August 30, 2004
TO:
Robert Mrtek, Chair
Senate Committee on Educational Policy
FROM:
Roger Nelson
Assistant Vice Chancellor for Academic Affairs
I am submitting for review and action by the Senate Committee on Educational Policy the attached
proposal from the College of Medicine to establish the Center for Lung and Vascular Biology.
In a memorandum dated February 12, 2004, Dr. Gerald Moss, Dean of the College of Medicine,
endorsed the creation of the center.
The proposal was originally submitted to the Graduate College Executive Committee in March 2004.
The Executive Committee postponed action until additional information could be supplied by the
proposed director, Dr. Asrar Malik. I am attaching to this letter Dr. Malik’s response to the
Executive Committee.
The proposal will be considered at the September 3 meeting of the Executive Committee, and I will
report to SCEP at its meeting on September 9 the action taken by the Executive Committee.
RN:
Attachment
Cc:
C. Hulse w/o attachment
R. Betts
G. Moss w/o attachment
M. Bailie w/o attachment
C. Desjardins w/o attachment
A. Malik w/o attachment
D. Martinez w/o attachment
D. Kellens w/o attachment
Principal Investigator/Program Director (Last, first, middle):
Malik, Asrar B.
Date: Wed, 25 Aug 2004 14:30:03 -0500
To: "Asrar B. Malik" <abmalik@uic.edu>
From: Clark Hulse <chulse@uic.edu>
Subject: Re: Center proposal
Cc:
Bcc:
X-Attachments:
One of the overall goals of the Center Lung and Vascular Biology as stated in the
OBJECTIVES document (as applicable to the Graduate College Executive Committee) is to
provide opportunities for the training of doctoral level students in the areas of lung and
vascular biology. There is an on-going NIH funded training program in Lung Biology
(Program Director A. B. Malik), which currently supports 5-6 doctoral level students trainees
(annual budget of $510,000). This NIH program fully supports the stipends, tuition, and
travel of these students who work in multiple labs under specific mentors. This program in
the future will be integrated within the Center, and as such the program (because it is NIH
supported) will provide the necessary infra-structure (such a administrative support) and
resources to set up the program in the context of the Center. It is expected being connected
with the Center will greatly help in making this program even more competitive and enabling
us to recruit trainees (such as MD's finishing their residency training) who may otherwise
would have been attracted to this program. Thus, only significant benefits can accrue from
having program associated with the Center.
Asrar
===================================================================
========
Asrar B. Malik, Distinguished Professor and Head, Pharmacology
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REQUEST FOR NEW ADMINISTRATIVE, RESEARCH
OR PUBLIC SERVICE UNIT
BACKGROUND
1. Name of Institution: University of Illinois at Chicago
1. Title of Proposed Unit: Center for Lung and Vascular Biology
3. CIP Code (6-digits):
4. Proposed Date for Initiation of Unit:
Fall 2004
5. Contact Person:
Dr. Charles Evans
5.1 Telephone
5.2 E-mail
5.3 Fax
(217) 333-3079
cevans4@uillinois.edu
(217) 244-5763
PURPOSE: OBJECTIVES MISSION AND PRIORITIES
6. Unit Objectives and Contributions
6.1. What are the goals and objectives of the new unit?
6.2. What is the relationship of the unit to the university's mission and priorities? Is the unit involved in
instruction and, if so, to what extent?
6.3. What specific needs and measurable contributions will the unit make to statewide priorities and needs?
6.4. What is the demand for the unit's services? What clients or population will the unit serve?
7. Organization
7.1. Describe the proposed unit's organizational structure.
7.2. Explain how the unit is organized to meet its objectives.
4 Temporary approval may be sought through reasonable and moderate extension for creation of a new, formally
organized, research or public service unit that bas a temporary mission up to five years. Following that time period, the
institution must seek permanent approval if the unit continues operation.
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PURPOSE: OBJECTIVES, MISSION AND PRIORITIES
6. UNIT OBJECTIVES AND CONTRIBUTIONS
6.1 Goals and Objectives
Overall Goals
The following are the overal1 goals of the Center Lung and Vascular Biology:
1. To develop a center of excellence in the thematic area of lung and vascular biology. This
will be accomplished by bringing together a multi-disciplinary group of investigators currently
working in this thematic area as well as the recruitment of outstanding tenure-track faculty at the
departmental and inter-departmental levels. To this end, the Center will recruit assistant professor
level faculty and provide a supportive environment and start-up resources needed to develop research
programs. Although the Center will be a new construct, its foundation will be the on-going vibrant
research programs in lung and vascular biology; thus, the underlying premise is that the Center will
capitalize on the existing strengths at UIC in lung and vascular biology research.
2. To have in place in the five-year time frame a nationally recognized and cohesive Center
for Lung and Vascular Biology in which investigators are extramurally funded through NIH grants
and through programmatic inter-disciplinary funding such as through the Program Project grant
mechanism.
