Date: June 23, 2008
Place: Natcher Building
Conference Room E1-E2
National Institutes of Health
Bethesda, Maryland
The 188th meeting of the National Advisory Dental and Craniofacial Research Council (NADCRC) was convened on June 23, 2008 at 1:00 p.m., in the Natcher Building, Conference Room E1-E2, National Institutes of Health (NIH), Bethesda, Maryland. The meeting was open to the public from 1:00 p.m. to 5:00 p.m. it was preceded by the closed session for Council business and consideration of grant applications from 8:30 a.m. until adjournment at 11:30 a.m. Dr. Lawrence A. Tabak presided as Chair.
CLOSED SESSION
This portion of the meeting was closed to the public in accordance with the determination that it was concerned with matters exempt from mandatory disclosure under Sections 552b(c)(4) and 552b(c)(6), Title 5, U.S. Code and Section 10(d) of the Federal Advisory Committee Act, as amended (5 U.S.C. Appendix 2).
OPEN SESSION
Members Present
Dr. Carole A. Anderson
Dr. Gilda Barabino
Dr. Augusto Elias-Boneta
Dr. Rene D’Souza
Dr. Cecile Feldman
Ms. Katherine Hammitt
Dr. Anne S. Lindblad
Dr. Laurie McCauley
Dr. Malcolm Snead
Dr. Philip Stashenko
Dr. Harold Morris (ex officio)
Members of the Public Present:
Mr. John Bresch, ADEA
Mr. Robert Burns, ADA
Ms. Aida Chohayeb, Women’s Network Collective
Mr. Chris DaCosta, PPD Inc.
Ms. Debra Darcy, ADEA
Dr. Raul Garcia, Boston University
Mr. David Guthrie, ADEA
Dr. Christopher Fox, AADR
Mr. Mike Kalutkiwicz, IADR/AADR
Ms. Monette McKinnon, ADEA
Ms. Myla Moes, ADEA
Ms. Erin Ransford, AAOS
Mr. Patrick Roe, University of the Pacific
Ms. Myla Moss, ADEA
Mr. Bjorn Steffensen, University of Texas Health Science Center
Ms. Kathy Svoboda, Baylor College of Dentistry
Mr. Richard Valachovic, ADEA
Ms. Joan Wilentz, TMJ Association
Federal Employees Present:
National Institute of Dental and Craniofacial Research:
Dr. Lawrence A. Tabak, Director, NIDCR
Dr. Isabel Garcia, Deputy Director, NIDCR
Dr. Norman S. Braveman, Executive Secretary, NADCRC
Dr. Alicia Dombroski, Deputy Director, Division of Extramural Activities (DEA)
Dr. Pamela McInnes, Director, Division of Extramural Research (DER)
Dr. Robert C. Angerer, Scientific Director, Division of Intramural Research (DIR)
Dr. Katherine Carbone, Deputy Scientific Director, NIDCR
Dr. Jane Atkinson, NIDCR, CCR
Dr. Albert Avila, NIDCR, RTCDB
Mr. Hong Cao, NIDCR, GMB
Ms. Michelle Culp, NIDCR, OCTOM
Ms. Mary Daley, NIDCR, GMB
Ms. Mary Daum, NIDCR, PIL
Ms. Ki-Cha Flash, NIDCR, GMB
Dr. Kevin Hardwick, NIDCR, DEA
Dr. Victor Henriquez, NIDCR, SRB
Dr. Jonathan Horsford, NIDCR, SRB
Ms. Mary Kelly, NICDR, SRB
Ms. Sooyoun Kim, NIDCR, SRB
Dr. Raj Krishnaraju, NIDCR, SRB
Dr. John W. Kusiak, Director, Molecular and Cellular Neuroscience Program, CIBID
Ms. Carol Loose, NIDCR, FM
Dr. R. D. Lunsford, NIDCR, DER
Ms. Rosemary McCown, NIDCR
Mr. Jeff Ortiz, DEAS
Dr. Melissa Riddle, NIDCR, BSSRB
Dr. Mario Rinaudo, NIDCR, SRB
Ms. Dede Rutberg, Grants Management Branch, NIDCR
Dr. Yasaman Shirazi, Program Director, Epithelial Cell Regulation and Transformation Program, CIBID
Dr. Lillian Shum, Program Director, Mineralized Tissue and Salivary Gland Physiology Program, CIBID
Mr. Romeo Tengey, NIDCR, GMB
Ms. Rebecca Wagonner-Miller, NIDCR
Ms. Traci Walker, Committee Management Assistant, OD
Mr. Robert Berendt, NIDCR Contractor
Ms. Alison Davis, NIDCR Contractor
Other Federal Employees:
Dr. William Maas, CDC
Ms. Mary Fran Deutsh, OEP/OPERA
OPEN SESSION
I. WELCOME AND INTRODUCTIONS
Dr. Lawrence Tabak called the 188th meeting of the Council to order and invited guests and Council members to introduce themselves. Updating recent personnel changes, Dr. Tabak mentioned that Dr. Mary Sue Hamann was appointed the new Evaluation Officer, Dr. Lillian Shum, was confirmed as permanent Chief, Integrated Biology and Infectious Diseases Branch; Dr. Rosemary McCown had joined the Office of Clinical Trials, Operations and Management; and Dr. Jonathan Horsford had assumed the responsibilities of Scientific Review Administrator. Several at NIDCR had left the Institute for other employment opportunities – Dr. Mostafa Nokta had moved to the National Cancer Institute; Dr. Albert Avila had accepted a position as training officer at NIDA; and Dr. Sangeeta Bhargava had joined the Center for Scientific Review. Dr. Eleni Kousvelari retired after 24 years of federal service as had Dr. Rochelle Small, who had been with NIDCR for 15 years.
