FY 98 INTER-AGENCY AGREEMENT

FY 98 INTER-AGENCY AGREEMENT

NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT

NATIONAL INSTITUTES OF HEALTH

and

OFFICE OF LIFE AND MICROGRAVITY SCIENCES AND APPLICATIONS

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION

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, 1998, 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.

1. Create a core facility at NIH for the NASA bioreactor three-dimensional tissue culture technology, equipped to allow efficient growing of multiple cell types simultaneously, and staffed to provide technical assistance to visiting NIH and NASA investigators wishing to evaluate the new 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. Augment the NIH three-dimensional tissue imaging facility to allow for critical measurements of intracellular calcium and pH in living bioreactor tissues, so as to judge tissue response to physiological and pathophysiological stimuli.

4. Grow cells obtained from human lymph tissue in the NASA Bioreactor, with full-time staff, and begin the development of an AIDS tissue model.

In 1998, the number of groups of intra- and extra-mural scientists that worked at the NASA/NIH Center for Tissue Culture increased from 11 to 13. These groups worked on 17 different projects related to cell-cell interactions, cell differentiation and function, and microbial-tissue interactions in NASA Rotating Wall Vessel Bioreactor (RWV). During the year, the following groups utilizing the new culture technology made the most significant progress in their research. (i) A group from National Institute of Dental Research (P.I. Dr. H. Kleinman) found that human salivary gland cells (HSG) cultured in RWV form acini within 24 hours in the presence of laminin-1. In addition to the earlier established AG73 active site on laminin for HSG cells, they have now identified a second site. Using HSG cells cultured in RWV and static cultures, they defined the involvement of beta-1 integrins and syndecan-1 in cell-matrix interactions during acinar differentiation. A paper was published in the Journal of Cell Biology describing these findings. (ii) A team of NASA/NIH Center Scientists (P.I. Dr. J. Zimmerberg) together with FDA scientists successfully replicated the human intestinal parasite Cyclospora in RWV-cultured CaCo-2 cells. CaCo-2 cells cultured in RWVs are more differentiated than cells grown in monolayers, and thus provide a target for ex vivo infection. Several stages of the life cycle never before described for this parasite have been witnessed in RWV-cultured CaCo-2 cells. (iii) NCI scientists (P.I. Dr. Paul Duray) successfully cultured benign human prostate tissue using the techniques developed at the NASA/NIH Center. They found that prostatic carcinoma cell lines were able to invade RWV-cultured benign prostate tissue explants. A paper describing this new system was recently accepted to J. Urology. (iv) A group of NCI/National Naval Medical Center scientists (P.I. Dr. B. Johnson) continued their research on bronchial metaplasia or dysplasia and the relation of these processes to the loss of heterozygosity at the APC gene locus. The Xillix bronchoscope at the National Naval Medical Center allows metaplastic and dysplastic tissue to be biopsied during bronchoscopy and post-histological tissue biopsy samples were cultured at the NASA/NIH Center. The RWV Bioreactor allows maintenance of metaplastic bronchial epithelia for as long as 21 days. This allows studying the genetic alterations in cultured samples and morphological changes consistent with normalization into a non-metaplastic phenotype using number of different retinoid compounds. Tissue biopsies from 35 patients have been cultured and microdissected. The work is in progress to analyze the loss of heterozygosity of APC and several other genes of interest.

In 1998 the Center’s scientists (Drs. L. Margolis and J. Grivel) became involved in studies related to the effects of microgravity on immune function. The randomized gravitional vector provided by the RWV Bioreactor creates an environment similar to the microgravity environment of space and also eliminates the other spaceflight-related factors that may impact immune function. Understanding the mechanisms of immune dysfunction in microgravity is critical for assessing the safety of long-duration space flights. In RWVs, human lymphoid tissues exhibit transient loss of their immune function. This transient immunodeficiency was caused by the inability of lymphocytes to be activated under conditions of microgravity.

The scientists of NASA/NIH Center made a significant progress in developing the dual-photon imaging technique to study the structure of cultured tissues. The first images of tissue lymphocytes have been obtained. In 1998 new studies have been launched at the Center to research endometriosis using RWV Bioreactor to culture endometrium biopsies, Lyme disease using innoculation of RWV- cultured human tissue, diabetes by maintaining human islet cells in the RWV, and arthritis by culturing synovial tissue from rheumatoid arthritis patients. Study of HIV mucosal transmission in RWV-cultured tissue was recently started in collaboration with French scientists.

