, Class II; Genes, HLA Class II; Genes, MHC Class I; Genes, MHC Class II; Genetic; Glycolipids; Goals; Granuloma; Granulomatous Lesion; Histopathology; Human; Human, General; Humoral Immunities; Hybridomas; Immune response; Immunities, Humoral; Immunity; Knockout Mice; Lead; Life; Lipids; M. tb; M. tuberculosis; M.tb; M.tuberculosis; MHC Class I; MHC Class I Genes; MHC Class II; MHC Class II Genes; Mammals, Mice; Man (Taxonomy); Man, Modern; Maps; Measures; Mediating; Mice; Mice, Knock-out; Mice, Knockout; Molecular Analysis; Mouse Strains; Multi-Drug Resistance; Multidrug Resistance; Murine; Mus; Mycobacterium tuberculosis; Null Mouse; Pb element; Peptides; Play; Population; Principal Investigator; Production; Programs (PT); Programs [Publication Type]; Proteins; Resistance to Multi-drug; Resistance to Multidrug; Resistance to Multiple Drug; Resistant to Multiple Drug; Resistant to multi-drug; Resistant to multidrug; Role; T-Cells; T-Lymphocyte; TB vaccine; Thymus-Dependent Lymphocytes; Tissues; Transgenic Organisms; Tuberculosis; Tuberculosis Vaccines; Vaccinated; Vaccines; Vaccines, Attenuated; Virulence; adipogenesis; anti-TB vaccine; antibody biosynthesis; antibody-based immunity; base; cytokine; disease/disorder; disseminated TB; disseminated tuberculosis; gene deletion mutation; gene product; heavy metal Pb; heavy metal lead; host response; immunogen; immunogenicity; immunoglobulin biosynthesis; immunoresponse; improved; in vivo; lipid biosynthesis; lipogenesis; live vaccine; member; multi-drug resistant; multidrug resistant; mutant; novel; prevent; preventing; programs; protective efficacy; resistant strain; response; social role; thymus derived lymphocyte; tool; transgenic; tuberculosis immunity; tuberculous spondyloarthropathy
Budget start date: 1-APR-2009
Budget end date: 31-MAR-2010
PFA/PA: PA-03-080
5P01AI063537-05_0002 (2009): $329392
Sponsored Links Excellgen http://Excellgen.com
Steven A Porcelli
Albert Einstein Col Of Med Yeshiva Univ, 1300 Morris Park Avenue, Bronx, Ny 10461
Grant 5P01AI063537-05_9002 from National Institute Of Allergy And Infectious Diseases
Abstract: Analytical flow cytometry is an essential technology for all of the investigators participating in this program project. This flow cytometry core will augment existing capabilities at our institution by providing the first flow cytometer to be housed and operated in our Biosafety Level 3 laboratories. Funds for the purchase of this instrument have already been committed by the institution, and we anticipate the purchase and installation of a Becton-Dickinson FACSCalibur analyzer with low speed sorting capability before the initiation of this program grant. This instrument will allow the performance of up to four color fluoresence single cell analysis of cell suspensions harvested from mice infected with virulent or potentially virulent strains of M. tuberculosis. Multicolor FACS analyses using a range of monoclonal antibodies to identify and quantitate specific cell populations will be carried out routinely by this facility, as will staining of specific T cell populations using peptide loaded MHC class I tetramer reagents. Analysis of apoptosis, cell cycle progression and calcium flux will also be available in a BSL-3 environment through the establishment of this core. Limited cell sorting capabilities that should be adequate for the proposed isolation of serologically modified mutants of M. tuberculosis will also by possible with this core facility. The core director, Dr. Steven Porcelli, was first trained in the use of analytical flow cytometers in 1989, and has been actively using these instruments in his own research continuously since that time. He also is currently the director of the flow cytometry facility of the NIH funded AECOM Center for Aids Research, which provides a BSL-2 environment for analytical flow cytometry as well as limited sorting capability using a Becton-Dickinson FACSort instrument. He is experienced in training and supervising technicians and other laboratory personnel to carry out flow cytometry procedures. In establishing this core facility, he will recruit and train an appropriate individual to serve as a dedicated flow cytometry technician to carry out the bulk of the day to day operation of the core
Keywords: Apoptosis; Apoptosis Pathway; Area; Cell Cycle Progression; Cell Death, Programmed; Cell Isolation; Cell Segregation; Cell Separation; Cell Separation Technology; Cells; Color; Commit; Containment; Core Facility; Cytofluorometry, Flow; Ensure; Environment; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Funding; Genes, Class I; Genes, MHC Class I; Goals; Grant; Harvest; Housing; Human Resources; Immune response; Individual; Infectious Agent; Infrastructure; Institution; Instruction; Instrumentation, Other; Investigators; Laboratories; Laboratory Personnel; M. tb; M. tuberculosis; M.tb; M.tuberculosis; MHC Class I; MHC Class I Genes; Mammals, Mice; Manpower; Methods and Techniques; Methods, Other; Mice; Microfluorometry, Flow; Moab, Clinical Treatment; Molecular Analysis; Monoclonal Antibodies; Murine; Mus; Mycobacterium tuberculosis; NIH; National Institutes of Health; National Institutes of Health (U.S.); Operation; Operative Procedures; Operative Surgical Procedures; Pathogenesis; Peptides; Performance; Phenotype; Population; Procedures; Programs (PT); Programs [Publication Type]; Quality Control; Reagent; Recruitment Activity; Research; Research Infrastructure; Research Personnel; Researchers; Safety; Sampling; Services; Sorting - Cell Movement; Speed; Speed (motion); Staining method; Stainings; Stains; Surgical; Surgical Interventions; Surgical Procedure; Suspension substance; Suspensions; T-Cells; T-Lymphocyte; Techniques; Technology; Thymus-Dependent Lymphocytes; Time; Training; United States National Institutes of Health; Update; Virulent; Work; calcium flux; calcium mobilization; cell sorting; cost; experience; flow cytophotometry; host response; immunoresponse; infectious organism; instrument; instrumentation; interest; mutant; mycobacterial; pathogen; personnel; programs; recruit; release of sequestered calcium ion into cytoplasm; single cell analysis; sorting; surgery; suspension; thymus derived lymphocyte; tuberculosis immunity
