FY 00 INTER-AGENCY AGREEMENT

The Microgravity Research Division (MRD), Office of Life and Microgravity Sciences and Applications (OLMSA) of NASA, and the Laboratory of Cellular and Molecular Biophysics (LCMB), National Institute of Child Health and Human Development (NICHD) of NIH, are collaborating on a joint project in three-dimensional tissue culturing using the bioreactor technology developed by NASA. This agreement supports the transfer of ground-based NASA bioreactor technology for three-dimensional tissue culture to NIH and the external research community. Dr. Joshua Zimmerberg of the NICHD is the Director of the joint NASA/NIH biotechnology project and provides scientific and administrative supervision of the project. This agreement is effective through September 30, 2002, and may be modified by mutual consent of both parties. Peer review for continued funding of this project will be continuous and rigorous by the scientific counselors of the NICHD.

The National Aeronautics and Space Administration, through its Microgravity Biotechnology Program, has achieved a breakthrough in the engineering of tissues which allows for cellular growth, aggregation, and the development of in vivo like tissues. The National Institute of Child Health and Human Development, through its Laboratory of Cellular and Molecular Biophysics, is conducting biomedical research to elucidate the mechanisms of cellular dysfunction which lead to morbidity and mortality. Specifically, LCMB has developed a project on the long-term culture of human tissue for biomedical research. NASA and NIH have jointly focused their research and expertise on several fundamental medical science issues. This joint NASA-NIH program is using the unique talents and experience of both agencies to make a breakthrough in the culturing of human tissues for biomedical research. The first goal will be to engineer a human lymph node model for AIDS research. The second goal is to evaluate a broad spectrum of tissues available at the NIH.
 

Project Tasks

1. The goal of the Center is to maintain a core facility at NIH for use of the NASA Rotating Wall Vessel Bioreactor for studying the basic mechanisms of normal and pathological intercellular interactions in multi-cellular systems. The Center provides NIH researchers an opportunity to develop new model systems for biomedical research and provides staff and access to the Bioreactor technology.

2. Develop tissue cultures for other NIH projects which require a higher level of organization than that available in monolayer or suspension culture by using the NASA bioreactor technology.

3. Use the NASA Bioreactor to develop an AIDS tissue model for use in understanding various aspects of HIV pathogenesis in human lymphoid tissue ex vivo.

4. Develop tests for drugs to control Cyclospora infection of human intestinal cells and the studying the life cycle of the parasite

5. Understand the cellular mechanisms of the development and metastasis of prostatic tumor in the context of benign tissue in RWV

6. Identify the genes involved in salivary gland cell differentiation.

7. Identify the cellular and molecular mechanisms underlying microgravity-triggered immunodeficiency

8. Develop a universal pathogen culture system in the RWV comprised of a multi-tissue equivalent that creates the necessary microenvironment for most common types of human pathogens

9. Develop a dual photon laser microscopy to monitor cells within the tissue architectures that are created using the NASA bioreactor.
 

FY 2000 Annual Report

I SUMMARY

One of the most important goals of the field of cell biology is to understand the normal and pathologic cell behavior within the organized tissue. To answer this challenge, in 1994 the NASA/NIH Center for Three Dimensional Tissue Culture was inaugurated, with the mission to develop and disseminate three-dimensional tissue culture techniques for studying cell biology in the context of tissue cytoarchitecture. Both NASA and NIH contributed to the Center organization in personnel, funds, and equipment. In less than four years, the NASA/NIH Center is fully operational and has attracted dozens of intramural and extramural scientists who have used NASA-developed techniques for their projects in Biomedicine. The Center facility is open to any intramural scientist who wishes to expand their project by using the RWV. Research teams from four Government agencies, including seven NIH Institutes, are currently work using the Center. Four projects have now produced results beyond the preliminary phase and are funded through the NASA/NIH Center intramural grants.

The scientific accomplishments of the research teams at the Center include: (i) Demonstration that the RWV Bioreactor is able to induce acinar differentiation when human submandibular cells are exposed to basement membrane matrix and the laminin-1 protein, giving new insight into the mechanisms of gland formation; (ii) Development of a system for studying HIV infection in human lymphoid tissue cultured in the RWV that can be used as a universal tool to study critical events in HIV infection; (iii) Use of the RWV for productive infection of human intestinal cells with human parasite Cyclospora, which poses a problem for public health in the United States and could not be propagated in other in vitro systems; (iv) Long term maintenance of human prostate tissue in the RWV Bioreactor, making possible the testing of the sensitivity of prostate tumors to various drugs; (v) The ability of the RWV bioreactor to maintain metaplastic phenotypes of bronchobiopsies for correlation of gene activity with various state of metaplasia; (vi) Induction of Kaposi-like lesions in human tissues co-cultured with HHV8-producing cells, providing conclusive evidence that HHV8 causes Kaposi's Sarcoma and contributing towards the understanding how this virus causes neoplastic transformation, and (vii) Assessment of microgravity effects on immunopathology in human lymphoid tissue cultured in RWV, and understanding the cellular mechanisms underlying this phenomena. This finding opens the ways to establish cellular and molecular mechanisms of the defects in immune system functioning in space flights.
 
 

II. INTRODUCTION

This report outlines the activity of the NASA-NIH Center for three-dimensional Tissue Culture in 1999. The Center was used by a number of scientists who work

on projects related mainly to cell-cell interactions and cell differentiation, as well as to the pathogenesis of infectious diseases. The Center facility is open to any intramural scientist who wishes to expand their project by using the RWV. We have established procedures for initiating projects. In Phase I, NIH scientists can launch pilot projects involving the RWV Bioreactor as extensions of their main intramural research projects. In addition, two Phase I projects were launched recently by extramural investigators from FDA and Johns Hopkins University. Based on these preliminary results a NIH group can submit a proposal for a study involving the RWV and get funding for purchasing a RWV, disposables, and post-doctoral fellow salaries. Four projects have now advanced beyond this preliminary phase: 1) Differentiation of salivary gland cells in the RWV Bioreactor, 2) A model of the lymphoid system for studying HIV infection in vitro, 3) RWV Bioreactor models of human squamous metaplasia, and 4) Long term maintenance of human prostate explants in the RWV.

The work of the NASA/NIH Center as a whole, and each funded scientific project is subjected to continuous and rigorous peer review by the Scientific Counselors of NICHD during their regular site visit. The most recent site visit took place in 1999. The Counselors had a high opinion of the scientific accomplishments and the strategy of the Center, and recommended continued funding with continued peer review.

Below are brief descriptions of the current projects at the NASA/NIH Center.

III. PARTICIPATING LABORATORIES AND PROJECTS

I. National Institute of Child Health and Human Development

1. Effect of microgravity on immune function of lymphoid tissue
2. Study of HIV transmission to lymphoid tissue
3. Dual-photon microscope for 3-D tissue analysis
4. Culture of endometrium biopsies

A. Prostate tissue proteomics

B. Study of Lyme disease in RWV Bioreactor

C. Maintenance of human preneoplastic lung lesions

III. National Institute of Allergies and Infectious Diseases

1. Growth of Norwalk-like viruses

A. Differentiation of salivary gland cells in the RWV Bioreactor

V. National Institute of Arthritis and Musculoskeletal and Skin Diseases

A. Culture of synovial tissue from rheumatoid arthritis patients

VI. Food and Drug Administration

A. Replication of human intestinal parasite in intestinal cells in the RWV Bioreactor
 
 

VII. Naval Medical Institute

A. RWV Bioreactor models of human squamous metaplasia in the lung

VIII. Howard University, Washington D.C.

A. Culture of hematopoeitic stem cells for gene therapy
 

IX. Institut Cochin de Genetique Moleculaire, Paris, France (in collaboration with LCMB, NICHD)

1. Study of HIV mucosal transmission

X. Rambam Medical Center, Haifa, Israel (in collaboration with LCMB, NICHD)

A. Differentiation of Rhesus embryonic stem cells
 
 

XI. Inst. of Mutagenesis and Differentiation, Pisa, Italy (in collaboration with LCMB, NICHD)

A. GM-CSF activity in tumor cells

IV. INDIVIDUAL PROJECTS: PROGRESS REPORTS AND PLANS

1. Role of laminin in differentiation of salivary gland cells.

 

 

Primary Investigators: Hynda Kleinman, Ph.D., Matthew P. Hoffman, D.D.S., Ph.D., and Dalit Hecht, Ph.D., Laboratory of Developmental Biology, National Institute for Dental Research, NIH.

Start Date: 4/96 Ending Date: Ongoing

Objectives: Determining the effects of the extracellular matrix on salivary gland cell differentiation. Defining the matrix requirements, determining the active site on laminin for acinar formation, the cellular receptors, the signaling events and induced genes.