Dr. Tabak invited Deputy Director, Dr. Isabel Garcia, to discuss the updating of NIDCR’s Strategic Plan. Dr. Garcia explained that the updating of the plan has begun with an information gathering phase. Mr. Robert Berendt, planning advisor, and Dr.. Alison Davis, science writer, will support the effort. There have been two listening sessions held to elicit comments from non-federal sources -- the first at the annual meeting of the American Association of Dental Research in Dallas; the second at the annual NIDCR Patient Advocates Forum. An invitation for comments was posted on the NIDCR web site and the response has been excellent. There will also be listening sessions at the meeting of the International Association of Dental Research in July. The invitation for public comment was published in the Federal Register. The final step in the information gathering process will be a review of the NIDCR scientific portfolio, and several internal meetings to invite comments from NIDCR staff.
II. APPROVAL OF MINUTES
Dr. Norman Braveman, executive secretary of the Council, invited approval of the minutes of the January 27, 2008 Council meeting. On motion duly made and seconded, the minutes were unanimously approved.
III. REPORT OF THE DIRECTOR AND UPDATE ON ASSESSMENT OF NIH PEER REVIEW
Dr. Lawrence Tabak
Dr. Tabak explained that the NIH Peer Review Assessment was commissioned because the increasing complexity of science in recent years and the need for peer review to be able to address increasing emphasis on multidisciplinary approaches to research. The assessment was designed in three phases. The first was a diagnostic process during which information and opinions were gathered from a wide variety of sources including a broad base of individuals with research interests at federal research agencies, non-federal research entities, research universities, foundations and the interested general public. An invitation for public comment was placed on appropriate web sites, and there were a number of meetings with NIH staff and funded researchers, as well as a series of regional meeting at which all were welcome to comment. From the wealth of information gathered, a draft recommendations report was developed and submitted to the NIH Director on February 29, 2008.
That report was posted on the web and significant response was received. In addition, there were focus groups conducted within NIH and in the private sector. By April 16th, an implementation plan was developed after consultation with leadership from the Center for Scientific Review, the Peer Review Advisory Committee, the NIH IC directors, and a number of study section chairs. Dr. Tabak stated that he recently presented the implantation plan to the Advisory Committee to the Director for consideration.
The implementation plan has four priorities.
The first is to improve the quality of the reviewers who are expected to participate in at least 12 review sessions over a period of 4 to 6 years. To accommodate the usually very busy schedules of these reviewers, the plan proposed allowing duty sharing and flexible review submission schedules, and the introduction of electronic review procedures as well as teleconference/videoconference. The latter should become more practical when band width improves. The process of recruiting reviewers will be improved through the actual selection process to identify qualified extramural and intramural researchers, and through a policy that would increase the involvement of the very best and most experienced scientists (e.g., those who receive Pioneer Awards, PIs whose research is funded from multiple grants (e.g., three or more RO1s), and PIs who receive grant renewals of $500,000 or more).
There must be formal acknowledgement of a reviewer’s positive service accomplishments, and positive programs to make the review experience personally satisfying and intellectually rewarding. Incentives for outstanding and sustained service (at least 18 full study section meetings) might include eligibility to apply for additional funding to an existing grant up to $250,000, and an invitation to compete for an NIH honorific award. Since there will be some changes in the traditional peer review process, there must be training and mentoring opportunities for new and veteran reviewers.
The second goal is to improve the quality of the review process by addressing issues relating to the measurement of scientific merit. For example, the current scale used to measure scientific merit has 41 points. Research on psychometric rating scales shows that 5 to 7 scale points provides ratings with the greatest validity and reliability. In addition to changing to a 7 point scale used to assess scientific merit, the rating system will be modified to focus on scientific merit in five major areas: impact of the research; the experience and qualifications of the investigative team; the project’s level of innovation and/or originality; the research plan and the feasibility of the concept; and the research environment. Each reviewer will provide a whole number global score (1-7) and the applications will be ranked in average global score order. In a break from past practice, applications that are not discussed by the review group will be provided feedback for each review criterion, including a brief comment about whether or not resubmission is encouraged as well as suggestions for improvement. Reviewers may take advantage of training and effective tools for generating the feedback. Finally, a new, shorter, application format will be developed.
The third goal is to be able to evaluate applications regardless of the area of science, the applicant’s career stage, or the level of innovation (and therefore risk) attached to the project. Dr. Tabak noted that the present system can process about 10,000 applications each year. In 2002, about 4,000 came from first-time investigators and 6,000 from established investigators. By 2007, the number of first-time applicants had decreased by about 500. In addressing the need to insure that we maintain a vibrant research workforce by funding new investigators, the third objective will include an effort to insure support for a minimum number of early stage investigators. Further, the review of applications from established investigators should focus on recent work within the last program period to insure that past productivity continues. Finally, innovative, transformative research must be encouraged by setting aside approximately one percent of RO1-like awards to research that has the promise of stimulating a paradigm shift within a given research area. Although innovative ideas can come from any area of science, several specific areas are noted in the implementation plan including: the science of behavior change; protein capture reagents; chronic pain; three-dimensional tissue engineering; and the mitochondriome.