This report outlines the activity of the NASA-NIH Center for three-dimensional Tissue Culture in 1998. 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. In Phase I NIH scientists can launch pilot projects involving the RWV Bioreactor as extensions of their main intramural research projects. Based on these preliminary results a research group can apply for Phase II by submitting a small grant proposal of a study involving the RWV and get funding for purchasing RWV, disposables, as well as for post-doctoral fellow salaries. This year one project entered Phase II, thus in total four projects have now produced results beyond the preliminary phase:

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

A. HIV infection in human lymphoid tissue in RWV

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

C. Development of an advanced dual-photon microscope for 3-D tissue analysis

D. Use of RWV Bioreactor to culture endometrium biopsies

E. Culture of Hermanski-Pudlak Syndrome fibroblasts

A. Long term maintenance of human prostate tissue in the RWV Bioreactor.

B. Study of Lyme disease in RWV Bioreactor

A. Culturing esophageal carcinoma cells in RWV Bioreactor

B. Culturing human islet cells in the RWV Bioreactor

C. Use of RWV Bioreactor for whole mouse embryo culture

Differentiation of salivary gland cells in the RWV Bioreactor

Culture of synovial tissue from rheumatoid arthritis patients

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

RWV Bioreactor models of human squamous metaplasia in the lung

Assessment of the RWV as a "Universal" pathogen culture system

Cryptosporidium infection of intestinal tissues and cells

Study of HIV mucosal transmission

Primary Investigators: Dr. Hynda Kleinman, Laboratory of Developmental Biology, National Institute for Dental Research, NIH.

Aim of Experiments: To use the RWV Bioreactor to study the effects of laminin-1 on three-dimensional human salivary gland (HSG) duct cultures and determine sites on laminin involved in acinar development.

BACKGROUND:

The HSG cell line is derived from salivary gland pluripotential stem cells. These HSG cells form three-dimensional acinar-like structures when cultured with basement membrane extracts. The RWV provides a better system for studying these differentiating cells since they can be cultured in suspension and cell interactions with the solid surface of the culture support are eliminated. The cells can be cultured in the RWVs with matrigel, laminin extracts, or synthetic peptides derived from the laminin chains. The RWV also has the advantage that lower amounts of peptides can be used to achieve the same results seen in static cultures.

Experiment Design: HSG cells will be cultured in RWVs with laminin-1 or laminin peptides previously identified as being involved in cell adhesion (AG73 and MG73) and assessed for the formation of acini or acini-like structures.

Continuing Results:

1. Acinar formation of HSG occurs in 24 hours in the presence of laminin-1 in the RWV, cells are polarized and form lumens. When cells are cultured in RWVwith AG73 or no peptide there is no acinar formation. Cells cultured with both AG73 and MG73 form large multicellular structures which lack organization; cells are not polarized and there is no lumen formation.

2. HSG cells cultured in RWVs with AG73, which has heparin-binding activity, do not form acini when heparin is added to the cultures. However, when heparin is added to cultures of HSG with the entire laminin molecule acini are formed. This suggests AG73 is not the only active site on laminin for HSG cells, but it does influence cell-cell interactions and morphological organization.

3. Peptides may be providing some but not all the necessary signals for the process of acinar differentiation. Using HSG cells cultured in RWV and static cultures has helped to define the involvement of beta-1 integrins and syndecan 1 in the cell-matrix interactions in acinar differentiation.

A paper was published in the Journal of Cell Biology describing these findings: Hoffman et al, J. Biol.Chem., 273(44), 28633-28641, October 30,1998.

Plans: Subtractive hybridization cloning will be performed on the cells in the Bioreactor, in both the presence and absence of laminin-1 and the peptides, to determine which genes are involved on this developmental process.

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

Aim of Experiments: Human intestinal cells will be cultured in the RWV Bioreactor system to obtain cells that are more differentiated than cells grown as monolayers. These cultures will provide an in vitro model in which we can study the interactions of Cyclospora sp. intestinal parasites and the infected cells and the mechanisms by which they cause disease.

BACKGROUND :

Little is known about the tissue interactions and pathogenic mechanisms involved in enteric infections of mammals with Cyclospora sp. Most in vitro models are based on infections of monolayers of poorly differentiated cells, which confounds comparison to the in vivo condition. The RWV Bioreactor will provide a high fidelity in vitro model of the mammalian epithelial gut wall. Using this model we hope to learn more about the kinetics of infection, the interactions between the parasites and infected cells, and the stages of life cycle occurring in the human gut.

Experimental Design: CaCo-2 cells, derived from human colon epithelial cells, will be cultured in RWV Bioreactors or in monolayers for 10-14 days. The cells will be incubated with Cyclospora oocysts and then cultured another 8-10 days. Medium will be collected from infected and control cultures for enumeration of oocysts in medium post-infection. Cells from infected and control cultures will collected for analysis with light and electron microscopy.

Continuing Results:

1. Electron microscopy studies have shown that RWV-cultured CaCo-2 cells are more differentiated than cells grown in monolayers, and thus provide a better model for in vivo infections.

2. Adherence and excystation of Cyclospora oocysts does occur in CaCo-2 cells cultured in RWV Bioreactors. Light microscopy reveals damage to cell cultures with infection by Cyclospora oocysts. Productive infection of cells is also suggested by increasing numbers of oocysts in the culture medium.

3. Cyclospora infected cells have been examined using transmission and scanning electron microscopy. Almost all stages of both the sexual and asexual life cycles have been witnessed in RWV-cultured CaCo-2 cells.