Background/Rationale: Cell-type specific interactions between laminins and multiple receptors and signaling pathways provide mechanisms for regulating the complex events of morphogenesis and the maintenance of differentiated adult tissues . We have used the HSG cell line obtained from a submandibular gland tumor to study acinar cell differentiation. We have found that when these cells are grown in the presence of the basement membrane glycoprotein laminin, they migrate towards each other and form acinar-like structures with polarized nuclei in 24 hours. On plastic they form a typical epithelial cell monolayer. In RWVs, the cells form acinar structures in the presence of laminin and undifferentiated clumps in the absence . We have identified an 8 amino acid site (LQVQLSIR) responsible for this morphological differentiation. The corresponding cellular receptor (syndecan-1), and PKC and MAPK pathways are important in the signaling process. Laminin and the active peptide both give nice acinar differentiation in RWVs (Figure 1). There are various diseases and certain drug treatments that result in loss of salivary gland function where the acini are lost or non-functional. Identification of the mechanisms involved in the formation of the glands will enable possible therapeutic intervention and/or gland regeneration.

Publications: Hoffman M, Nomizu M, Roque E, Lee S, Jung, DW, Yamada Y, Kleinman H.(1998), Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line. J. Biol. Chem. 273:28633-41.

Progress Report: Our previous experiments using HSG cells cultured in RWV have helped to define the involvement of beta-1 integrins and syndecan 1 in the cell-matrix interactions in acinar differentiation. These studies have also shown that the peptides identified so far may be providing some but not all the necessary signals for the process of acinar differentiation. We have defined the active site on laminin using a systematic screening of synthetic peptides. Once identified, we used affinity chromatography, antibodies, and SDS gels to identify the key cellular receptors. We are now using commercially obtained cDNA arrays to identify genes induced by laminin in RWVs. We have screened some 10,000 cDNAs and identified several that are induced by laminin in salivary glands cultured in RWV. We have confirmed by northern analysis that one is upregulated some 5-fold.

Specific Aims:

Search for additional laminin-regulated genes: We are continuing to screen commercial filters from Research Genetics with mRNA from HSG cells grown in RWVs in the presence and absence of laminin for various times. Each filter will be screened at least twice, once with the mRNA from cells exposed to laminin and once with mRNA from cells not exposed to laminin, and each experiment will be done twice to reduce false positives. We will select only for genes that are changed five-fold or more.

Characterization of laminin-regulated genes: All positive clones will be checked by northern analysis using RNA from HSG cells at different time points in the presence and absence of laminin in the RWV and in regular tissue culture. This will provide a profile on the size of the gene and its timing of expression as well as to confirm that it is regulated either up or down by laminin. We will next check by northern analysis if this effect is specific for HSG cells by looking at mRNA levels in endothelial, melanoma, and fibrosarcoma cells that have been exposed to laminin. Next, we will use antisense and sense oligonucleotides in cell culture to determine the functional importance of the selected and interesting genes. We also will obtain antibodies to study the biological role of these genes in vitro and their localization in the developing gland in vivo. We anticipate identifying several regulatory genes important for cell differentiation. We will select the ones that show the greatest differences at various time points. We also hope to identify certain salivary gland cell-specific genes. A possible problem is that many of the genes that would be most interesting could be very low abundance genes that are much harder to identify and work with. We are trying to increase the sensitivity of our screen to find these low abundance genes.

2. The effect of microgravity on the immune function of human lymphoid tissue.

 

 

Primary Investigators: Jean-Charles Grivel, Ph.D., Leonid Margolis, Ph.D., and Joshua Zimmerberg, M.D., Ph.D., Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, NIH.

Start Date: 12/97 Ending Date: Ongoing

Objectives: To determine the effect of microgravity on the immune function of human lymphoid tissue and lymphocytes.

Background/Rationale: Astronauts have demonstrated transient immune dysfunctions that may be due to microgravity experienced in space . However, exposure to other space flight-related factors such as launch-related hypergravity, psychological stress and cosmic radiation may also affect immune function. In an attempt to investigate the isolated effects of microgravity, we studied the immune competence of human lymphoid tissue in the simulated microgravity generated in the NASA-designed Rotating Wall Vessels (RWVs).

Publication: Fitzgerald W, Grivel J-C, Margolis L. Modeled microgravity in the NASA Rotating Wall Vessel causes immune suppression in human lymphoid tissues ex vivo. (submitted)

Progress Report: Unlike static cultures, RWV cultures produce antibodies neither in response to recall antigen, tetanus toxoid (Figure 2), nor to the polyclonal activator pokeweed mitogen (PWM). Isolated lymphocytes lose responsiveness to polyclonal activators when cultured in RWVs (Figure 3). In RWVs, despite a rapid decline in immunoglobulin production, lymphocytes residing inside the tissue blocks are activated by PWM as measured by proliferation, suggesting that the early steps of this response take place in RWVs. Indeed, when blocks are activated in RWVs and transferred to static culture, antibody production resumes, whereas tissue blocks challenged in static culture stop producing antibodies when transferred to RWVs. Tissue microenvironment protects lymphocytes from microgravity since, as opposed to isolated lymphocytes, tissue resident lymphocytes are activated in RWV and responses are restored with return to static condition. We suspect that after they have differentiated into plasma cell precursors, B cells leave the protective niche of lymphoid tissue and become fully exposed to the detrimental effects of microgravity. Indeed, both antibody production and viability of plasma cells are decreased in RWVs.

Specific Aims:

Identifying patterns of membrane reorganization in isolated lymphocytes activated in RWV: We will investigate early events in cell membrane reorganization involved in lymphocyte activation. Specialized detergent-insoluble, glycosphingolipid-enriched membrane domains (DIGs), also known as "rafts" or membrane microdomains, serve as the site of attachment for a variety of lipoproteins involved in cell signaling . Disruption of DIGs attenuates signal transduction in T cells. Following TCR stimulation, DIGs are redistributed in plasma membrane and become enriched in several enzymes involved in signal transduction such as ZAP70 and phospholipase C?1 (PLC?1) . Since PMA restores mitogen response in RWVs, this suggests that DIGs are altered such that PLC?1 is not optimally activated in lymphocytes. We will test whether DIGs are altered in RWVs by isolating DIGs from lymphocytes cultured in RWV, analyzing their composition by western blot, and comparing it to that of lymphocytes cultured in non-rotating vessels or static cultures. In addition, we will compare DIG composition in both tissue resident lymphocytes and lymphocytes that have left the tissue. We anticipate finding differences in DIGs from isolated lymphocytes cultured in RWV and control lymphocytes. If no differences in DIGs composition are found, we will evaluate the next step in the response to receptor-mediated activation, the reorganization of cytoskeletal elements. We expect that in contrast to isolated lymphocytes, DIGs from tissue resident lymphocytes will not be different from controls. This could explain why lymphocytes inside the tissue respond to mitogens. In addition DIG redistribution will be followed either by flow cytometry or confocal microscopy using FITC-labeled cholera toxin B subunit, which reports on GM1 glycosphingolipid distribution.

Cytoskeletal changes upon activation: TCR engagement results in a polarized reorganization of cytoskeleton and the formation of caps . This process involves a series of depolymerization and polymerization of F-actin and myosine filaments which results in a complete reorganization of co-receptors and enzymes necessary to sustain cellular activation. Microgravity has been shown to alter cytoskeletal organization . We hypothesize that RWV culture interferes with cytoskeleton reorganization leading to abnormal signaling. We will address this issue by studying cytoskeleton structure in lymphocytes cultured and activated in RWVs, or cultured in RWVs and activated in static cultures. Activation will be performed using beads coated with both anti-CD3 and anti CD28 antibodies. The actin filament cytoskeleton will be visualized by confocal microscopy using Rhodamine-labeled phalloidin either alone or in association with components of the TCR signaling complex such as CD3.

Proving microgravity is the cause of immune suppression: Our experiments demonstrating lymphoid tissue loses its ability to maintain an immune response in RWVs suggest that microgravity is the cause of immunosuppression, however, alternative interpretations are possible. Particular features of RWV design, such as the form of the vessel, the speed of rotation etc. may be responsible for the observed effects. Although we reported on important controls for some of these factors, there are significant difficulties in controlling all of them. For example, we designed an experiment to control for the effect of rotation on cultured cells by incubating them in a non-rotating RWV, however, for tissue blocks such a control experiment is not feasible. When all the proper controls are designed, there is still a point of discussion as to what extent and in which aspects the RWV mimics microgravity conditions . The ultimate proof of microgravity as a cause of immunosuppression can be obtained only in experiments in the microgravity of space. In case such an opportunity is provided, we are currently in the process of designing a series of experiments and controls feasible to perform in the framework of the International Space Station.

3. HIV transmission to human lymphoid tissue.

 

 

Primary Investigators: Leonid Margolis Ph.D., and Jean-Charles Grivel, Ph.D., Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, NIH.

Start Date: 4/95 Ending Date: Ongoing

Objectives: Investigation of early events in HIV transmission to human lymphoid tissues.