An overarching objective of modifying the peer review process is to reduce the burden of work on applicants, reviewers and NIH staff. Dr. Tabak explained that much of that work involved resubmissions. In 1998, about 60% of all NIH applications were funded at the first review, about 30% after the first amendment and 10% after the second amendment. By 2007, less than 30% of applications are funded at the first review. The NIDCR history is similar, though not as dramatic. From another view, in 1998 over 90% of applications that fell within the 20 percentile score or better were funded on the first review; by 2006 that number was about 30%. In terms of numbers, in 1998 NIDCR funded 1,600 out of 8,900 first time applications; by 2006 there were 14,000 applications and less than a thousand were funded. The effect is a sharply increased number of applications that are resubmitted. In fact, about 80% of the applications are eventually funded after the second or third submission. Therefore, the implementation plan suggests a policy review that would enhance the possibility of funding applications earlier in the process, and would discourage those applications, that in reality will not get funded, from moving through the resubmission process.
Finally, the fourth goal is to ensure a continuing evaluation and improvement of the peer review process. That would involve introduction of pilot programs to address ongoing issues. For example, alternative models (e.g., editorial board model) for peer review should be constantly assessed as should the use of a “prebuttal,” alternative ranking schemes, tests of high bandwidth electronic review processes, and developing a metric to monitor the review process.
Concerning researcher remuneration, there was a perception that NIH was shouldering the lion’s share of compensation. In fact, a review of the programs revealed that two-thirds of NIH-funded principal investigators are expending 50% or less of their effort on research grants. That means that the rest of their effort is dedicated to non-NIH enterprise and is therefore not compensated by the NIH programs. There was also discussion during the information-gathering process about establishing a minimum effort requirement to be a principal investigator. Because of the wide variety of business models in the non-federal research community, establishing a minimum percentage effort is impractical. There is an alternate proposal that if a PI has cumulative support of more than a million dollars, the PI must justify the reason for any additional resources requested.
Dr. Tabak noted that the next step will be the formation of an Ad Hoc Peer Review Task Force, headed by the NIH Deputy Director, to finalize the details of the implementation plan and initiate that implementation. A new entity within the Division of Program Coordination, Planning and Strategic Initiatives will oversee the continuous review process.
During discussion, Dr. Tabak explained that there would be a significant effort, especially in the fall, to inform the research community about the new aspects of the peer review process. Concerning the implementation process, there are some aspects of the system that can be implemented across the board; other changes may require further evaluation and a more gradual implementation. Asked about the continuity of the program as leadership may change at NIH, Dr. Tabak offered assurance that typically new leadership continues to support the existing programs and change would be gradual. In addition, as the metrics are developed and applied, the effectiveness of the new peer review system will become visible and influence future revisions to the program.
IV. REPORT AND DISCUSSION: EVALUATION OF NIDCR’S RUTH L. KIRSCHSTEIN NRSA TRAINING PROGRAMS
Dr. Kevin Hardwick
Dr. Hardwick reported the results of a study of individuals who received NRSA support from the NIDCR between 1995 and 2003, including both institutional trainees and individual fellows. Funding for trainees derives from a T32 grant awarded to a research institution, which then selects and funds the trainee. Funding for fellows, under an F grant mechanism, requires that the individual apply directly to NIH for an award , undergo a peer review evaluation process, and then receive the grant funds directly from NIH. The purpose of the study was to identify the professional outcomes of all the trainees and fellows who could be identified; that is, the individual’s current employment, area of professional endeavor, and subsequent NIH grant activity.
There were 691 individuals who received NIDCR-supported training during the time period of the study; 601 were T32 trainees, 90 were F fellows. About two-thirds were in postdoctoral status during the training period. In the T32 category, there were 259 dentists (141 of which were DDS/PhD or equivalent). Dr. Hardwick and Albert Avila were able to identify current location and job information for 612 of the grantees. Deducting those still in supported-training, the final number of individuals in the analysis was 566.
The results of the study: Among the analysis cohort, 75% are presently in full-time research or academia (whether or not they are directly involved in research). Comparing dentists with non-dentists, about 55% of the former are in academic or industry research compared to 90% of the latter. The dentists are more likely to be in private practice. Among the dentists, approximately 55-65% of those with research doctorate or MPH degrees are in research or academia, while only about 35% of those with a dental degree only are in full-time research or academic positions.
Of the analysis cohort, only 5% have received an R01 research grant after completing their NRSA training. Non-dentists have been more successful than dentists.
Dr. Hardwick reminded the Council of a previous study of all NIH postdoctoral trainees and fellows who trained from 1975 to 1992, which found that the fellows were more successful than the trainees in obtaining subsequent NIH R01 grants. An analysis of NIDCR data from that same time period revealed that NIDCR trainees had similar results.