Plans: RWV growth conditions will continue to be optimized to aid in better infection rates. Electron microscopy studies will continue to better understand the process of Cyclospora infection and this knowledge will be applied to more effective treatment strategies.

Primary Investigator: Dr. Paul Duray, Department of Pathology, NCI, NIH.

Aim of Experiment: To utilize the previous experience of the NASA/NIH Center with human prostate tissue explants in the RWV Bioreactor, to expand our studies on basic prostatic epithelial tumorigenesis. The specific aims of the study are to:

1. To continue biological studies on effects of prostate epithelium and supporting stroma over time in RWV culture. Expression of prostatic specific antigen (PSA), a major serum protein kallikrein, will be assessed over time using immunohistochemistry and northern blotting to assess PSA mRNA at baseline and at four weeks of culture time. Vimentin, TGF beta, and prostatic acid phosphatase will be determined by immunohistochemistry.

2. To assess feasibility of maintaining prostatic adenocarcinoma tissue in RWV culture. Cultures of tumor will be cultured under the same conditions used in normal tissue trials without adding exogenous androgens. We will test the hypothesis that some androgen-independent carcinomas can be maintained in ex vivo tissue culture.

BACKGROUND:

Various hypothesis for the increased incidence of invasive prostatic adenocarcinoma in the United States and the development of benign prostatic hypertrophy, have been proposed. The ability of the RWV Bioreactor to facilitate normal three-dimensional cell-cell and cell-extracellular matrix interactions in normal human prostate tissue explants, makes this an ideal culture system to explore the applicability of these various hypothesis.

Experiment Design: Needle biopsy derived prostate samples will be obtained from patients with prostate cancer under a separate NCI protocol which allows the exploration of newer cultivation techniques (prostate protocol T95-0038) and placed in RWV culture. Samples will be cultured a minimum of 30 days followed by histologic examination in the pathology laboratory. Benign prostate tissue samples will be cultured in RWVs to further determine effects of long term maintenance of prostate epithelium. Tissue samples will be removed at various intervals and snap frozen for RNA extraction or they will be fixed for immunocytochemistry. Microdissection will be used to isolate prostate epithelial cells for further analysis. PSA will be analyzed by reverse transcriptase PCR with actin as a control using random hexamers as primers.

Continuing Results:

1. Prostatic tissue in long term RWV cutlure was shown to have down-regulated PSA while vimentin and TGF beta 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. We did not expect any changes in TGF beta expression, and none were found.

2. After multiple attempts with patient biopsies, it was found that 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.

Plans: Work will continue on long term culture of androgen dependent and androgen independent prostatic carcinomas. Additionally cultures of benign prostate will be cutlured for a better understanding of changes in prostatic epithelium during tumorigenesis and the effects of androgens on this process.

Primary Investigators: Dr. Paul Duray, Department of Pathology, National Cancer Institute, NIH., Dr. Bruce Johnson, NCI, National Naval Medical Center.

Aims of Experiments: Tissue biopsies with a pre-existing bronchial epithelial metaplasia within the context of an otherwise normal tissue environment will be maintained in long duration culture. The cultures will aid in determining if a loss of heterozygosity at the APC gene locus is responsible for the irreversible progression of bronchial metaplasia into dysplasia. Biopsies from patients with bilateral metaplasia or dysplasia will be assessed to determine if gene alterations are similar in lesions in both lungs.

BACKGROUND:

Histological studies suggest there is a successive progression from metaplasia to dysplasia with eventual invasive adenocarcinoma, but there is no direct evidence that metaplastic epithelia are clonal precursors to this or other types of epithelial cancer. The Xillix bronchoscope at the National Naval Medical Center allows metaplastic and dysplastic tissue to be biopsied during bronchoscopy and post-histological tissue biopsy samples are sent to the NASA/NIH Center for culture. Recent studies have implicated APC gene alterations as one of the earliest genetic changes that occur during progression from a histologically benign to a malignant epithelial phenotype. Cells of interest can be microdissected out of tissue samples and subjected to PCR to look for APC gene alterations in different cells representative of the stages of progression from the metaplasia to dysplasia and overt carcinoma. This technique will be useful for studying patients with bilateral disease to determine if lesions arise from the same gene alterations, such as that seen in retinoblastoma.

Experiment Design: In addition to normal tissue culture methods, the RWV Bioreactor will be used to for long duration culture of small blocks of bronchial biopsy. Maintenance of metaplastic tissue during culture will be assessed by immunohistochemical staining of tissue sections using a defined panel of epithelial-associated cell surface antigens. Microdissection of metaplastic and dysplastic areas within the RWV cultured tissue and PCR techniques will be used to assess APC heterozygosity and clonality.

Continuing Results:

1. The RWV Bioreactor allows maintenance of metaplastic bronchial epithelia for as long as 21 days.

2. Tissue biopsies from 35 patients have been cultured. Samples from most cultures were sectioned and microdissection and PCR are in progress to look for loss of heterozygosity of APC and several other genes of interest.

3. Tissue biopsies from patients with bilateral disease are in the process of being analyzed to look for clonality between lesions.