Background/Rationale: Critical events of HIV disease occur in lymphoid tissue . The use of cultured human lymphoid tissues enables a better understanding of the pathogenesis of HIV disease. In previous stages of this study we have shown that the RWV system allows maintenance of blocks of human lymphoid tissues for three weeks, and these tissue blocks can be infected with laboratory strains of HIV-1 .

Publications: Margolis LB, Fitzgerald W, Glushakova S, Hatfill S, Amichay N, Baibakov B, Zimmerberg J. (1997) Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor. AIDS Res Hum Retroviruses 13:1411-20.

Progress Report: Previous work on HIV pathogenesis using lymphoid tissue cultures in RWVs was published. Work has not yet started on the mucosal transmission model in the RWV. Vessel design has been approved and the vessels have been ordered.

Specific Aims:

Studying mechanisms of HIV transmission: A newly developed dual-chamber RWV will be used to explore early events in HIV infection. Lymphoid tissue and/or lymphocyte suspensions will be cultured in the first chamber. Intestinal or vaginal cells or biopsies will be cultured in the second chamber on the permeable membrane that separates the two chambers. Culture conditions in both chambers will be independently optimized. This design will mimic the mucosal barrier that HIV first encounters before being transmitted to lymphoid tissue. We anticipate that in the dual-chamber RWV, HIV will cross the mucosal barrier into the second chamber and infect target cell populations. HIV strains of different tropism will be studied and transmission of the viruses to specific cell types will be examined. Specifically we will test whether there is a difference in transmission between CCR5- and CXCR4-tropic HIV-1 variants (R5 and X4, respectively). If R5 are transmitted in this setting more efficiently than X4 variants this could explain why in vivo the former transmit infection. We also plan to use this model of transmission to test methods of preventing vaginal/rectal transmission of HIV infection at the point of entry.

Dual Chamber RWV for Study of other Pathogens: Applications of the above described model could be expanded further to include the study of early infection events and pathogenesis of other sexually transmitted pathogens such as human papilloma virus (HPV), herpes simples virus (HSV), cytomegalovirus (CMV), Neisseria gonorrhoeae, and Chlamydia trachomatis.
 

4. Study of HIV mucosal transmission.

 

 

Primary Investigators: Morgane Bomsel, Ph.D., Signalling, Inflammation and Cellular Transformation, Institut Cochin de Genetique Moleculaire, Paris, France.

Start Date: 2/99 Ending Date: Ongoing

Objectives: Studying the spread of HIV infection in the mucosa using cultures of intestinal and rectal mucosal biopsies. Neutralizing HIV spread in mucosal biopsies with anti-HIV envelope protein antibodies (IgA and IgM).

Background/Rationale: HIV infected mononuclear cells spread HIV infection across mucosal epithelial cells. HIV virions bud off the mononuclear cells and are internalized by epithelial cells via the epithelial transcellular vesicular pathway. After transcytosis, the infection is spread to other mononuclear cells at the basolateral side of the epithelial barrier . Details of this process are difficult to follow in vivo thus a high-fidelity in vitro model is useful for these types of studies. If this transcytosis process can be mimicked in vitro, it would provide a useful model for analyzing the ability of polymeric IgA anti-HIV envelope to neutralize the spread of HIV. It is believed that induction of mucosal immunity to HIV envelope proteins impairs the transcytotic route of HIV mucosal transmission .

Progress Report: HIV infection of mucosal biopsies was performed in a polarized manner from the luminal pole of the biopsy using Ussing chambers. After infection, the biopsies were cultured in RWVs for two weeks. Samples were taken periodically to analyze for HIV content by p24 assay kits. Mononuclear cells on the basolateral side of the epithelium were analyzed to look for productively and non-productively infected cells.

Specific Aims:

Difficulties have been encountered in maintaining viability of the biopsies for extended periods of time. Improvements are being made to culturing techniques to increase culture length for these biopsies. Alternate methods, such as using cell lines or looking at events within a shorter time frame, are also being researched. Once culture conditions are improved then analysis of underlying lymphoid associated cells will begin and neutralization assays will be performed. Tissues will be infected and transferred to RWV culture in the presence of anti-HIV envelope dimeric IgA to try to neutralize HIV transcytosis.

5. Differentiation of rhesus embryonic stem cells.

 

 

Primary Investigators: Abraham Lightman, M.D. and Joseph Itskovitz-Eldor, M.D., Department of OB/GYN, Rambam Medical Center, Haifa, Israel, Joshua Zimmerberg, M.D., Ph.D., Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, NIH, and Cynthia Dunbar, M.D., Division of Intramural Research, National Heart Lung, and Blood Institute, NIH.

Start Date: 5/99 Ending Date: Ongoing

Objectives: Analyzing the factors promoting or preventing differentiation of Rhesus embryonic stem cells.

Background/Rationale: Embryonic stem (ES) cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers . ES cells can be used to study the mechanisms controlling differentiation of specific lineages. Rhesus ES cells offer a better model of human development than other stem cells, such as those from the mouse . Since ES cells have the developmental potential to give rise to all adult cell types, any disease resulting from failure of specific types would be potentially treatable through transplantation of differentiated cells derived from stem cells. Since the RWV has been useful in obtaining more differentiated phenotypes of many cell types, this device will be used to allow differentiation of rhesus ES cells. The RWV may allow the stem cells to differentiate into cell types that are not easily obtained in two-dimensional culture.

Progress Report: The rhesus ES cells were just recently received from Haifa. The NASA/NIH Center technician was trained by the Haifa team to work with these cells, and they are being expanded in culture. The ES cells are typically cultured on feeder layers of mouse embryonic fibroblasts (MEF) which have been irradiated or treated with mitomycin C. MEF have also been obtained by the Center and they have been cultured successfully in RWVs on microcarriers either with or without mitomycin C treatment.

Specific Aims:

Spontaneous differentiation of ES cells: ES cells will be allowed to spontaneously differentiate for several weeks in RWVs, either with or without feeder layers. When these cells are allowed to spontaneously differentiate in standard tissue culture, they typically express markers of trophoblast or endoderm cells . We will examine cells from these RWV cultures for evidence of ectoderm and mesoderm derived cells. Live cell samples will be evaluated by confocal microscopy and histology will be analyzed on fixed cell samples. Enzyme linked immunosorbant assays will also be used for analysis of culture supernatants to aid in identification of cell types.

Controlled differentiation of ES cells: Experiments will focus on directed differentiation of ES cells into specific lineages. Through the use of cocktails of growth factors and cytokines, we anticipate to be able to drive differentiation of cells into specific lineages. Emphasis will be on analyzing signals and factors required for differentiation of ES cells into the various hematopoietic cell types.

Identification of the factors that prevent differentiation: No factors have yet been identified which allow the rhesus ES cells to proliferate and remain undifferentiated in the absence of fibroblasts. Neither conditioned medium nor matrix from fibroblasts prevents differentiation . We hypothesize that contacts between ES cells and MEF are critical for maintenance of the undifferentiated status. ES cells will be cultured in RWVs in presence of MEF and the mixed cell aggregates will be removed from the vessels over a set time course during which the ES cells will progress from an undifferentiated to differentiated phenotype. The contact areas between MEF and undifferentiated or differentiated ES cells will be quantified and their morphology will be analyzed using laser confocal and electron microscopy. We anticipate that in aggregates with differentiated ES cells, we will see a decrease in areas of contact. If this is not the case, then we will study whether the ultrastructure of area where ES contacts MEF is altered. Also we will search for MEF-associated factor involved in ES cell differentiation. We will compare using chip technology MEF gene expression in MEF-ES cell aggregates at early stage of co-culture, when ES cells remain undifferentiated and at the late stage when MEF failed to support undifferentiated ES cell status.

6. Developing an advanced dual-photon microscope for 3-D tissue analysis.

 

 

Primary Investigators: Paul Blank, Ph.D., Laboratory of Cellular and Molecular Biophysics, NICHD, NIH.

Start Date: 5/95 Ending Date: Ongoing

Objectives: Design, optimization, and application of a two-photon excitation fluorescence microscope for tissue imaging.

Background/Rationale: Optical microscopy and spectroscopy are indispensable techniques for the study of cells and cellular processes. Optical probes for a variety of intracellular environments can be detected and localized with fluorescence microscopy. Aspects of cellular biochemistry can be manipulated with photoreactive "caged" compounds and recently, fluorescently modified versions of endogenous proteins can be expressed and monitored in situ. The use of a wide variety of different microscope technologies has expanded the concept of the traditional microscope image. Any time and/or spatial variation in a signal representing an underlying process of interest can be considered as an image map of this process. A key problem in biology is how cellular organization influences the development, function, and activity of individual cells. This problem is best approached when there exists the ability to monitor functioning cells within the organized structure, i.e. tissue. Optical techniques are powerful tools for studying living cells but imaging tissue with high spatial and temporal resolution remains a challenge. One approach to resolving the difficulties inherent in optically thick specimens is to use the technique of two photon fluorescence excitation microscopy. This optical technique relies on the non-linear properties of fluorophores coupled with laser scanning microscopy to produce an intrinsic, three-dimensionally resolved (confocal) image map of the specimen. A two-photon excitation fluorescence microscope has been designed, constructed, and tested and image processing tools have been developed to facilitate 4-D imaging of (3-D time lapse) isolated and organized cells.