Dr. Hardwick compared the performance of NIDCR postdoctoral trainees and fellows who were supported between 1975 and 1992 and between 1995 and 2003. With regard to applying for R01 grants, the fellows in the two time periods were similar (48% and 41%), but applications from trainees dropped significantly (from 33% to 11%). Of those receiving awards, both cohorts declined, the fellows from 26% to 17% and the trainees from 17% to 4%. Finally, looking at dentists versus non-dentists in the 1995 to 2003 cohort, the R01 application rate of dentists is significantly less than the non-dentists. Only 9% of T32-trained dentists in that cohort applied for R01 grants, and only 3% have received an R01 - a poor rate of return on the investment involved.
In summary, Dr. Hardwick noted that 75% of all NRSA trainees in the 1995-2003 population are in full-time positions in research or academia, although 45% of the dentists in that group are not -- most of these are in private practice. Five percent of those supported in the NSRA program have received an R01. Similar to the NIH, NIDCR fellows are more likely than T-32 trainees to both apply for and receive NIH R grants, although the numbers in both categories are slightly less than NIH-wide.
Dr. Hardwick discussed several questions that merit consideration. What criteria should define a successful outcome for the NRSA training program? What training programs should the NIDCR support and what should be the balance of funding in the programs selected? . Should training become more interdisciplinary? Should some part of the program be devoted to non-citizen dentist applicants? Why are dentists less competitive than PhD non-dentists? Would integrating an oral biology component in the PhD programs be appropriate? Until these questions are examined and a strategy for improved outcomes is developed, the NIDCR decided to temporarily cease accepting comprehensive T32 applications.
During the discussion, Dr. Hardwick stated that the NIDCR sees the NRSA program as a foundation for building a future research cadre, not as a program to support dental school research infrastructure or to enhance an individual’s educational qualifications. Clinical dentists should be grounded in science, but that is not a primary objective of the NRSA training program.
There was a suggestion that the study might look at educational experience (e.g., courses taken) to see if there are any factors that enhance future career choices. Noting that such an effort would require looking at academic records of the individual trainees, Dr. Hardwick suggested that a more feasible approach might be to look at which schools generate more research-oriented trainees. Asked about whether the sharp difference in success between dentists and non-dentists has a parallel in the NIH among programs involving MDs and PhDs, Dr. Hardwick stated that comparative data was not available,.
There was a suggestion that examination of NRSA alumni publications might be a good indicator of involvement in research. Dr. Hardwick agreed, and that form of analysis is being considered for the future.
Dr. Hardwick noted that there are discussions in progress about the ramifications of focusing T32 grants on institutions with major research programs versus making awards to smaller institutions that could identify two or three candidates. In fact, across NIH, T32 grants have a median of four trainees.
Asked about using the Medical Scientist Training Program (MSTP) as a model, Dr. Hardwick noted that MSTP requires the participating medical schools to pay all costs for students selected, which has not been required of dental schools thus far. Dr. Tabak added that the DSTP should more closely model the MSTP. Current DSTP trainees will be invited to campus for a September workshop to discuss training and career issues for dentist scientists. He noted that the typical dental student looks forward to a career in small business -- private practice – and the old model of recruiting dentist scientists from students already enrolled in dental school has not been very successful. A few dental schools are trying to recruit a different type of student, students whose interests include research, and in the future the DSTP will place a greater emphasis on identifying and including that type of student. Dr. Hardwick noted that the NIDCR is currently supporting 82 individuals in DSTP programs, 61 through the T32 programs, and 21 on F30s. Another 24 are graduate dentists pursuing PhDs. However, it is not certain that there are a sufficient number of appropriate dental school positions waiting for them when they complete their training.
There was a suggestion that there be some assessment of best practices in the various university-based dentist scientist training programs. Dr. Hardwick noted that there had already been a proposal to hold a DSTP best practices workshop to look at that issue. Asked about how the NIDCR would gather feedback on the issue, Dr. Hardwick stated that the open Council meeting was the first step and that he would anticipate feedback from Council members and other interested parties. Nonetheless, consideration of the issue will be a continuing effort for the NIDCR.
V. SCIENTIFIC PRESENTATIONS
In the introduction to four scientific presentations of research conducted within the Division of Intramural Research (DIR), Dr. Robert Angerer, Scientific Director, explained that the DIR has six laboratories and branches, with 31 principal investigators who head laboratories, and a staff of more than 300, including post docs, staff scientists and clinicians, and support staff. The DIR program has relatively stable funding from one year to the next which allows investigators wide latitude in developing protocols consonant with the NIDCR mission and encourages risk-taking and innovative approaches to research. The entire program is reviewed quadrennially by a board of scientific advisors and ad hoc reviewers.
Dr. Angerer gave a short overview of the studies currently underway in DIR. The first involved research on taste discrimination. Traditionally it was held that taste buds individually detected a variety of sensations by sorting out the components of a substance by receptors on the taste bud that discriminate various tastes (bitter, salty, sour, sweet, umami). Then the taste buds send an integrated signal to the brain. A project in the Laboratory for Sensory Biology identified a dedicated subset of taste bud cells that sense the sour taste, confirming that taste is a function of the cell type, not the substance being tasted or the taste bud receptor. Therefore, the information generated in these hardwired cells is apparently integrated at a higher level in the central nervous system and not in the taste bud. That mechanism is the subject of a current study.