Plans: Microdissection/PCR will continue on remaining samples. New studies will involve experimentally trying to induce morphological changes consistent with normalization into a non-metaplastic phenotype using number of different retinoid compounds.

Primary Investigators: Leonid Margolis PhD., and Joshua Zimmerberg, MD, PhD. Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, NIH.

Aims of Experiment: To study the effects of HIV infection on tissue and circulating cells in human lymphoid tissue ex vivo.

BACKGROUND:

Understanding the pathogenesis of HIV disease is best accomplished by studying the progression of disease in intact tissues rather than in cell lines or suspensions of stimulated peripheral blood lymphocytes. In previous stages of this study we have shown that the RWV system allows maintenance of blocks of human lymphoid tissues for up to three weeks, and these tissue blocks can be infected with laboratory strains of HIV-1. The RWV system also enables lymphocytes to traffic between the tissue blocks and the surrounding medium. This unique environment provided by the RWV allows us to study the effects of HIV infection in two compartments of the lymphoid system – the lymphoid tissue itself and the circulating lymphocytes. We will determine if viruses of different tropism cause different patterns of activation of cells and/or different patterns of migration of specific subsets of cells.

Experiment Design: HIV-infected and non-infected lymphoid tissue explants were placed into the RWV. HIV infection was monitored by measuring p24 in the culture medium and by measuring infectious titer of the produced virus. Cells were collected from tissue blocks and from the surrounding medium from the RWVs and proliferation was measured in each fraction by 3H-thymidine incorporation. Cells from each compartment were also stained for various surface markers and analyzed by flow cytometry.

Continuing Results:

1. Previous results on infection of RWV cultured human lymphoid tissues with HIV-1 and on circulatory traffic of lymphocytes in the RWV were published: Margolis, L. et al, AIDS Research and Human Retroviruses, 1997, 13(16): 1411-1420.

2. Experiments are in progress to look at effects of infection on tissue cells and circulating lymphocytes – no preliminary results available.

Plans: Experiments will continue with HIV infection of RWV cultured human lymphoid tissues. Affects of HIV on activated tissues will also be analyzed.

Primary Investigators: Jean-Charles Grivel and Wendy Fitzgerald, Laboratory of Cellular and Molecular Biophysics, NICHD, NIH.

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

BACKGROUND:

Various impairments of immune function during spaceflight have been reported. Several factors may play a role in spaceflight anomalies including microgravity, stress, cosmic radiation, and launch-related hypergravity. Determining the exact role of microgravity on immune function is difficult with space shuttle missions, but may be possible using the RWV Bioreactor. The randomized gravitional vector provided by the RWV Bioreactor creates an environment similar to the microgravity environment of space and eliminated the other factors that may impact immune function. Understanding the mechanisms of immune dysfunction in microgravity is critical for assessing the safety of long-duration space flights.

Experiment Design: Blocks of human lymphoid tissue or lymphocytes isolated from these tissues will be cultured in the RWV or in static cultures. Cultures will be challenged with recall antigens and polyclonal activators. Viability, changes in lymphocyte subsets, proliferative responses, and antibody production will all measured.

Continuing Results:

1. Blocks of tissue cultured in RWV remain viable and metabolically active for three weeks of culture. However, unlike static cultures, the RWV cultures do not respond to recall antigens with production specific antibodies and do not respond to polyclonal activation with increased immunoglobulin production, in fact immunoglobulin production rapidly declines.

2. If responses are first initiated in static cultures and then the tissues are transferred to RWVs, the responses are aborted. However, if responses are initiated in RWVs and then transferred to static culture, the responses are restored. Thus the microgravity effect is transient.

3. Neither T nor B lymphocyte cell suspensions can be activated in RWV Bioreactors, but they can be activated in non-rotating RWVs, suggesting a direct role of microgravity in the impairment.

4. If lymphocytes are challenged in RWVs and then transferred to static culture, the cells do not regain their ability to be activated. If cells are first activated and static culture and then transferred to RWVs, they remain activated.

Plans: Results are being compiled for upcoming publication. Future studies will concentrate on determining where defects in immune responses of both lymphoid tissues and lymphocyte cell suspensions are occurring. We will analyze B cell differentiation and T cell-B cell cooperation in tissues and we will look at early activation events and cytoskeletal changes in lymphocytes.

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

Aim of the Experiment: To design, optimize, and apply a novel two-photon excitation fluorescence microscope for tissue imaging.

BACKGROUND:

Optical microscopy and spectroscopy are indispensable techniques for the study of cells and cellular processes. Optical probes for a variety of intercellular environments can be detected and localized with fluorescence microscopy and some aspects of cellular biochemistry can be manipulated with photoreactive "caged" compounds. The use of different microscopy technologies has expanded the concept of the traditional microscopic image. In this respect, a two-photon excitation fluorescence microscope has been designed, constructed, and tested. An argon ion pump laser and tunable titanium-sapphire laser are used as an excitation light source to produce a train of mode-locked pulses. A standard confocal microscope has been modified for use with this two-photon light source and the system operates in the de-scanned mode using single channel detection.