Progress Report: A Leica TCS microscope was modified for two-photon excitation fluorescence microscopy. An argon ion pump laser and tunable titanium-sapphire laser were used as the excitation light source to produce the required train of mode-locked light pulses responsible for the non-linear generation of fluorescence. The instrument was further modified to allow direct detection of fluorescence collected by the objective with two photomultiplier tubes. At this time, two preparations were examined in the microscope: monolayers of human lymphocytes from tonsils and cultured blocks of human lymphoid tissue. Two photon evoked images were obtained using DiOC5(3), an endoplasmic reticulum sensitive indicator, and the calcium sensitive indicators Indo-1 and Fura-2 in monolayers of cells and tissue cultures. However, lymphocytes loaded with calcium green, Fluo-3, or Fluo-4 and monitored using conventional one-photon microscopy all showed increases in intracellular calcium concentration in response to 100 uM ATP, but when these cells were loaded with Indo-1 and Fura-2 then observed using two-photon excitation they did not respond to ATP with an increase in calcium. These observations illustrate the difficulty and requirement for optimizing this technique for studying cells. Our working hypothesis was that the excitation intensity levels were too high. To reduce the level of excitation, an optical compressor was incorporated into the device. The use of an optical compressor maintains the required pulse width at the sample by compensating for the inherent pulse spreading properties of the optical elements which the laser light must propagate through. The use of the optical compressor has increased our excitation efficiency such that detection of Indo-1 fluorescence requires ~ 20 fold less light at the sample. Images generated previously with excitation levels of 20 - 100 mW can now be collected using 1 - 5 mW at the sample. Our goal is to reduce the excitation power to ~ 1 mW when possible in order to create as large a margin between efficient generation of signal and detrimental phototoxicity. Functional studies to evaluate the reduction in excitation power are ongoing.

Specific Aims:

Optimization of the two-photon excitation fluorescence microscope: We will continue to improve the 4-D tissue imaging and increase of the spectral range available for two-photon excitation. The availability of two fluorescence detection channels will now allow collection of ratio images derived from one or two fluorophores or direct detection of the laser for use in noise reduction. Modification of the Ti-sapphire laser with a new optics set will allow continuous excitation wavelength selection from ~ 700 to 1000 nm. Two-photon excitation in the IR can be used with Green Fluorescent Protein (GFP) or similar spectral analogues.

Application of two-photon excitation fluorescence microscope: One application of this technology will be the imaging of the early events of immune responses in lymphoid tissue ex-vivo. The development of an immune response takes place in the highly organized secondary lymphoid tissue. This organization optimizes the spatio-temporal interaction of the antigen with the different cells involved in the generation of a successful immune response. The biochemical and cellular basis of T cell and B cell antigen recognition are well understood, however, despite a large body of literature on the anatomical organization of lymphoid tissue, a clear picture of these interactions as they appear in real time is still lacking. The development of methods for tracking individual T cells and B cells of known antigen specificity will shed some light on the cellular interactions that occur after the introduction of an antigen. The experiment we plan to perform will take advantage of the existence of several lines of mice transgenic for T cell receptor genes and B cell receptor genes that confer these cell types a pre-defined specificity for a well described antigen, Hen Egg Lysozyme (HEL). Mice transgenic for the T cell receptor of the hybridoma 3A9 are a homogeneous population of T cells that recognize the peptide 46-61 of HEL in the context of the class II MHC molecule IA-k. B cells from the mice transgenic for the HYHEL-10 encoding genes contain a unique population of B cells that only recognize HEL. In addition to their unique antigen specificity, other tools have been designed to characterize these cells. A monoclonal antibody C4H3 specific for the MHC peptide complex I-Ak/HEL46-61 recognizes the same epitope as the TCR of the T cell transgenic mice and therefore allows the monitoring of antigen processing by antigen presenting cells. Anti-idiotypic monoclonal antibodies specifically recognize the B cell receptor of HYHEL-10 transgenic B cells, enabling their tracking in tissues. These tools will enable us to monitor the most important phase of a nascent immune response: the antigen (labeled HEL), antigen presentation (antibody against MHC-peptide complex), T cell activation (transgenic T cells), B cell activation (transgenic B cells) and distribution (anti-idiotype antibody). We will monitor ex-vivo cultures of lymph node from these transgenic animals with our two-photon microscope. In B cell transgenic tissue, Fluo-3 labeled T cells will be introduced in the ex-vivo culture and their activation following perfusion with HEL (the antigen) will be monitored, as well as antigen presentation. In T cell transgenic tissue, Fluo-3 loaded B cells will be added and their response to HEL perfusion will be monitored. Finally the whole response will be monitored in lymphoid organs obtained from a naïve recipient mouse reconstituted with both labeled HEL-specific B and T cells, and the initiation of immune response will be monitored upon addition of HEL in these cultures.

Alternatively, we would like to develop sets of transgenic mice expressing both immune receptors of known specificity such as the one previously described and green fluorescent proteins (GFP). There are now several mutants of GFP that fluoresce at different wavelength, enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP) and enhanced yellow fluorescent protein (EYFP). Transgenic mice can now be constructed with these different fluorescent proteins under the control of various promoters. We will establish line of double transgenic mice for immunoglobulin genes (HyHEL10 mice) and ECFP under the control of the immunoglobulin promoter. Similarly mice double transgenic for T cell receptor genes (3A9 mice) and EBFP under the control of CD 4 promoter will be constructed. Finally a transgenic mice for EYFP under the control of the alpha actin promoter and CMV enhancer promoter will be constructed. These three mice will label the major three major cellular comparments involved in the development of immune response. T cells taken from the mice transgenic for T cell receptor and EBFP, together with B cells taken from mice transgenic for HyHEL-10 and ECFP will be introduced in animals transgenic only for EYFP that will provide the lymphoid tissue background (dendritic cells, follicular dendritic cells, stromal elements), allowing us to visualize an ongoing immune response.

7. Replication of a human parasite in intestinal cells.

 

 

Primary Investigators: Darcy Hanes, Ph.D., Food and Drug Administration, Laurel, MD, and Joshua Zimmerberg, M.D., Ph.D., Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, NIH.

Start Date: 8/96 Ending Date: Ongoing

Objectives: Establishing a differentiated intestinal cell model that mimics the in vivo condition to study the kinetics of infection of intestinal parasites, in particular Cyclospora sp., the interactions between the parasites and infected cells, and the stages of life cycle occurring in the human gut.

Background/Rationale: Little is known about the tissue interactions and pathogenic mechanisms involved in enteric infections of mammals with Cyclospora sp., as well as about the life cycle of this parasite . Most in vitro models are based on infections of monolayers of poorly differentiated cells, which confounds comparison to the in vivo condition, and in many cases do not support parasite replication. The RWV provides a high fidelity in vitro model of the mammalian epithelial gut wall.

Progress Report: CaCo-2 cells, derived from human colon epithelial cells, were cultured in RWVs in order to obtain a higher degree of differentiation of the cells. The cultures were then incubated with a known amount of Cyclospora oocysts and cultured another 8-10 days. The number of oocysts the culture medium post-infection was quantified by light microscopy. Cells from infected and control cultures were collected for analysis with light and electron microscopy. These types of evaluation demonstrated that C. cayetanensis has been successfully cultured in differentiated CaCo-2 cells in the RWV. Enumeration of oocysts from cells and supernatant of infected RWV cultures demonstrated a four-fold increase in the number of oocysts when compared to the initial inoculum (Figure 4). This productive infection has made it possible to study the life-cycle of this parasite ex vivo. Several stages of the asexual portion of the C. cayetanensis life-cycle, that had been previously described only in intestinal biopsies from infected individuals, have been identified in infected RWV cultures of human intestinal cells. Early events in infection including attachment can also be followed using electron microscopy (Figure 5).

Specific Aims:

Improvement of the current culture model: One focus of future research will be to increase the number of oocysts produced during infection. A new cell line, BBE, will be evaluated for this purpose since they are reported to be more differentiated than CaCo-2 cells. Since the differentiation status of the cells is thought to be crucial for C. cayetanensis infection, this cell line may increase productivity of oocysts. A cell-adapted C. cayetanensis oocyst line may also increase productivity. Oocysts will be continually passaged through RWV culture in an attempt to produce a parasite line adapted to RWV tissue culture. Harvested oocysts have been used for re-infection studies; data from these pilot experiments indicate that oocysts produced in the RWV are infectious and initiate further productive infection in CaCo-2 cells. Data from a related parasite, Cryptosporidium parvum, indicate that cell mediated immunity plays an important role in infection . Certain cytokines such as tumor necrosis factor alpha (TNF-?) enhance infection, and preliminary data shows that CaCo-2 cells are capable of binding TNF-?. Cytokines will be evaluated for their ability to increase the oocyst production of C. cayetanensis in RWV tissue culture.