In the Clinical and Skeletal Diseases Branch, the first identification of a tendon stem/progenitor cell was accomplished. These cells were shown to grow to form a colony (clonogenic cells) that can differentiate into cartilage, skeletal tissue or tendon, which makes them multi-potent. Finally, it was shown that the stem cells can be removed from a mouse, placed in culture, returned to the mouse, again removed and placed in culture, and finally placed in a different mouse in which the stem cells differentiate on a suitable substrate, which shows that the stem cells are self-renewing.
In the Mucosal Immunity Laboratory, research on oral pharyngeal cancer led to the identification of cancer cells that produce a signaling molecule, vascular endothelial growth factor (VEGF), which signals blood vessels, which then internalize a cell surface molecule, vascular endothelial cadherin, which causes a vascular breakdown that results in “leaks,” providing a point of entry into the vasculature for cancer cells to metastasize. By identifying these steps in that process, the investigators may then identify potential cancer therapy targets.
The salivary gland effects differentiation by way of a process called “branching morphogenesis.” This process creates secretory acini (an epithelial cell) leading into ducts that drain the products of salivation. The objective of the morphology is to create a very large surface area in a very small body. Work in the Laboratory of Cell and Developmental Biology identified two distinct mechanisms required to produce this morphology. The investigators showed that, unlike most epithelial cells that are typically adherent, rigid structures, the acinar epithelial cells move extensively relative to each other, even though the structure does not appear to change. The salivary gland has a grape cluster-like structure in which the “grapes” are separated by clefts that form at the gland surface. The investigators found that fibronectin, a high-molecular-weight glycoprotein, is secreted in the cleft and creates a wedge in the tissue (which increases surface area). In the morphogenesis, the acinar cells’ movement provides plasticity permitting the fibronectin to drive a wedge separating the grapes.
Finally Dr. Angerer shared some examples of the clinical research program at NIDCR, carried on mainly at the NIH Clinical Center. The program includes a natural history study with a therapy component for Sjogren’s syndrome; a gene therapy approach to reversing the loss of salivation caused by radiation therapy for head/neck cancer; an assessment of fibrous dysplasia of the bone and McCune Albright syndrome (a “stem cell” disease); a study of the natural history of hypoparathyroidism, and a study of clinical applications of resiniferatoxin, which may become part of an effective therapy for chronic pain.
In summary, he noted that NIDCR is involved in patient research and treatment, conducts an extensive training program, produces over 200 publications a year involving more than 200 colleagues, facilitates the technical transfer of new therapies to the public marketplace, and the shares of resources with independent research entities (tissue samples, DNA clones, mouse models, etc.).
Dr. Angerer then invited four NIDCR investigators to share their research.
1. Stem Cells: What We Know, What We Don’t Know, and How We Might Use Them
Dr. Pamela Robey, Chief, Craniofacial and Skeletal Diseases Branch, DIR
Dr. Robey explained that a stem cell is a self-renewing cell that is able to reconstitute an entire tissue. When a stem cell divides it may produce two identical adult stem cells, or it may produce one adult stem cell and a progenitor cell that continues to divide until the cells differentiate into a different type of cell. These stem cells may be capable of extended proliferation in an undifferentiated mode.
The first stem cell in a human organism is the mother’s fertilized egg that forms all of the cells and tissue structures of the embryo and the placental membranes. The first stage of that process is the formation of the blastocyst, whose outside “shell” forms the placenta, and whose inner cluster of cells are embryonic stem cells which go on to form the embryo. The next phase involves the development of the gastrula (ectoderm, mesoderm and endoderm) composed of fetal stem cells that are dedicated to forming specific tissues. After birth almost every tissue contains adult and progenitor stem cell populations.
Bone marrow is a rich source of hematopoetic stem cells (which form blood cells) and mesenchymal (or skeletal) stem cells that form the bed on which blood cells form. Research has demonstrated that a single stem cell, cultured in a specific manner, can be implanted in a mouse and the cell will for a complete bone marrow organ (bone, the stroma that supports blood formation, and marrow). This stem cell, which forms four distinct cell phenotypes, is called a multi-potent stem cell. Using techniques developed for establishing these stem cells in culture other potential sources of mesenchymal stem cells were investigated. Pulp from baby teeth was used to obtain single cells, which were placed in an in vivo transplantation system, and primary dentin was formed, as well as dental pulp-like complex. The cells were able to form two tissues. In another process, single cells from cementum were transplanted and formed new cementum and reformation of the periodontal ligament necessary to link the cementum to the alveolar bone.
This research suggested questions -- where do the various mesenchymal cells come from and are they similar or dissimilar? A large number of markers associated with these cells have been investigated. However, these markers are found on almost all connective tissue and are therefore non-specific to mesenchymal cells. Of the markers assessed only CD146+ cells proved to be colonizing cells. If these cells are implanted in a mouse model they make lamellar bone or globular dentin. Therefore, the CD146+ cells do not mark a single mesenchymal stem cell, but provide a pericyte that is the source of the local progenitor. The research question is to determine the characteristics of the specific cells that produce these different results. Dr. Robey noted that no single mesenchymal cell is the basis of all connective tissues and that there is no common mesenchymal stem cell distributed throughout the body. Therefore, stem cell nomenclature should be based on the tissue of origin rather than simply being designated “mesenchymal.”