Continuing Results: Two preparations have been examined in the microscope – monolayers of human lymphocytes from tonsils and cultured blocks of human lymphoid tissue. Three different dyes have been evaluated for their use in two-photon microscopy: the endoplasmic reticulum sensitive indicator DiOC5(3), and the calcium sensitive indicators Indo-1 and Fura-2. Two-photon evoked fluorescence images were obtained using all three indicators in both monlayers of cells and cultures tissues. Lymphocytes loaded with calcium green, Fluo-3, or Fluo-4 all showed increases in intracellular calcium concentration in response to 100 uM ATP when monitored using conventional one-photon microscopy. However, lymphocytes loaded with either Indo-1 or Fura-2 then observed using two-photon excitation did not respond to ATP with an increase in calcium. The origin of this discrepancy is under investigation.

Plans: Both an optical compressor and an auxiliary photo-multiplier will be added to the existing system. The use of narrower pulses will allow a marked reduction in the overall illumination intensity used and the auxiliary detector will be used in the absence of descanning for more efficient detection of fluorescence.

Primary Investigators: Dr. Steven Hatfill, Laboratory of Cellular and Molecular Biophysics, NICHD, NIH and USAMRIID, Dr. Paul Duray, Department of Pathology, NCI, NIH, and Dr. Michael Bray, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Ft. Detrick, MD.

Aim of Experiments: To demonstrate the potential ability of the RWV Bioreactor to act as a "universal" pathogen culture system for the primary isolation of previously unrecognized pathogens during outbreaks of emerging disease. By demonstrating the ability of the RWV to culture a variety of known pathogens of different classification, i.e. RNA viruses, retroviruses, DNA viruses, parasites, spirochetes, etc., we hope to demonstrate the applicability of the RWV system for de novo primary pathogen isolation protocols.

BACKGROUND:

At present, the de novo isolation of previously unknown or unrecognized emerging disease pathogens requires elucidating the proper culture media or permissive cell line applicable for growth of the pathogenic agent in culture. A few examples serve to illustrate the difficulty inherent in this process using conventional technology. The 1970’s outbreak of Legionaries disease required months to identify the causative agent as a bacterium. The HIV-1 retrovirus required over 2 years to isolate and in the 1993 outbreak of Myuro Canyon disease in the four-corners region of the United States it took 7 weeks to grow the Hantavirus in culture. Clearly these timelines are unacceptable with respect to many public health threat scenarios for emerging disease agents. By utilizing the ability of the RWV to maintain a normal three-dimensional cytoarchitecture and microenviroment for a number of tissues, the possibility of using human tissue explants for primary pathogen isolation, becomes a distinct possibility.

Experiment Design: Attempts will be made to culture a variety of infectious pathogen in the same simple medium (RPMI-1640, 15% FCS) containing human tonsil tissue explants maintained in the RWV Bioreactor. In addition, a human liver and epithelial tissue equivalent will be formulated from established cells lines grown on Cytodex 3 microcarrier beads. These will be co-cultured with the tonsil explants. Known pathogens which have proved to be difficult to isolate by normal protocols, will be introduced into the RWV culture and allowed to incubate with the tissue and tissue equivalents for two weeks.

Continuing Results:

1. It has been previously demonstrated that the spirochetal organism Borrelia burgdorferi, the agent of Lyme disease, grows and proliferates in this system. The system has been shown to support productive infection with HIV-1 strains. Ebola virus was also shown to the amplified in this culture system.

2. The RWV Bioreactor system has been used to study differences between human Ebola strains and Ebola Reston, a macaque strain. Human exposure to Ebola Reston causes no apparent illness, but has an almost 100% mortality rate in non-human primates (NHP). The system has allowed the demonstration that in human tissues unlike in NHPs, Ebola Reston is unable to infect the intact epithelial ling of human blood vessels and this presumably accounts for the non-pathogenicity of the strain in humans.

3. The RWV Bioreactor has also been shown to support productive infection of Monkeypox orthovirus in human lung tissues. This model will hopefully shed light on the pathogenesis of not only monkeypox but also the related smallpox.

Plans: The system will be used to further explore the tissue tropism of Ebola strains, Reston, Zaire, Sudan, and Gabon. RWV Bioreactor cultures will be used to explore the basic pathogenesis of monkeypox in both human and NHP tissues, with particular reference to postulated cytokine production. If possible, in the scientific and political context, this work will be extended to study smallpox infection in human tissues since this type of experiment has never been done before. The RWV will be used in an attempt to culture Treponema pallidum, the causative agent of syphilis. This organism is notorious for its resistance to culture by conventional methods.

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

Aim of Experiments: To use the RWV Bioreactor to culture Borrelia burgdorferi, the etiologic agent of Lyme disease, in tissues. Proliferation of the spirochete in tissues is desired so that the infection process and ensuing disease progression can be studied in an in vitro model. The model could be used to study the infection process and understand basic questions about the parasite, such as whether it actually infects cells or whether it replicates in intercellular spaces.