Production of polyclonal antibodies to C. cayetanensis: Anti-C. cayetanensis antibodies are not readily available. Oocysts derived from the RWV will be used to generate antibodies to C. cayetanensis. Intact and excysted oocysts will be used to produce polyclonal antisera in rabbits. The specificity of this antisera will be characterized using oocysts derived from RWV tissue culture. Such an antisera has potential for diagnostic testing.

Further application of culture system: The FDA is developing several strategies such as irradiation and freezing to inactivate C. cayetanensis oocysts on contaminated produce . RWV tissue culture will be used to assess the viability of oocysts following treatment. Initial studies suggest that frozen oocysts are less viable than untreated oocysts. Several parameters of freezing and thawing will be evaluated using RWV tissue culture. Irradiation studies will begin once the freezing experiments are completed. Similar experiments will be conducted with irradiated Cryptosporidium parvum, in hopes of replacing the mouse-lethality assay. We also hope to propagate C. parvum in the RWV to provide material for research since the major source of C. parvum is laboratory infected cattle, which is expensive and available only at specialized facilities. Eimeria tenella, a chicken parasite, is a major concern for the poultry industry. This parasite is also difficult to propagate in conventional tissue culture . Growth of this parasite in CaCo-2 cells grown in the RWV is being evaluated in collaboration with Merck Industries. Initial studies indicate that E. tenella does not complete its life cycle in these cells. However, several different cell lines will be tested in the RWV for propagation of this parasite.

8. Modeling of Lyme disease.

 

 

Primary Investigators: Paul Duray, M.D., Department of Pathology, National Cancer Institute, NIH.

Start Date: 3/98 Ending Date: Ongoing

Objectives: Development of an in vitro model of tissue infection by Borrelia burgdorferi, the etiologic agent of Lyme disease, for studying the infection process and ensuing disease progression and identifying the genes involved in the spirochete's survival.

Background/Rationale: Lyme disease is a multisystem inflammatory disease caused by infection with the tick-borne spirochete Borrelia burgdorferi and is the most common vector-borne infection in the United States. Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response and is able to "adapt" to distant human tissue such as the brain, liver, and joint. It is not known how the organisms survive immune attack in the mammalian host. The recent isolation of p100, a glycoprotein expressed in borrelial spirochetes upon invasion of human brain tissue supports our hypothesis that this spirochete undergoes genetic alteration with association of protein expression . We hypothesize that newly expressed proteins correlate with tissue specificity, tissue tropism, and invasion. There is not an appropriate in vitro model in which to study the progression of Lyme disease in humans or an animal model that presents all the symptoms of human disease.

Progress Report: We have conducted multiple trials to explore the feasibility of cultivating the Borrelia burgdorferi, under ex-vivo conditions in the RWV utilizing human tissue substrates. We have shown that RWV cultures: a) allow maintained growth of low passage Borrelia burgdorferi spirochetes in RWVs (manuscript in preparation), b) permit the colonization of human tissue samples co-cultivated with the microorganisms (Figures 6 & 7) and c) allow exponential increases in the numbers of spirochetes in far greater numbers compared to current methods existing in the literature. Numerous experiments were done utilizing controls, inhibition steps, and culture medium trials with and without the RWV, with the best results obtained using human tonsillar tissue substrates and RPMI medium for growth up to 28-30 days. Tissue biopsies from patients with Lyme disease have been cultured in RWVs to look for proliferation of spirochetes in primary infected tissue. The RWV has also been used to culture normal human tissues with Borrelia, to study the infection process.

Specific Aims:

Identification of the genes involved in Borrelia invasion and persistence: Our current hypothesis that in the course of Lyme disease, Borrelia expresses certain genes to generate peptides that enable it to invade tissue. This is done in such a manner that cell immunity fails to recognize these protein antigens, or if it does, it triggers an autoimmune response to similar antigens on normal cells . We need to isolate these putative genes and protein products that the spirochete expresses or uses in order to understand how Borrelia survives in the skin following a tick bite, how it survives exposure to host inflammatory cells and immune mechanisms, and how it develops the capacity to disseminate and invade distant tissue sites such as brain, heart, and other organs. The entire genome for B. burgdorferi has been sequenced by TIGR, Rockville, MD, thus enabling the generation of a cDNA library for the spirochete, exclusive of a few genes on extra-chromosomal plasmids that remain to be sequenced. Since we have established that spirochete is able to invade and grow in the RWV cultures, the next step will be to isolate the proteins and cDNA at different time points from lymph node cultures infected with the spirochete and generate expression libraries. In this way, we should be able to identify cDNA involved in B. burgdorferi tissue adaptation. The first step will be to conduct protein fingerprinting using the SELDI method, a system referred to as tissue proteomics, and compare the protein profile at baseline and at different time points post-infection in the RWV. This may lead to identification of genes involved in human tissue invasion and persistence, and could provide information to design target molecules for future vaccine development.

9. Growth of Norwalk-like viruses.

 

 

Primary Investigators: Kim Y. Green, Ph.D., and Albert Kapikian, M.D., Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH.

Start Date: 3/99 Ending Date: Ongoing

Objectives: Establishing an in vitro system that supports replication of the Norwalk-like viruses.

Background/Rationale: Norwalk and Norwalk-like viruses, members of the Caliciviridae family of single-strand, positive-sense RNA viruses, are important etiologic agents of acute, nonbacterial epidemic gastroenteritis. A major limitation in the study of the prototype Norwalk virus has been the failure to propagate it in cell culture, despite numerous attempts since its discovery in 1972 . Chimpanzees are the only laboratory animals in which Norwalk virus replication has been demonstrated . Thus, an inexpensive and practical animal model is not available. The ability to grow Norwalk virus in cell culture would be a major advance in the study of these human pathogens. A cell culture system would allow the development of a neutralization assay and provide an important tool in the study of the biology and replication of the virus. It is possible that the growth restriction for Norwalk virus in conventional cell culture is related to the absence of appropriately differentiated cells. The RWV may allow the growth of differentiated (or less de-differentiated) cells that are susceptible to Norwalk virus infection.

Progress Report: Preliminary experiments involved Norwalk virus infection of two intestinal cell lines (CaCo-2 and Intestine 407) cultured in the RWV. Cell and medium samples were collected and assayed for evidence of viral amplification. Assays used included reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, immune electron microscopy, Western blotting, and enzyme-linked immunoassay. Norwalk viral RNA was detected by RT-PCR in these cultures for up to 7 and 13 days, for CaCo-2 and Int407 cells, respectively, following the initial inoculation. Medium from these infected cultures was serially passed to fresh cultures in an effort to adapt the virus to growth in cell culture. However, these attempts have not been successful. Infection of intestinal cell co-cultures (epithelial/fibroblast) is currently underway.

Specific Aims:

Establishing a cell culture model for Norwalk-like virus infection: We will continue to screen various RWV-cultured cell lines, cell line co-cultures, or primary isolated small intestine cells for their susceptibility to Norwalk virus infection. Several culture conditions will be varied in an attempt to improve infection conditions, including addition of proteolytic enzymes or intestinal fluids and manipulation of pH and temperature. Recent studies showed that porcine enteric calicivirus grows in cultured porcine cells only in the presence of intestinal content fluids from a gnotobiotic (germ-free) pig . We will continue to examine whether serial passage of Norwalk virus in the RWV will select variants that are better adapted to growth in cell culture. Susceptibility of cells to infection will be monitored by at least two of the above methods in order to decrease the likelihood of false positives or negatives. In addition to screening cell lines, we will examine whether fresh human chimpanzee or human intestine tissue explants support the propagation of Norwalk virus in the RWV. These explants may help in identifying which cells in the small intestine are permissive to infection. An important goal of our study is to identify the point at which infection and replication of Norwalk virus is blocked in cultured cells. Evidence for entry of virions into cells would suggest that the block is not at the receptor level, but rather, inside the cell. Perhaps specific enzymes or factors involved in Norwalk viral RNA transcription or viral protein processing are missing in many cultured cell lines. If a cell/tissue culture model can be devised, we will use this model system for growth of other related strains such as Hawaii Virus and Snow Mountain Virus.

Utilization of the cell culture model: A cell culture system would be invaluable to basic research of the Norwalk-like viruses and would also have a major impact on the development of prevention and control measures for these important, but poorly characterized, human pathogens. Several applications include the establishment of antigenic relationships by neutralization, mapping of major antigenic sites, determination of the role of neutralizing antibodies in immunity, mapping of the cellular receptor, uncovering the mechanisms for virus protein trafficking and processing in infected cells, and detailed analyses of RNA transcription and replication. In addition, a cell-culture system would allow the possibility of developing live, attenuated vaccines.