Dr. Robey turned to the effect skeletal stem cells may have on skeletal disease. These cells are basic to the homeostasis of bone metabolism, and if there is a genetic mutation in the cells or a change in the metabolism caused by the bone microenvironment, bone disease may result (e.g., fibrous dysplasia or McCune-Albright syndrome). These diseases are currently being studied in the lab in mouse models with the expectation that in vivo transplantation of human disease will allow determination of methods for silencing the mutation and achieving resolution of the lesion.
Dr. Robey discussed the use of bone marrow stromal cells in regenerative therapy. It is well established that ex vivo expansion of a stem cell population (often with molecular engineering to silence gene mutations), and subsequent placement of the stem cells on an appropriate scaffold in bone will result in regeneration of damaged areas. Research is ongoing to determine if the cells can be injected percutaneously and reach the appropriate target to effect the same regenerative result. This would eliminate the need for invasive surgery.
Dr. Robey commented on unconventional uses of bone marrow stromal cells (BMSC), noting that there is an extensive literature on transgermal differentiation -- for example, skeletal stem cells from the mesoderm that apparently form cells of another germ layer (such as nerve cells) Despite the large literature, there is evidence that this transdifferentiation does not actually occur. However, there is evidence that BSMC can have a positive effect in supporting the initiation of tissue repair. BSMC does not persist at an injury site, but they play a role in “nursing” endogenous cells to begin repair. This effect has been reported for neuronal disease, lung fibrosis and cardiovascular disease. Dr. Robey noted that BSMC may be immunomodulatory and/or immunosuppressive, which is being studied in the lab in a mouse model. Immunosuppresion has been demonstrated by neutralizing lymphocyte cell division in one mouse strain and mixing in another normal responder strain, which results in proliferation of the responder cells. But if BMSC is added there is a significant prevention of proliferation of responder lymphocytes, indicating an immunosuppression.
In a second rat model, a high dose of an anti-inflammatory drug induced intestinal ulcers. When BSMC was added, the ulceration was significantly reduced, making the study a preclinical model for inflammatory bowl disease.
Finally, Dr. Robey discussed comparative stem cell biology, an important area of research. The mechanism that support unlimited cell replication and pluripotency (the ability of embryonic and fetal stem cells to form different tissues) needs to be defined. Conversely, differentiation in post-natal stem cells is predictable and defining the genes that induce this control is an appropriate research area. That control characteristic is not present, for example, in embryonic stem cells, which can lead to the formation of teratomas. These issues are important in research related to comparative stem cell biology.
2. Regulating Immunity in the Oral-Mucosal System
Dr. Wanjun Chen, Tenure Track Investigator, Oral Infection and Immunity Branch, DIR, NIDCR
Dr. Chen explained that the oral mucosal system discriminates between “good” and “bad” antigens and is the major entry route for antigens found in foods, pathogens in the environment, and commensal (generally beneficial) bacteria. The importance of dealing with these antigens is suggested by the fact that 60% of the peripheral lymphoid tissue is located within the gut mucosa and its job is to tolerate dietary antigens and expel infections pathogens. The oral mucosal system relies on T-lymphocytes (T-cells) to tolerate or resist an antigen. When foreign antigens attempt to enter the mucosal system, the T-cells decide whether to tolerate the antigen or to mount an immune response.
The objective of research in this area is to determine who the immune system decides how to respond to an antigen and the definition of the mechanism of action which results in an immune response or the establishment of a tolerance level. With that knowledge it may be possible to manipulate the immune system to develop more effective immune responses in humans.
Dr. Chen suggested the Chinese philosophy of Yin and Yang as an analogy to the balance between immunity and tolerance. When that balance is disturbed, the body is more vulnerable to infection (when the immune system weakens) or the effects of autoimmune diseases (when the tolerance level is reduced). This can be demonstrated in humans and mouse models when a reduced tolerance results in a disease such as Sjogren’s syndrome, periodontal disease, inflammatory bowel disease, allergies and transplant rejection.
Dr. Chen noted that only about 10% of T-cells are associated with tolerance. These cells, CD4 regulatory T-cells (Treg), are critical to immune tolerance because if they are removed in mice, the mice develop massive inflammation and die within four weeks. These cells regulate inflammation related to a number of diseases, but they may also have a dark side in that the majority of individuals with oral cancer have an increased frequency of Tregs in blood and in tumor tissue. Again, a better understanding of how Tregs are generated and how they function may provide a way to manipulate them for positive results.
Dr. Chen stated that he believed transforming growth factor beta (TGF beta) plays an important role. TGF beta is a cytokine found in most organisms and appears to be one of the most important immunoregulatory cytokines. Again, removal of TFG beta in mice has a rapid fatal consequence. Dr. Chen’s lab looked at mechanisms to characterize the role of TGF beta in the regulation of Treg in mouse models by deleting the gene for TGF beta only in T cells. The mouse model was a knockout with a T-cell specific deletion of the TGF beta receptor. The result was a rapid inflammation in the mice leading to death within seven weeks. This supported the hypothesis that TGF beta signaling is essential for normal Treg production. That signaling plays an essential role in the induction of Tregs. Without it, the generation of T cells is impaired, immune tolerance is compromised, and autoimmunity and inflammation results.
With the importance of TGF beta established, the next research question is the source of TGF beta. In the body a certain number of healthy cells are routinely programmed for death (apoptosis) and those apoptotic cells may be attacked by macrophages, which consume them, and research has shown that one product of the process is TGF beta. To see if it could be linked to the production of Treg, Dr. Chen’s lab tested the process in mice by exposing them to CD3-specific antibody treatment, which induces immune tolerance in vivo.