BACKGROUND:

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. It is not known how the organisms survive immune attack in the mammalian host. 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.

Experiment Design: Tissue biopsies from patients with Lyme disease will be cultured in RWV Bioreactors to see if proliferation of spirochetes in primary infected tissue can be seen. The RWV will also be used to culture normal human tissues with Borrelia, the Lyme disease parasite, to study the infection process. Media other than BSK will be used to determine whether Borrelia is replicating in the medium in cultures or in the tissue itself, since it is known that Borrelia does not replicate in media other than BSK.

Preliminary Results:

1. Very good replication of Borrelia has been seen tonsils infected with the spirochetes. Infection has been confirmed by silver staining of tissue sections and by PCR.

2. Cultures of tonsils infected with Borrelia in media other than BSK show good replication, suggesting that the spirochete replicates in the tissue blocks and not in the medium.

Plans: Methods of quantitation of spirochete load in infected tissues are being researched so that better methods of comparison are available. With this model of infection we hope to begin studying the differences between infectious and non-infectious Borrelia. We plan to look for genetic variation of infectious spirochetes during a long-term culture.

Primary Investigator: Dr. Douglas Clark, Johns Hopkins University School of Medicine.

Aim of experiment: RWV Bioreactors will be used to culture both intestinal biopsies and intestinal cell lines to try to obtain an in vitro model for Cryptosporidium infection. These models would be helpful for determining tropism of certain species of Cryptosporidia and for replication of the parasite in human rather than animal intestinal cells for a better understanding of the life cycle and infection process.

BACKGROUND:

Cryptosporidiosis is common opportunistic infection in AIDS patients but relatively little is known about the parasite and its mechanisms of infection. Although some species can be replicated in other animals, it does not cause disease in most other hosts. Most in vitro studies are performed on monolayers of intestinal cell lines, which may not be representative of the human host. Human intestinal tissue is difficult to culture in vitro for any length of time, but the unique properties of the RWV Bioreactor may be helpful their culture. In addition the RWV bioreactor will be used to culture three dimensional aggregates of highly differentiated intestinal cell lines.

Experiment Design: Small and large intestine biopsies will be cultured in static and RWV cultures to attempt to maintain viability of tissue for at least several days. Intestinal cell lines will be cultured in RWVs on microcarriers to establish aggregates of differentiated cells. These cultures will be infected with Cryptosporidium oocysts and analyzed by staining of thin sections.

Preliminary Results:

1. Intestinal cell lines do form differentiated cell aggregates in the RWV. However, analysis of infection of cells is difficult due to presence of microcarriers, which make sectioning difficult.

2. Intestine biopsy cultures can be maintained for several days and infection of cultures is now beginning.

Plans: New support materials, such as polygalactic acid scaffolds, will be tried for culturing cell lines in the RWVs in place of microcarriers to eliminate problems with sectioning. Three dimensional cell agregates on these scaffolds will then be used for infection with Crytposporidium. Improvements continue to be made to intestine biopsy culture and to infection procedure to begin studying infection in human intestinal tissue.

Primary Investigators: Lynette Nieman MD and Pam Stratton MD, Department of Endocrinology Branch, NICHD, NIH.

Aim of Experiments: Human endometrium biopsies will be cultured as air-interface static cultures or as tissue explant cultures in RWV Bioreactors. Once cultures of tissue biopsies can be maintained in culture, the tissues will be used to gain an understanding of the role of growth factors and their receptors in normal and abnormal endometrial growth.

BACKGROUND:

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 the normal endometrium growth but also in abnormal endometrial growth, or endometriosis. Incresed 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 cultures are difficult to maintain since epithelial structures are typically overgrown by the stromal elements. A better model would employ the use of endometrial biopsies in a culture system that allows both cell types to survive. Such a model could then be used to study regulation of HGF and c-MET in normal and abnormal endometrium.

Experiment Design: Endometrium biopsies as well as menses will be collected from normal volunteers during various phases of the menstral cycle. These specimens will be cultured as small blocks of tissue in static or RWV cultures for several weeks. Tissues will fixed weekly and sectioned and stained for morphologic analysis.

Preliminary Results:

1. Culture conditions and medium continue to be improved and tissue blocks have been successfully cultured for 3 weeks with survival of both cell types.

2. Tissue biopsies from menstral and proliferative phases of the cycle have been cultured.

Plans: Viable tissue cultures will begin to be used to look at expression of HGF and c-MET 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 hopefully provide insight into the processes leading to endometriosis.

Primary Investigator: Lance Ferrin MD, Genetics and Biochemistry Branch, NIDDK, NIH.

Aim of Experiments: Biopsies from patients with various types of esophageal carcinomas, in particular Barrett’s esophagus, will be cultured in RWV Bioreactors. Proliferation of cells is needed in order to obtain enough cell DNA to look for rearranged regions to find tumor suppressor genes or oncogenes.