10. Maintenance of human preneoplastic lesions in the lung.

 

 

Primary Investigators: Bruce E. Johnson, M.D., and R. Ilona Linoilla, M.D., Medicine Branch at the Navy, DCS, National Cancer Institute and National Naval Medical Center; Melissa Means-Markwell, M.D., Department of Medicine, National Naval Medical Center.

Start Date: 1/97 Ending Date: Ongoing

Objectives: Determining if RWV culture maintains the histology of biopsy samples from the airways of patients at risk for a second lung cancer. Comparing of the genetic make-up of cultured tissues with the patient biopsy and their original tumor. Applying this culture system to testing of chemotherapeutic agents.

Background/Rationale: To identify which of a panel of chemotherapeutic agents is the most potent for a particular cancer, it is necessary to develop a test system in which the cancer tissue either retains its original morphology and genetic makeup or develops in the same manner as it would develop in vivo. We will attempt to develop such a system. Patients successfully treated for both small cell and non-small cell lung cancer remain at risk for developing second lung cancers. Patients surviving small cell lung cancer are typically treated with chemotherapy and chest radiotherapy while most patients with non-small cell lung cancer are treated with surgical resection. Patients treated with chest radiotherapy are at 2-3 fold higher risk of developing a second lung cancer than patients treated with surgical resection alone . A new technology, the Xillix bronchoscope, analyzes the airways of patients at high risk for second cancers using the principles of tissue autofluorescence to detect subtle differences in epithelial tissue pathology. This increases the sensitivity of bronchoscopy two fold for detecting areas of moderate dysplasia, severe dysplasia, and carcinoma in situ . These abnormal lesions are biopsied and sent for histological examination by a pathologist and for RWV culture. The histologic examination of the original and cultured biopsies are reviewed by an anatomic pathologist and identified as showing metaplasia, mild, moderate, or severe dysplasia.

Progress Report: Forty patients at risk for lung cancer have been studied from 1997 through 1999. The biopsy specimens have all been reviewed by two anatomic pathologists. The biopsies of the airways have shown metaplasia in 9 biopsy specimens, moderate dysplasia in 2, severe dysplasia in 1 and carcinoma in situ in 1. All the specimens with dysplasia or carcinoma in situ have come from patients treated with chest radiotherapy with or without chemotherapy. The biopsy specimens sent to the Center for culture were grown in RWVs for 2-6 weeks. RWV specimens from 11 patients have been reviewed at this time. The normal specimens maintained morphology in RWV culture of 2-3 weeks, but longer culture resulted in changes of normal pseudostratified columnar epithelium into squamous-like epithelium. RWV specimens from two patients with carcinoma in situ and moderate dysplasia maintained the histologic phenotype. The additional RWV specimens are undergoing histologic review. The initial diagnostic pathologist specimens from these patients have been retrieved and DNA from these specimens is being analyzed for K-ras and p53 mutations and loss of heterozygousity.

Specific Aims:

Genetic analysis of all biopsy specimens: We will initially perform analyses on two patients with areas of initial tumor, normal epithelium, moderate dysplasia, and carcinoma in situ. The areas of epithelium will be microdissected (500-1,000 cells) using Laser Capture Microscopy (LCM) to obtain relatively pure populations of tumor, atypical epithelium, and normal epithelium from the original specimens and from matched RWV-cultured specimens. The DNA from these specimens will be studied for mutations and loss of heterozygousity (LOH). Genomic DNA isolated from freshly collected peripheral blood leukocytes served as matching normal DNA in the LOH studies. K-ras and p53 mutation analysis will be performed by PCR amplification followed by direct sequencing with automated DNA sequencer. For LOH analysis we choose 10 microsatellite markers to cover 6 chromosomal arms (2q, 3p, 5q, 9p 13p and 17p) which were previously described to have LOH in at least 20% of non-small cell lung cancers. Following PCR amplification, the samples will be run on a Genetic Analysis System. The LOH pattern, KRAS1, and TP53 mutation patterns will be compared in the primary tumor, biopsy specimen, and RWV culture established from the same area as the initial airway biopsy specimen. We expect that the histology of the airway specimen and biopsy specimens will be concordant in more than 80% of the examined cases. If less concordance is seen in the airways and RWV, we will consult the visual record of the bronchoscopy to see if the biopsy site of the atypical epithelium was too small to give rise to two specimens. We expect the concordance of the LOH pattern, p53, and K-ras pattern in the RWV specimen to be similar to the airway specimen. We do not anticipate ras and p53 mutation in the preneoplastic lesions since these are relatively late events in the development of lung cancer. If the LOH pattern is different, the histology of the lesions will be reviewed and noted if the degree of dysplasia/carcinoma is different. If the histologies are similar, further areas of epithelium will be microdissected and tested for LOH. We also anticipate that areas of normal epithelium will have LOH as well, particularly alleles on chromosome 3p and 9p, as has been previously documented. We anticipate that the histology and genetic changes in the atypical and normal areas will match the RWV specimens. If this is the case, this system will provide an extremely valuable tool for screening chemoprevention agents in vitro. Patients with dysplastic lesions in the airways can have the lesion placed in RWVs with a candidate chemoprevention agent. This is particularly important since there are few intermediate biomarkers for patients at risk for lung cancer.

11. Prostate tissue proteomics.

 

 

Primary Investigators: Paul Duray, M.D., Department of Pathology, National Cancer Institute, NIH.

Start Date: 5/97 Ending Date: Ongoing

Objectives: Determining protein profiles in actively growing prostate tissues, both benign and malignant, in the presence of exogenous androgens.

Background/Rationale: Embryologic development of the human prostate gland is complex with contributions from several anlage . The biochemistry is accordingly complex with scores of enzymes and kallikreins produced during sexual maturity. Despite the known functions in human fertility, the complete physiologic and biochemical functions of the prostate are still incomplete and the biologic determinants of prostate cancer and progression of it are not fully understood.

Publications: Margolis L, Hatfill S, Chuaqui R, Vocke C, Emmert-Buck M, Linehan W, Duray P.(1999) Long term organ culture of human prostate tissue in a NASA designed rotating wall bioreactor. J Urol 161(1): 290-7.

Progress Report: Prostate needle biopsies were cultured in RWVs for a minimum of 30 days followed by histologic examination in the pathology laboratory. Tissue samples were removed at various intervals and used for RNA extraction, immunocytochemistry, and microdissection. We generated cDNA libraries of total RNA from given tissues via microarray and large-scale sequencing. Prostate specific antigen (PSA), vimentin, TGF-?, and TGF-?2 receptor and ligand were analyzed by RT-PCR. Prostatic tissue in long term RWV culture was shown to have down-regulated PSA while vimentin and TGF-?2 receptor and ligand were maintained . Results are in accord with the regulation of androgens (which were not exogenously added) upon biosynthesis of kallikreins such as PSA. No changes were seen or expected in TGF-? expression. Prostatic carcinoma samples show marked tissue necrosis after long term culture in RWVs. All patients had received androgen hormone blockade therapy prior to coming to NCI for biopsy, and this may influence their ex vivo behavior in the RWV .

Specific Aims:

Protein fingerprint profiles: Prostate tissue will be obtained from operative samples. Half of the biopsy tissue will be cultured in RWVs and the other half will be snap frozen, cryostat sectioned and the epithelium captured by Laser Capture Microdissection (LCM). Protein lysate will be prepared for Surface Enhanced Laser Desorption Ionization Spectroscopy (SELDI) and applied to the SELDI protein chip. The mixture of bound proteins will be separated by laser desorption and ionization followed by time-of-flight mass analysis by a molecular weight (MW) detector. The peaks and bands reflect MW of the proteins and permits comparisons. SELDI profiles of RWV cultured tissue will be compared to baseline (cryostat obtained cells). We will compare cancerous prostate with benign, hyperplastic prostate. Experiments will then shift to addition of exogenous androgens. We will compare the protein fingerprints under androgen stimulation with the profiles obtained in the first phase experiments. We will not have sequenced or conducted detailed analyses of generated proteins from the RWV experiments in the first phase of this proposed work, but we anticipate gaining insight into those profiles of known proteins that may be lost, gained, or changed in the fingerprints or microscale patterns. This has potential of revealing classes of proteins previously not known to be important under androgenic stimulation of both neoplastic and non-cancerous prostate epithelium. Since epithelium is influenced by extracellular matrix we will also obtain stromal samples by LCM to see if protein profiles are altered in parallel with the epithelium. The advantages of combining the technologies of RWV culture under hormonal stimuli, LCM, and SELDI protein analysis promise to increase our understanding of prostate carcinogenesis.
 
 

12. Culture of endometrium biopsies.

 

 

Primary Investigators: Lynnette Nieman, M.D., Department of Endocrinology Branch, NICHD, NIH.

Start Date: 12/97 Ending Date: Ongoing

Objectives: Establishing long term cultures of endometrium for ex vivo experiments to gain an understanding of the role of growth factors and their receptors in normal and abnormal endometrial growth.