Budget start date: 1-APR-2009
Budget end date: 31-MAR-2010
PFA/PA: PA-03-080
5P01AI063537-05_9002 (2009): $139090
BINDING AND PRESENTATION OF LIPID ANTIGENS BY CD1B
Steven A Porcelli, Professor
Yeshiva University 500 W 185th St New York, Ny 10033
Grant 5R01AI045889-06 from National Institute Of Allergy And Infectious Diseases IRG: ALY
Abstract: Adapted from Investigator s ) Recent studies have identified the CD1 family of cell surface glycoproteins as novel antigen presenting molecules encoded by genes located outside of the major histocompatibility complex. Identification of naturally occurring antigens presented by CD1 has revealed the surprising finding that these prominently include a range of foreign lipids and glycolipids, such as several that are known to exist in the cell walls and membranes of pathogenic mycobacteria. Among the currently known CD1-presented antigens, the best structurally characterized are the mycolic acids and their monoglucosylated analogue, glucose monomycolate (GMM). These compounds have a lipid structure that is unlike any found in mammalian tissues, and occur abundantly as a major outer cell membrane component in a variety of pathogenic bacteria, including Mycobacterium tuberculosis. Recognition of these lipids appears to be a frequent feature of CD1b-restricted M. tuberculosis-specific T cells in humans, suggesting that T cell responses to mycolic acids and GMM may play a significant role in the protective host response to this important pathogen. Studies in the current proposal will assess whether mycolic acids or GMM are among the immunodominant T cell antigens of M. tuberculosis in humans infected with this bacterium, and will seek to clarify the molecular basis for the presentation and recognition of these novel T cell antigens. A combination of preparative and synthetic chemistry will be used to generate a range of structural analogues of mycolic acids and GMM. These will be studied for their ability to be recognized by CD1b-restricted T cells, and for their ability to bind directly to CD1b proteins. In addition, the structural basis for the binding of these antigens to the CD1b protein will be analyzed by carrying out site directed mutagenesis of residues forming the predicted ligand binding groove of CD1b. These studies will lead to a detailed understanding of the fundamental rules governing the interaction of a newly recognized class of T cell antigens with their antigen presenting molecule. The identification and detailed analysis of this newly recognized pathway for T cell antigen recognition is likely to be important for understanding the human immune response to M. tuberculosis and related pathogens, and has potential implications for future vaccine development efforts.
Keywords: CD1 molecule, Mycobacterium tuberculosis, antigen presentation, bacterial antigen, chemical binding, T lymphocyte, binding site, chemical structure, hydroxy fatty acid, microorganism immunology, chemical synthesis, clinical research, human subject, site directed mutagenesis
Project start date: 1999-07-01
Project end date: 2004-06-30
5R01AI045889-06 (2003): $271738
ANTIGEN PRESENTATION BY CD1 PROTEINS
Steven A Porcelli, Professor
Yeshiva University 500 W 185th St New York, Ny 10033
Grant 5R29AI040135-06 from National Institute Of Allergy And Infectious Diseases IRG: ZRG2
Abstract: Adapted from s ) The CD1 family of cell surface glycoproteins have recently been proposed to be a third lineage of antigen presenting molecules that have diverged markedly from the MHC class I and II families in their structure, function and pattern of expression. Remarkably, the antigens currently known to be presented to human CD1 restricted T cells are not peptides, but are unique lipid and glycolipid molecules found in bacterial cell walls. The CD1 system thus mediates recognition of an important set of nonpeptide antigens by T cells, and as such is likely to perform an important role in innate or acquired immunity to infectious pathogens. In this application, the antigen presenting function of CD1 will be demonstrated directly by using radiolabelled microbial glycolipid antigens as probes for antigen binding to CD1 proteins. A powerful new mass spectrometry technique (electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry) will be used to determine the structure of processed foreign glycolipid antigens bound to CD1. Since previous studies implicate an endosomal pathway for antigen processing in CD1 restricted responses, a variety of subcellular imaging and fractionation methods will be used to establish the particular endosomal compartments to which CD1 molecules localize, and the mechanisms that control the sorting of CD1 proteins to these sites will be determined. CD1 restricted T-cell lines specific for bacterial lipid and glycolipid antigens will be used to assess the functional importance of endosomal localization of CD1 proteins for presentation of exogenously or endogenously acquired antigens. These studies will help to establish a new paradigm for cellular immune recognition that expands our overall appreciation of how T cells see foreign antigens, and should have broad potential implications for the host response to foreign and self antigens in health and disease.