3. To promote the development of synergistic and inter-disciplinary research programs.
Synergism is necessary so that the whole is greater than the sum of its parts and inter-disciplinary
research is necessary because this will be a philosophical underpinning of the Center; that is, we
believe that major and fundamental discoveries will be made at the boundary areas of science where
two disciplines interact. Thus, the Center will foster interactive research in lung and vascular biology
through multi-disciplinary and multi-departmental efforts. The Center will strive to develop
interactions between basic science disciplines but also between basic sciences and clinical
departments such as with the Division of Pulmonary and Critical Care Medicine in the Department of
Medicine.
4. To train doctoral level students and post-doctoral fellows (Ph.D.s and M.D.s). The current
NIH funded training program in Lung Biology (Program Director A. B. Malik), which supports 6
doctoral level students and 6 post-doctoral trainees (annual budget of $510,000) will provide the
infra-structure and resources for the training programs of the Center.
Specific Research Areas
What kinds of research will the Center foster? The Center’s underlying approach will be to
address clinically relevant or mechanistically-driven questions at molecular and cellular levels as well
as at the integrated functional level. We believe that such a approach will be needed to provide a
clear picture of the physiological as well as the disease processes; thus, the intent of the Center would
be to go beyond the reductionist view. The emphasis of Center’s research programs will be to
increase our understanding of specific disease processes at an integrated level. This will require
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advanced experimental approaches in molecular and cell biology, molecular genetics, imaging, and
integrative physiology and pharmacology as well as ultimately clinical studies in patients.
At its inception, the Center for Lung and Vascular Biology will focus on the following specific
scientific aims or research objectives, although it is expected that as the Center takes shape other
research focuses will emerge:
 To define mechanisms of lung microvascular injury, a hallmark of acute lung failure, as
mediated by various cells such as polymorphonuclear leukocytes
 To define mechanisms which oxidants generated by vascular endothelial cells, the cells lining
blood vessels, orchestrate the diseases processes of tissue injury and inflammation
 To define the regulation of vascular endothelial barrier function, the blood vascular barrier
comprising the layer of endothelial cells, so as to understand the mechanisms by which
dysregulation of barrier function leads to tissue edema and lung failure
 To define mechanisms by which the vascular endothelium is activated resulting in its
hyperadhesivity to blood leukocytes, and to ultimately tissue injury
 To define mechanisms by which blood leukocytes infiltrate tissues via migration across the
vessel wall under normal states as well as various diseases such as asthma and lung
inflammation.
 To define mechanisms of how polymorphonuclear leukocytes can be activated via specific
signaling pathways under normal conditions to combat infection, but also can be activated
inappropriately to produce tissue injury and inflammation.
The applied research focus of the Center will be in diseases of the lungs and the vasculature. The
Center will provide opportunities for basic scientists and physician scientists to interact in the spirit of
inquiry. The research to be carried out in the Center will have the potential to lead to improved
clinical care and better prognosis of patients suffering from diseases such as acute lung injury (ALI),
asthma, inflammatory diseases, pulmonary hypertension, diabetes, and acute transplant rejection. All
these diseases have a significant lung and/or vascular components; e.g., the vascular complication of
diabetes is the primary cause of morbidity and mortality in these patients. These diseases are often
complex, involve multiple organs, and have high levels of morbidity and mortality. For some
diseases (e.g., ALI or acute transplant rejection), there are no current reliable therapies and other
diseases (e.g., asthma) therapies are often inadequate. Some of these diseases such as ALI have a
mortality of greater 50% even in Intensive Care Units, whereas others such as asthma, pulmonary
hypertension, and multiple inflammatory diseases are chronic. These diseases in toto account for tens
of billions of dollars in health expenditures. Asthma is now recognized as a major developing
problem in urban areas, especially among minority children. In these populations, it has been
characterized as an “epidemic”. Acute transplant rejection is the primary reason for the failure of
transplants, and thus the process of transplant rejection needs to be addressed systematically by
harnessing the approaches and strategies of multiple disciplines so that the organs of these patients
can be salvaged. The investigators in the Center will address these important diseases by providing
new insights into their mechanisms so that as to better understand their pathogenesis. We believe that
with this information, it will possible to design more rational and up-to-date therapeutics with the
hope that this will open up new avenues for therapy.
6.2 Relationship to the University's Mission
The Center’s focus on research and teaching in health and medical sciences as related to lung
and vascular biology is consistent with the UIC Focus Statement (Attachment #1). The Center’s
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goals in research and training as outlined above are in full accord with the UIC mission.
To accomplish its mission, the Center will work with academic departments in the College of
Medicine to appoint faculty to the Center and to recruit new outstanding faculty members. The
Center will recruit faculty at the assistant professor level and will provide them the supportive and
nurturing environment and start-up resources needed to develop independent, vibrant research
programs. Although the primary appointments of the faculty will be in College’s academic
departments, the Center will serve as their intellectual home.
In addition, the Center will recruit Ph.D. and post-doctoral research trainees supported
training such as NIH funded training grant entitled “Training Program in Lung Biology and
Pathobiology” (Training Program Description; Attachment #2). In this context, the Center faculty
will be participate in advanced research training at the bench and will also be involved in instruction
of the trainees through course work. An example of the graduate level course to be directed by the
Center faculty is provided in Attachment #3 (course title “PHARMACOLOGY AND VASCULAR
BIOLOGY”). These aspects of the Center's mission directly parallel the institutional missions and
priorities.