CD3 is a molecule on all T-cells and CD3 antibody depletes about half of the total T-cells, thereby inducing a long-term immune tolerance, which extends beyond the time when stoppage of CD3 antibody treatment allows restoration of T-cells. Dr. Chen’s lab developed the hypothesis that CD3 antibody injection stimulated apoptosis and during the process macrophages digested the cells and TGF beta was a byproduct, resulting in generation of regulatory T-cells which then increases immune tolerance. To test the theory, CDS antibody was injected into normal mice and 24 hours later there was a significant increase in systemic TGF beta, which lasted a few days. This supported the theory.
Dr. Chen’s lab then tested the process in a rodent disease model, a model for human multiple sclerosis, a T-cell-mediated autoimmune disease. SJL mice were injected with proteolipid protein, which induced disease in ten days. The mice were divided into ten groups -- one control untreated; a second CD3 antibody-treated group and a third CD3 antibody treated a group that also was macrophage depleted. CD3 antibody treatment significantly reduced the disease. Macrophage depletion completely negated the benefit CD3 antibody treatment, the mice faring far worse than even the control cohort.
In conclusion, Dr. Chen stated that programmed cell death results in a macrophage-related byproduct, TGF beta, which induces regulatory T-cell production that, in turn, improves immune tolerance. The positive effect of this process is the ability to develop immune therapies for diseases of the mucosal system (e.g., Sjogren’s syndrome) and inflammatory disease (e.g., osteoporosis). A negative aspect is that immune tolerance interferes with anti-tumor immunity in cancer patients. Worse, the process can deplete CD4 cells in HIV-AIDS patients with very detrimental results.
Dr. Chen stated that future research will focus on enhancing the positive effects, controlling the negative effects, and hopefully eliminating the exceptionally negative effects.
3. Mechanism and Function of Ca2+ Entry into Salivary Glands
Dr. Indu Ambudkar, Chief, Molecular Physiology and Therapeutics Branch, DIR
Dr. Ambudkar, representing the Molecular Physiology and Therapeutics Branch, explained that salivary gland function is an important research area in the Branch. The salivary gland is a system of the main glands, the parotid, the submandibular, and the sublingual, plus a number of minor glands that line different parts of the oral cavity. The gland system produces a complex fluid, saliva, which is composed of proteins, enzymes and water. Saliva performs reparative and lubricating functions, contributing to the enamel pellicle, dissolving tastants for taste bud response, repair and maintenance of the mucosa, and it serves as an antimicrobial and antifungal medium.
Any salivary gland dysfunction impairs these important contributions to oral and overall health. One of these is Sjogren’s syndrome, an autoimmune disease that mainly affects females. Its prevalence in the U.S. population may be up to 5%. The cause of the disease is believed to be a high level of lymphocytic infiltration and it results is the loss of salivary function.
Dr. Ambudkar noted that a second area of research is radiation-induced loss of salivary gland function, which affects up to 40,000 individuals in the U.S. (400,000 worldwide) who undergo head/neck radiation therapy. Even one treatment can cause irreversible loss of salivary function, which can lead to increased risk of oral infections, caries, and inflammation mucous membrane linings of the digestive tract, and generally a poor quality of life. There are no adequate therapies available to address this condition. Branch research is focused on understanding the physiology and molecular biology of the salivary gland, the mechanisms involved in dysfunctions, and developing diagnostic and treatment options.
Dr. Ambudkar explained the structure of the salivary gland, each of which looks like a cluster of grapes in which the grapes are composed of acinar cells. Saliva is generated in these clusters and drains into a graduated duct system that supplies saliva to the mouth. She explained the mechanism of saliva production, which begins in the basolateral (acinar cell) region through stimulation of a number of receptors that create two basic signals -- a calcium (fluid) signal and a cyclic AMP (protein) signal. As the fluid moved through the ductile cells, biochemical changes occur that form the final salivary product, a hypertonic low-salt solution.
The calcium signal leads to water secretion. The mechanism relies on calcium’s ability to regulate the ion transport process that regulates the accumulation of sodium and chloride in the lumen of the ductal system, which are required for water to move through the primary water channel, aquaporin 5. Without the calcium elevation the process shuts down and there is no water movement. The calcium function is also important to fertilization, cell cycling, apoptosis and gene regulation. A research problem is that calcium entry into the salivary gland is different from calcium channel functions in other parts of the body. Only recently has data improved the understanding of how the salivary calcium channels work.
About ten years ago a “super family” of cation channels was defined, the transient reception potential (TRP) family, which is a unique groups of cation channels, all of which are calcium permeable and found in most animal species. Three components of this family are pertinent to salivary gland research in terms of fluid secretion, volume regulation and morphogenesis.
The lab has correlated the amount of TRPC1 in a cell with the level of calcium, which indicates that TRPC1 may be important in regulating calcium entry into cells. A mouse model was developed in which TRPC1 was deleted, resulting in a decrease in salivary gland function. There was also decreased calcium entry into salivary acinar cells in these knockout mice.