Keywords: CD1 molecule, T lymphocyte, antigen presentation, Mycobacterium, bacterial antigen, glycolipid, confocal scanning microscopy, electrospray ionization mass spectrometry, immunoelectron microscopy, tissue /cell culture
Project start date: 1996-06-01
Project end date: 2001-05-31
5R29AI040135-06 (2000): $126183
5R29AI040135-04 (1999): $122462
5R29AI040135-02 (1997): $188089
5R29AI040135-03 (1998): $117751
Immunoregulatory NK T Cell /systemic Lupus Erythematosus
Steven A Porcelli, Professor
Yeshiva University 500 W 185th St New York, Ny 10033
Grant 1P01AI051392-010003 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Abstract: A population of T lymphocytes known as NK T cells recognizes specific lipid ligands in the context of MHC class I-like CD1d molecules and has recently been found to contribute to the regulation of immune responses and the maintenance of immunological tolerance to self antigens. Numerous published reports have linked the loss of NK T cells and changes in their function to the progression of autoimmune diseases, including systemic lupus erythematosus (SLE) and mouse models of this disease. The central hypothesis of the current proposal is that NK T cells constitute an important regulatory arm of the immune system that normally assists in preventing the development of aggressive autoimmunity such as that which occurs in full-blown SLE. The proposed studies will evaluate changes in NK T cells during the progression of spontaneous lupus-like disease in NZB/W F1 mice, and will determine the influence of NK T cells on SLE-associated autoantibody production in murine models of spontaneous and induced SLE-like disease. These studies will build on preliminary findings that strongly implicate NK T cells as a major factor in the regulation of marginal zone B cells, a distinct B cell subset with inherent autoreactivity that has recently become a significant focus for research into the origins of autoantibody production in SLE. Methods that should allow the direct stimulation of NK cells in vivo will be investigated as potential approaches to therapy of SLE that could take advantage of the natural functions of these regulatory T cells. The program project format provides an optimal environment for these studies by providing numerous collaborative interactions with the other members of the program who bring it a wealth of expertise and resources for the analysis of murine models of SLE that are highly relevant to this proposal. These studies will also benefit from extensive usage of all of the Program s proposed core facilities.
Keywords: T lymphocyte, immunoregulation, natural killer cell, systemic lupus erythematosus, B lymphocyte, CD1 molecule, aging, antinuclear autoantibody, autoantibody, autoimmunity, cell growth regulation, cell mediated cytotoxicity, cell population study, cyclophosphamide, cytokine, cytotoxic T lymphocyte, disease /disorder model, immunogenetics, immunopharmacology, immunosuppressive, pathologic process, remission /regression, enzyme linked immunosorbent assay, flow cytometry, laboratory mouse, serology /serodiagnosis
Project start date: 2002-04-01
Project end date: 2007-03-31
Steven A Porcelli
Albert Einstein Col Of Med Yeshiva Univ
Project start date: 2011-03-01
Project end date: 2016-02-29
Steven A Porcelli, Professor
Yeshiva University 500 W 185th St New York, Ny 10033
Grant 5R01AI048933-05 from National Institute Of Allergy And Infectious Diseases IRG: ZAI1
Keywords: CD1 molecule, antigen presentation, bacterial antigen, cellular immunity, T cell receptor, glycolipid, phospholipid, synthetic antigen, Mycobacterium avium, Mycobacterium tuberculosis, cell line, human tissue
Project start date: 2000-08-01
Project end date: 2006-07-31
5R01AI048933-05 (2004): $272401
5R01AI048933-04 (2003): $343058
Sponsored Links Excellgen http://Excellgen.com
5R01AI048933-02 (2001): $253573
1R01AI048933-01 (2000): $263114
NON PROTEIN ANTIGEN PRESENTATION And CHARACTERIZATION
Steven A Porcelli, Professor
Boston University Medical Campus 715 Albany St, 560 Boston, Ma 021182394
Grant 5P41RR010888-020049 from National Center For Research Resources
Keywords: bacteria, biological product, biomaterial, biomedical resource, carbohydrate, communicable disease, growth factor, human tissue, immunology, lipid, lymphatic system, structural biology
ANTIGEN PRESENTATION BY CD1 PROTEINS
Steven A Porcelli, Professor
Brigham And Women s Hospital Research Administration Boston, Ma 02115
Grant 1R29AI040135-01 from National Institute Of Allergy And Infectious Diseases IRG: ZRG2
Project start date: 1996-06-01
Project end date: 2001-05-31
1R29AI040135-01 (1996): $110127