Background/Rationale: Human endometrial regeneration is modulated by several growth factors. However, little is known about the detailed mechanisms involved in the repair of the endometrium during the menstrual period. Hepatocyte growth factor (HGF) and its receptor c-MET are believed to play an important role not only in normal endometrium growth but also in abnormal endometrial growth, or endometriosis . Increased production of HGF may be a characteristic of endometriosis and may be involved in the pathophysiology of the disease . An in vitro model of human endometrial tissue requires co-culture of both epithelial and stromal cells since HGF is produced by stromal cells and c-MET is expressed on epithelial cells . Such a model could then be used to study regulation of HGF and c-MET in normal and abnormal endometrium.

Progress Report: Endometrium biopsies were collected from normal volunteers during various phases of the menstrual cycle. These specimens were cultured as small blocks of tissue in static or RWV cultures for several weeks. Tissue blocks were fixed weekly then sent out for paraffin embedding, sectioning, and H&E staining. Stained sections were analyzed by a pathologist and the health of tissue biopsies was assessed. Tissue biopsies from menstrual and proliferative phases of the cycle have been cultured for as long as 8 weeks, but cell survival in the biopsies declines by one to two weeks and no regeneration has been seen with increased culture. Various types of medium and medium supplements have been tested in an effort to promote survival of both cell types, and various culture techniques in both static and RWV culture have been attempted.

Specific Aims:

Establishing an endometrium tissue culture model: Several new approaches are being proposed using new static culture techniques as well as RWV culture. Previous attempts to culture the biopsies on gelfoam sponges were complicated by tissue digestion of the sponge leading to tissue submersion. Use of Cytopore matrixes will be resistant to such degradation and therefore will support the tissue better and hopefully prompt better survival. Morphologic analysis of sections of previous cultures revealed that stromal cells are not surviving well. Changes to medium formulations, including the addition of growth factors or conditioned medium from stromal cell cultures, may increase stromal cell survival. Culture of endometrial biopsies in RWVs may be improved by culture with support scaffolds, such as microcarriers or biodegradable Vicryl mesh, or the biopsies may be embedded in various gels to help keep tissue intact. Embedding tissues in collagen was complicated by digestion of the collagen by the tissues, so alternate gels, such as agarose and alginate, can be tested. If these experimental approaches to culture tissue biopsies are unsuccessful, we plan to use the RWV to co-culture primary epithelial and stromal cells. Several established techniques are available for isolating the epithelial and stromal cells from biopsies, and these cells can then be cultured in RWVs on microcarriers or Vicryl mesh. Once we are able to maintain tissue biopsies or co-cultures, we will use hormone cycling to simulate in vivo conditions, and we will examine HGF and c-MET by immunohistochemistry, or in situ hybridization in normal endometrium during several phases of the cycle. Exogenous HGF will be added to cultures to look for effects on the tissue and on expression of c-MET. This will provide insight into the processes leading to endometriosis.

13. Culture of synovial tissue from rheumatoid arthritis patients.

 

 

Primary Investigators: Raphaela Goldbach-Mansky, M.D. and Hani El-Gabalawy M.D., Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH.

Start Date: 8/98 Ending Date: Ongoing

Objectives: Studying the aggressive phenotype of synovial tissues from patients of rheumatoid arthritis (RA), compared to those of spondyloarthropathies, undifferentiated polyarthritis, and with evidence of Chlamydia infections under controlled conditions ex vivo. Studying the role of cytokines in the aggressive phenotype of the tissue

Background/Rationale: Synovial tissue from patients with RA assumes an aggressive phenotype that leads to bony erosions and joint destruction. Other forms of inflammatory arthritis, which are histologically undistinguishable from those in RA, do not cause bony erosions and severe functional disability. Previous experiments in the ARB laboratory showed good potential for growing synovial tissue from needle biopsies. By studying ex vivo synovial cultures and co-cultures of synovial and cartilage tissues, we hope to understand the mechanisms of the unique characteristics of RA tissues that lead to the more aggressive phenotype. We believe that cytokines play a key role in determining the phenotype of synovial tissue. A three dimensional tissue system which includes both synovial and cartilage tissues is necessary to understand these mechanisms and to test the role of various cytokines in synovial tissue behavior.

Progress Report: Biopsies of RA synovial tissues and cartilage tissue were cultured in RWVs and in static cultures for up to one month periods. Tissues were cryosectioned on a weekly basis to determine viability and maintenance of all cell types. Cultures of both types of biopsies have been successful and the tissues remain viable and are structurally maintained for up to four weeks. Co-cultures of inflamed synovial tissues and cartilage were accomplished by embedding the tissues together in collagen. Invasion processes were tracked by cryosectioning and studying the morphology of the tissues.

Specific Aims:

Studying the aggressive phenotype of RA synovial tissues: RA synovial tissues will be compared to normal controls in RWV cultures, as well as compared to synovial tissues from other inflammatory arthritic conditions. We will begin using use serum free medium for these cultures so we will be able to evaluate whether there is correlation between synovial tissue invasion and cytokine production . Endogenous cytokine production, as measured by enzyme linked immunoassay, will be examined in synovial cultures from patients with various inflammatory arthritis conditions, and in invasion studies of synovium and cartilage. We will also examine the roles of exogenously added proinflammatory cytokines in disruption of organized growth.

14. Culture of hematopoietic stem cells for gene therapy of hemoglobinopathies.

 

 

Primary Investigators: Seigo Ohi, Ph.D., Dept. of Biochemistry & Molecular Biology and Center for Sickle Cell Disease, Howard University College of Medicine, Washington, D.C.

Start Date: 2/99 Ending Date: Ongoing

Objectives: Use of the RWV to grow and expand mouse hematopoetic stem cells (mHSCs) and assessment of the cultured mHSCs to cure ?-thalassemic mice by HSC transplantation and gene therapy.

Background/Rationale: Gene therapy offers a hope to cure/alleviate genetic and acquired diseases, and our ultimate goal is to develop protocols for gene therapy. The genetic hemoglobinopathies (thalassemias and sickle cell anemia) are well-characterized molecular diseases and may be amenable to gene therapy. Exploiting non-pathogenicity of an adeno-associated virus, serotype 2 (AAV2), the human parvovirus, Dr. Ohi's laboratory has constructed recombinant AAVs that harbor human globin genes for gene therapy of hemoglobinopathies . In addition, toward ex vivo gene therapy via bone marrow transplantation (BMT), his laboratory established methods for purification of HSCs and developed a novel culture system for growing/expanding HSCs without stromal cells. The human globin genes were efficiently expressed from the rAAVs in the mHSCs in culture. An ability to expand HSCs in culture readily will assist gene therapy. Our experience indicates that these cells require constant low shear mixing and co-location of cell populations and growth factors, and the RWV should provide such conditions.

Progress Report: Several preliminary experiments have been conducted to establish conditions for growing/expanding the mHSCs in RWV culture. Subsequently, experiments were carried out to compare static and RWV cultures of mHSCs with various cytokines and growth factors. The mHSCs were cultured for several weeks and the ability of cells to self renew or differentiate was analyzed using cell counts and flow cytometry. Initial results were encouraging and indicate some phenotypic differences in cells in static and RWV cultures, possibly indicating beneficial effect of the RWV.

Specific Aims:

Optimization of HSC cultures: The expansion of mHSCs in the RWV culture will be optimized (adjusting rotation rates, medium change, etc.). In addition, systematic analysis of cells retrieved from the RWV cultures will be conducted. Lineage-specific antibodies and flow cytometry analysis will be used to characterize differentiation. Recruitment of a hematologist is under way for additional analysis of cells.

Bioassay of the cultured HSCs: The RWV-cultured wild type or heterozygous HSCs will be allotransplanted to ?-thalassemic mice to examine their ability to cure the disease and compared to HSCs grown in static culture. Cystamine-cellulose acetate electrophoresis system will be used to determine hemoglobin types of the mice, as well as conversion of hemoglobin types in the recipient following HSC transplantation. Studies with stationary grown HSCs indicate that chimeric hemoglobins are produced in the peripheral blood of transplant recipients.

Development of protocols for gene therapy: Following transduction of donor HSCs with rAAVs and subsequent transplantation, efficient, controlled, and sustained gene expression in the recipient are critical factors in ex vivo gene therapy. These parameters will be studied using autologous HSC transplantation in the ?-thalassemic mice. The HSCs will be prepared from ?-thal mice, expanded in RWV culture, transduced with rAAV that harbor human hemoglobin (Hb) genes and then marrow transplanted to isologous ?-thal mouse. The expressed human Hb in the ?-thal mice will be analyzed by cystamine cellulose acetate electrophoresis. Using this system, gene expression from the integrated rAAV in vivo will be optimized.