BACKGROUND:

Barrett’s esophagus is characterized by a switch of normal squamous epithelium in the lower esophagus to glandular epithelium containing goblet cells. Barrett’s esophagus is a premalignant condition that progresses to esophageal adenocarcinoma in a large percentage of patients. The molecular genetic mechanisms underlying the progression of disease are poorly understood. Identification of tumor suppressor genes and oncogenes in cells from Barrett’s esophagus would help to understand disease progression and would also serve as objective means for diagnosis and grading of lesions.

Few attempts at culturing Barrett’s esophagus in vitro have been successful. RWV culture will be employed to determine if cells have increased proliferative capacity in this environment as compared to standard tissue culture techniques.

Experiment Design: Biopsies of Barrett’s esophagus and esophageal adenocarcinoma will be cultured in RWV Bioreactors. Biopsies will be minced then cultured with microcarrier supports to promote cell adherence and proliferation. Cultures will be monitored to look for proliferation of cells. Cell samples will be fixed and processed for histologic evaluation. If proliferation of cells is achieved, the cells will harvested and DNA will be used to identify genes involved in the pathogenesis of the tumors.

Preliminary Results:

1. Several biopsies have been cultured. Cells are surviving but there are currently no signs of massive proliferation. Several types of growth medium are being tested to determine optimal conditions.

2. The microcarriers currently being used may not be conducive to growth. New types of support will be tried to obtain better results.

Plans: Improvements will continue to be made to culture conditions. If attempts continue to be unsuccessful, we will transform cells using human papilloma virus (HPV) in order to obtain enough cells to work with.

Primary Investigators: Teresa Jones MD, Metabolic Diseases Branch, NIDDK, NIH and Alberto Hayek, Whittier Institute, Department of Pediatrics, UCSD.

Aim of Experiments: To maintain long-term culture of fetal islet cells to study their growth and differentiation.

BACKGROUND:

Diabetes is a disease of inadequate insulin to maintain euglycemia. For Type I diabetics, the disease is primarily one of a loss of pancreatic islet beta cells which secrete insulin. Replacing the islet cells, instead of just insulin, would be a cure for diabetes. At this

time, organ transplantation has not proven to be a feasible option for several reasons. Another treatment option would be to grow and transplant a sufficient number of beta cells. In traditional cell culture, these cells have been maintained in culture for less than a month. Moreover, mature beta cells lose their ability to secrete insulin in culture. Fetal

islet cells show growth and some differentiation into islet secreting cells with short term culture. For this reason we have decided to work with this material.

Experiment Design: Islet cell clusters (ICCs) will be cultured in the RWV bioreactor over a two week period and compared to cells grown in suspension or monolayer. Cultures will be analyzed for morphological appearance, proliferation, insulin secretion, and insulin content. Later experiments will involve long-term culture of the ICCs to try to obtain first proliferation of cells and then differentiation into insulin secreting cells.

Preliminary Results:

Short term cultures of cells in the RWV revealed excellent morphology, secretion of insulin and insulin located within the cells. The cells in monolayer were dead and those in suspension had a poor appearance but did secrete insulin. The major problem with the result was a loss of islet cells in the bioreactor condition.

Plans: We plan to make modifications to decrease the loss of cells and try a long-term culture of 8-12 weeks. We will test for the amount of growth and differentiation. If we find the correct conditions that mimics the growth and differentiation seen when the islet cells are transplanted into nude mice, we will then investigate whether the role of

soluble factors versus intrinsic programming in the cell.

Primary Investigators: Raphaela Goldbach-Mansky, MD and Hani El-Gabalawy MD, Arthritis and Rheumatism Branch, NIAMS, NIH.

Aims of Experiments:

1. Biopsies of synovial tissue from patients with rheumatoid arthritis (RA), spondyloarthropathies, undifferentiated polyarthritis, and with evidence of Chlamydia infections will be cultured in static and RWV cultures.

2. Ability of different tissues to grow on synthetic matrices in disaggregation experiments will be tested.

3. Effects of proinflammatory cytokines (IL-1 and TNF-) will be tested on the growth and potential invasiveness of various synovial lesions.

4. The capacity of inflammed synovial tissues to invade explanted articular cartilage and matrices will be evaluated.

BACKGROUND:

Synovial tissue from patients with rheumatoid arthritis resumes an aggressive phenotype and leads to bony erosions and joint destruction. Other forms of inflammatory arthritis histologically undistinguishable from those in RA do not cause bony erosions and severe functional disability. Previous experiments in this 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 more about the unique characteristics of RA tissues that lead to the more aggressive phenotype.

Experiment Design: Culture conditions will be worked out for biopsies of RA synovial tissues and cartilage tissue. Tissues will be cryosectioned on a weekly basis to determine viability and maintenance of all cell types. Disaggregation cultures will be performed and their ability to reaggregate in synthetic matrices will be tested.

Preliminary Results:

1. Several biopsies have been cultured. Tissue biopsies remain viable and are structurally maintained for up to four weeks.

2. Reaggregation experiments in collagen 1 are in progress. Disaggregated cell suspensions are now being expanded in static culture before reaggregation is attempted to try to overcome limitations due to small sample size.