TRPV4 affects salivary gland acinar cells, which are the cells that expand (accumulate water) and contract (expel water into the ductal system). TRPV4 is regulated by increases in acinar cell volume and appears to play a role in the reversal of the process, the contraction of the cell and expulsion of water. In other experiments it has been shown that TRPV4 and AQP5 may work together to regulate that process.
Finally, Dr. Ambudkar discussed the function of TRPM7, which is involved in cell metabolism. Looking at salivary gland buds in culture there was normal cell growth. Staining revealed that TRPM7 was present in the cells and related to the presence of fibroblast growth factor, which plays a central role in cell growth. FGF was shown to up regulate TRPM7 in the cell growth process. In conclusion, Dr. Ambudkar stated that the lab’s research has demonstrated the importance of calcium entry mechanisms in mediating fluid secretion, volume regulation and branching morphogenesis. The studies have identified critical TRP channels that affect fluid secretion (TRPC1), cell volume regulation (TRPV4), and cell growth (TRPM7). Future research will look at potential targets for treatment of salivary disorders.
4. Erecting Barriers in the Oral Cavity: Balancing Proteolysis and Anti-Proteolysis
Dr. Thomas Bugge, Senior Investigator, Proteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer Branch, DIR
Dr. Bugge explained that epithelial cells, which in most of the body’s organs serve to exclude pathogens, facilitate excretion and absorption, macromolecular transport and maintenance of ion gradients. The cells are very tightly packed to form a barrier which has an apical membrane on one side and a basolateral membrane on the other. The oral cavity is lined with epithelial tissue, and the salivary gland is composed of epithelial cells.
Dr. Bugge pointed out that his lab’s focus is on a class of enzymes called proteases, enzymes capable of cleaving other proteins. In humans, 566 distinct proteases have been identified (mice have 644). When proteases are misregulated, expressed at the wrong time or place, they contribute to a large variety of human ills -- bleeding gums and tooth loss, tumorogenesis, cardiovascular disease and neurological disorders, among others. But proteases also contribute to processes such as blood clotting and resolution of blood clots, promotion of digestion, growth, memory, killing bacteria and cancer cells, etc.
Research at NIDCR contributed to identifying a previously unknown protease function, the ability to make epithelium tight. Around 2000, a new family of proteases was discovered, type II transmembrane serine proteases. Dr. Bugge said that his lab is focusing on one of the 20 proteases in that family, matriptase. One reason for the late discovery of this protease is that there was apparently very little in tissue. However, in a mouse model a marker gene was inserted in a matriptase gene so that it could be identified in any tissue in which it existed. The lab was able to identify matriptase in the epithelial cells of the salivary gland and subsequently in a large number of tissues in other parts of the body.
The research question was, what is its function and its mechanism of action? Since matriptase was present in most mouse tissue, a mouse model was developed that would prevent the expression of matriptase. The result was that the epithelial barrier function was lost and the epithelium was no longer tight. This could be demonstrated by immersing membrane (first a mouse tongue, and then an embryo) in a blue dye solution. In the control mouse with normal matriptase, the epithelium resisted the penetration of the dye. In the matriptase deficient mouse, both the tongue and the embryo showed penetration of the epithelium - that is, the tissues were dyed blue. Because these mice died immediately after birth, a second mouse model was developed, using a Cre enzyme that allowed deletion of matriptase embryonically, specifically in the salivary gland. Over a period of time the mice deprived of matriptase in utero were unable to salivate normally.
A more sophisticated mouse model was then developed that involved insertion of a patented Cre-ER enzyme, which fused to a mutated estrogen receptor and prevented Cre-ER entry into the cell nucleus. Therefore, the mouse was born with normal matriptase. However, later adult administration of tamoxifen released the Cre ER enzyme which inactivated the matriptase, and shortly thereafter there was a massive breakdown of the mouse’s internal organs. The conclusion was that matriptase is required for the embryonic development of epithelial tissue and for its subsequent maintenance throughout life. The mechanism of action remains a mystery and a subject of future research.
Subsequently a Swiss group published a report on a mouse model that was deficient in a different enzyme, prostasin, and the phenotype of that mouse model was nearly identical to the phenotype of the NIDCR matriptase-deficient mouse model. Dr. Bugge stated prostasin must be cleaved by a protease to become active, and further investigation showed that matriptase and prostasin are expressed at the same place and time in various tissue sites, making this relationship a subject of further research.
Dr. Bugge noted that most proteases are regulated by endogenous protease inhibitors. The recently discovered inhibitors HAI-1 and HAI-2 appear to be inhibitors of matriptase. To test this, a mouse model was developed that compared embryonic development in a mouse with normal HAI-1 and a test mouse which was deficient in HAI-1. The HAI-1 deficient mouse embryo failed to develop, suggesting that a lack of the inhibitor produced an overload of matriptase that resulted in a lethal outcome similar to a lack of matriptase.
ADJOURNMENT
The meeting was adjourned at 5:00 p.m.
CERTIFICATION
I hereby certify that the foregoing minutes are accurate and complete.
________________________ _________________________
Dr. Lawrence A. Tabak Dr. Norman S. Braveman
Chairperson Executive Secretary
National Advisory Dental and National Advisory Dental and
Craniofacial Research Council Craniofacial Research Council
ATTACHMENTS
I. Roster of Council Members
II. Table of Council Actions
III. Director’s Report to the NADCRC, June 2008