15. GM-CSF activity in tumor cells.


Primary Investigators: Roberto P. Revoltella, M.D., Ph.D., Institute of Mutagenesis and Differentiation, CNR, Pisa, Italy, and Leonid Margolis, Ph.D., Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, NIH.

Start Date: 11/99 Ending Date: Ongoing

Objectives: Investigate the effects of recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) on keratinocyte stem cells transformed by H-ras which constitutively express functional GM-CSF receptors and are sensitive to this factor.

Background/Rationale: GM-CSF is a natural glycoprotein that controls many activities of hematopoietic cells. GM-CSF and its specific cell membrane receptor can also be constitutively expressed in non-hematopoietic cells, including endothelial cells, fibroblasts, and keratinocytes. Moreover, GM-CSF seems to play a regulatory role in controlling proliferation and migration in vivo and in vitro, of tumor cells of different tissue origin. Tumor progression is a complex, multi-step process, leading to highly malignant cells capable of continuous cell renewal. The uncontrolled growth regulation of malignant tumor cells might correlate with an abnormal production of potent pleiotropic or multiple growth factors, leading to abnormal tumor angiogenesis and growth of selected subsets of tumor cells expressing functional receptors for such growth factors.

Progress Report: Anticipated start date is 11/99. Dr. Revoltella will visit the Center to begin this project.

Specific Aims:

Development and testing of model systems: We will investigate the stimulatory role of recombinant human GM-CSF using organotypic models of human skin carcinogenesis. Phenotypically stable clonal variants of immortalized human keratinocytes, will be analyzed for their capacity to express functional GM-CSF receptors (GM-Ra ). These tumor cells or normal skin keratinocytes will be grown in monolayer culture and in RWV organotypic cultures. We will examine the expression of functional GM-CSF receptors in these cultures and correlate these findings with the protein level, assessed by immunohistochemical assays. We plan to analyze whether transformed keratinocytes that constitutively express specific GM-CSF receptors proliferate at higher rates compared to normal keratinocytes that do not express the receptors. Effects of exogenous recombinant GM-CSF, such as proliferation, will be evaluated. We will also analyze the specificity of the growth factor-receptor interaction, using a panel of both neutralizing and non-neutralizing monoclonal antibodies raised in Dr. Revoltella's laboratory against GM-CSF or its receptor . We will also use a panel of synthetic peptides homologous to different overlapping peptide fragments along the primary amino acid sequence of GM-CSF or its receptor a subunit . Some of these synthetic peptides are powerful antagonists and agonists of proliferation of myeloid leukemia cell lines in vitro. This approach should identify critical domains on GM-CSF important for binding of this growth factor to its specific receptor a chain subunit. This would provide insight into the molecular mechanism of triggering of inductive signaling pathways in these non-hematopoietic target cells. We anticipate that RWV cultures may be suitable models for the analyzing critical features of progression of tumor skin carcinogenesis. To understand mechanisms of tumor progression we will analyze kinetics of cell proliferation; cell migration; tumor cell differentiation and death; and production and release of multiple stimulating and/or differentiation or inhibitory factors, released by either malignant epithelial cells or stromal cells. This information might turn out to be clinically relevant, considering the increasing use of recombinant GM-CSF in adjuvant tumor therapy.

Scope

In accordance with the Memorandum of Understanding between the National Aeronautics and Space Administration (NASA) and the National Institutes of Health (NIH) regarding Biomedical and Behavioral Research, signed July 21, 1992, the Microgravity Research Division (MRD), Office of Life and Microgravity Sciences and Applications (OLMSA) of NASA, and the Laboratory of the Mechanisms of Ocular Diseases (LMOD) of NEI/NIH, agree to collaborate on joint projects in the area of Biofluids Research and Technology developed by NASA. NASA and the NIH will share research data from these joint projects and publish related data in the open scientific literature.

This agreement shall support the transfer of ground-based NASA laser light scattering technology for the early detection and diagnosis of eye diseases to NIH and the external research community. NASA shall provide funding to create, maintain, and support testing of the laser light scattering eye diagnostic instrument (includes upgrades to prototype instruments) at NIH for collaborative NASA-NIH research on biofluids research and technology. NASA support includes funding for personnel, supplies, and equipment. The LMOD of the NEI/NIH shall provide laboratory space, access for NASA or NASA-sponsored professional personnel to NIH resources, intramural NIH researchers to perform studies and serve on review panels/boards, and accessibility to NASA-developed instrument(s). NASA shall administer grants, provide experienced professional(s) to fully participate in this collaborative program, assist new personnel in instrument operation and participate in this collaborative program, assist new personnel in instrument operation and procedures, and protocol development for effective technology transfer from the NASA MRD. Dr. Sam Zigler (Chief of LMOD) of the NEI/NIH shall be the Director of and shall provide scientific and administrative supervision of the above-mentioned joint NASA/NIH specific collaborative project under this grant, and Dr. Manuel B. Datiles III of the NEI is a Co_Investigator. Dr Rafat R. Ansari of the Glenn Research Center is the NASA Principal Investigator for this inter-agency collaboration.
 
 

Specific Collaborative Project: The use and laboratory/clinical evaluation of a novel fiber optic probe technology developed by NASA for the early detection and diagnosis of eye diseases such as cataracts.

Purpose of the Agreement

The purpose of this agreement is to enhance the capabilities of NASA developed technology to better serve the needs of the medical research and service community and advance the transfer of that technology to those persons/organizations who can best apply its capabilities to improve the quality of life in our Nation and around the world.

Background:

Cataract is a leading cause of blindness all over the world. Since this disease has no medical cure except the surgical removal of the opacified lens, it is proving to be a major public health burden. It is estimated that more than 70 million Americans will have cataract in year 2030 compared to 34 million figure today. The problem in Africa and India is already out of control with cataract extractions taking place at relatively young age (in the 40's). It is estimated that in 20 years, in spite of surgical efforts by ophthalmologists, and because of the aging of the world population, there will be 200 million eyes legally blind worldwide from cataract. The social implications of visual impairment due to cataract are growing. In a recent Salisbury Eye Evaluation (SEE) project, it was shown that 1/3 of all vision impairment in patients (³ 65 years) is due to cataract. This could lead to dependency on others, reduced physical activity, social isolation, and even death. For example in many communities in the United States driving an automobile is the primary means of transportation and is considered an integral part of one's mobility and therefore quality of life. Cataract as the leading cause of vision impairment in older adults, affecting almost half of those over 75 years of age. It is estimated that older drivers are the fastest growing group of drivers in the United States. By the year 2024 one out of every four drivers on the road will be over the age of 65, and for every 100,000 miles driven, the crash rate of older drivers is twice than that of younger drivers. Thus the need to control this disease is much greater today than ever before. NEI/NIH estimates that a delay in cataract formation of about 10 years would reduce the prevalence of visually disabling cataract by about 45%. This scenario has raised enthusiasm and optimism among various groups of biochemists, e.g., suggesting both high-tech and low-tech solutions such as the use of drugs like glutathione, pentethine, aldose reductase inhibitors, vitamin supplements, and drinking red wine and (green) tea. But the methods in clinical use for quantifying cataract remain quite primitive and at best elusive at the level of early detection. To this date the methods in clinical have failed to detect and grade cataracts reliably and quantitatively at their early stages of formation and hence no accurate method is available to test the efficacy of possible medical cure for cataract. NASA has developed new fiber-optic based compact and extremely sensitive instrumentation for the early detection of cataract, vitreous modalities, and corneal structures. In these instruments DLS was used in slit-lamps and Scheimpflug cameras. Recently Ansari et al. have shown that a growing cataract can be detected using DLS at the molecular level; much earlier than any state-of-the-art clinical methods in use today. However, the success of these methods in clinical use depends upon the precise control of the scattering volume inside a patient's eye and especially during repeat visits. This is of great importance in the longitudinal studies of cataract and during anti-cataract drug screening. This is important because the scattering volume inside the eye is very small (few microns) in dimension. Slight variation in the measurement site inside a patient's lens can cause significantly different values in measured parameters of interest.

FY 00 Progress:

This year we focused our attention to circumvent these problems by fabricating a new DLS fiber optic probe with a working distance of 40 mm and by mounting it inside a cone of a corneal analyzer. A corneal analyzer (Optikon 2000 Keratron, Italy) was modified by placing a DLS fiber optic imaging probe inside its cone. This allows plenty of room to work with and an enormous comfort to a patient during the measurements without touching the sensitive corneal tissue. A further advantage is the alignment feature of this device. The front-end eye piece is equipped with two fiber optic position sensors on extreme (equatorial) ends. An infrared (IR) beam of light passes through one fiber and collected by another. When this IR beam is intercepted by the cornea the instrument is aligned with the apex of the cornea. The IR beam does not pass through any other ocular tissue. Thus the actual laser light used during the 2-5 second DLS measurement is never used to align the instrument, making it much safer for human use. This new instrument is being tested on patients at NEI/NIH. The alignment an repeatability tests have already been successfully completed at NEI on a volunteer human subject.