Plans: Future experiments will involve co-cultures of synovial and cartilage cultures to study invasion. Cultures of biopsies will be tested in serum-free medium for use in cytokine studies to determine if exogenously added proinflammatory cytokines disrupt organized growth.

Primary Investigator: Dr. Morgane Bomsel, Signalling, Inflammation and Cellular Transformation, Institut Cochin de Genetique Moleculaire, Paris, France.

Aim of Experiments: Intestinal and rectal mucosal biopsies will be infected with HIV and cultured to study the spread of infection in the mucosa after transcytosis across the epithelial barrier. Additionally, the effects of anti-HIV envelope proteins (IgA and IgM) will be used to study neutralization of HIV spread in mucosal biopsies.

BACKGROUND:

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.

Experiment Design: HIV infection of mucosal biopsies will be performed in a polarized manner from the luminal pole of the biopsy using Ussing chambers. After infection, the biopsies will be cultured in RWV Bioreactors for two weeks. Samples will be taken periodically to analyze for HIV content. Mononuclear cells on the basolateral side of the epithelium will be analyzed to look for productively and non-productively infected cells. Tissues will also be infected and transferred to RWV culture in the presence of anti-HIV envelope dimeric IgA to try to neutralize HIV transcytosis.

Preliminary Results:

Project is in early stages, no results available.

Primary Investigator: William Gahl, Heritable Disorders Branch, National Institute of Child Health and Human Development, NIH.

Aim of Experiments: The RWV Bioreactor will be used to culture three-dimensional organizations of fibroblasts from Hermansky-Pudlak Sydrome patients to study the defects in defective vesicular trafficking seen in these cells.

BACKGROUND:

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by pigment dilution, nystagmus, decreased visual acuity, a bleeding diathesis, and lysosomal accumulation of ceroid lipofuscin. Electron microscopic evidence demonstrating lack of platelet-dense bodies provides the sine qua non for diagnosing HPS. Ceroid lipofuscinosis is considered to cause several serious complications, including progressive pulmonary fibrosis leading to death in the fourth or fifth decades. Currently, only symptomatic treatment can be offered. Although rare in the general population, HPS occurs in northwest Puerto Rico with a prevalence of 1 in 1800. HPS1, the first gene found to be responsible for HPS, has no homology to any known protein. HPS1 mutations result in truncated proteins. The two mutations in the mouse pale ear gene (ep), which is the murine homology of HPS1, cause similarly truncated proteins. The pathologic nature of these truncation mutations may result from unstable mRNA. Two mouse models, pearl and mocha, suggest there is defective vesicular trafficking, specifically cargo packaging, vesicle formation, vesicle docking, or membrane fusion, which may comprise the basic defect in HPS. An appropriate in vitro model with human cells would be helpful for studying the proteins involved in intercompartmental transport for melanosomes, platelet-dense bodies, and lysosomes and would lead to a better understanding of the mechanisms of organellogenesis and to more effective therapies for HPS.

Experiment Design: Fibroblasts from normal patients or HSP patients will be cultured in RWV Bioreactors with microcarrier supports for several weeks to allow formation of aggregates of cells. Thin sections of the cell aggregates will be analyzed for histology. Differences between normal and HSP fibroblasts will be identified using various staining techniques. If HSP fibroblasts, in vitro, maintain their in vivo characteristics, these cells will be used for more detailed studies on vesicular trafficking.

Preliminary Results:

Cultures of normal and HSP fibroblasts have been cultured in RWVs. Cells remain viable and proliferate in culture for at least one month. Thin sections of the cell aggregates have been prepared and are ready for staining and analysis.

Primary Investigator: Constance T. Noguchi, Laboratory of Chemical Biology, NIDDK, NIH.

Aim of Experiment: RWV Bioreactor will be used to culture whole mouse embryos. If cultures with normal embryos are successful, the system will be used to culture embryos from erythropoietin receptor knockout mouse.

BACKGROUND:

Erythropoietin receptor (EpoR) on erythroid progenitor cells is the primary target for erythropoietin (Epo) binding resulting in proliferation and differentiation along the erythroid lineage and is critical for normal erythroid development. In addition, expression of EpoR has been observed on cells of non-hematopoietic origin, such as the embryonic mouse brain during mid-gestation. It is not known what role EpoR may play in the development of non-hematopoietic tissues. EpoR knockout mice are embryonic lethals (death at day 13.5) due to the hematopoietic defects, so studying the role EpoR in other tissues is not possible. Culturing of embryos in vitro may allow embryos to survive longer , without hypoxia due to no blood, and perhaps other complications from the Epo knockout would be apparent.

Experiment Design: Mouse embryos will be collected at day 11 and cultured in RWVs in a high oxygen environment. Embryos will be removed from culture each day and assessed for viability. Optimally we would like to culture the embryos for several days past their typical life span (13.5 days). Analysis of thin sections of embryos at each time point will be used to assess defects possibly due to EpoR absence, such as neural defects.

Preliminary Results:

Experiments are very preliminary, no results available.