6.3 Specific Need and Measurable Contributions to Statewide Priorities and Needs
The Center will contribute to statewide priorities as outlined in the document “CITIZEN’S
AGENDA FOR ILLINOIS HIGHER EDUCATION”. Specifically, the Center will do its part
helping to sustain and foster strong economic growth through the Center’s emphasis in biotechnology
and drug development. The Center’s research programs have specific relevance to biotechnology and
drug development. Thus, as an example the Center’s research efforts may lead to the development of
specific antibodies that could be important in diagnosis or therapy. With the unraveling of the
mechanisms mediating diseases processes at their molecular levels, it is likely that new patents would
be filed and drug targets and/or drugs would be developed in concert with pharmaceutical and
biotechnology companies.
An important contribution to state priorities specifically relevant to the biotechnology industry
in the State of Illinois will be through the development of molecular therapeutics targeted specifically
for lung and vascular diseases. The Center will operate on the premise that these therapeutics can be
most effectively developed as the direct result of an advanced understanding of the disease processes
at their cellular and molecular levels; that is, on the basis of solid basic research underpinnings and
interactions of multiple disciplines.
Besides drug discovery, the Center’s investigators will be involved in developing novel
approaches for delivery of drugs. This is particular limiting factor with many protein-based drugs
today, because these proteins cannot be ingested and must be taken by direct injection into the blood
or at other sites. Many protein-based drugs are being developed as the result of the genomic
revolution but their clinical usefulness is hampered because they cannot be easily delivered. Even
older protein drugs such as insulin need be injected. An important emphasis of the Center in this area
will be to develop new approaches for the delivery of these proteins across airway lining epithelial
cells following inhalation of the drug. We have an important lead on the basis of a recently issued
patent (Attachment #4), but our state of knowledge is now even more sophisticated and thus other
patents would be in the offing as the direct result of the work carried out in the Center. Thus, we
believe that it will be possible through this inter-disciplinary and concerted research effort to develop
new solutions for delivery therapeutic proteins by inhalation. But obviously much more research is
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needed in an integrated setting such as the proposed Center, which will be an incubator for such
developments.
Another aspect of the Center’s contribution to statewide priorities will be in its ability to
garner significant extramural research and training grant funding as well as major center and program
grants. These programs will not only employ and provide opportunities for technical staff but also
serve as magnets for other programs, funding, and the recruitment of outstanding faculty and trainees.
Thus, a visible and successful Center based on entrepreneurial thinking is likely to serve as an
“amplification system” for the development of other research activity. As an example of
programmatically based funding which we wish to replicate is the currently funded Program Project
grant entitled “Signaling of Endothelial Permeability and Lung Vascular Injury” (Description of this
program’s synergism is provided in Attachment #5). This program is currently supported for total
budget of $6,493,464, and the pending budget for the next five-year cycle is $11,229,000, almost
doubling of the budget. Because of the clear focus of the Center, the leadership to be provided, and
the quality of the faculty, it is reasonable to expect that there would be other significant programmatic
funding in the future as the result of direct “spin-off” from the Center .
Because the Center will provide opportunities for basic and physician scientists to interact, the
research carried out in the Center has the potential, and indeed as its key objective, of improving
clinical care and better prognosis of patients suffering from diseases such as acute lung injury (ALI),
asthma, inflammatory diseases, pulmonary hypertension, diabetes, and acute transplant rejection. As
the Center investigators will address these lung- and vascular-related diseases by providing novel
insights into their pathogenesis, it will possible to design more rational and up-to-date therapeutics
that will open up new treatments. Because these diseases represent a major drain on the economy,
even small advances in decreasing morbidity and mortality are likely to have a major benefit.
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6.4 Demand for Unit Services
The Center will serve the needs of researchers across the University of Illinois at Chicago
with specific interests in lung and vascular biology in both basic sciences and clinical medicine. The
Center will provide opportunities for faculty investigators from multiple disciplines to interact on the
basis of research interests and for training of Ph.D. and postdoctoral level research trainees. Besides
the specific research interactions and training of students and post-doctoral fellows, the Center will
sponsor seminars by visiting professors and senior scientists as a part of its academic programs.
The Center will appoint specific existing faculty members (to be selected on the basis of
research expertise, discipline relevant to lung and vascular biology, and overall excellence as
scholars). The Center will also recruit new faculty by contributing salary support and start-up funds
conjointly with academic departments. Besides these formal affiliations with the Center, other
constituencies will also be involved with the Center. For example, pharmaceutical or biotech
companies interested in the results of the research in development of novel drugs (such as directed
towards chronic inflammation, transplant rejection, or asthma) and ways of efficaciously delivering
these drugs will work with the Center’s staff in translating the research findings to practical results.
Thus, it is expected that research ideas and products of the Center will attract the interest of
businesses and industries outside the university as well as of non-governmental foundations and
organizations supporting research such as the American Lung Association and American Heart
Association.
7. ORGANIZATION
7.1 Proposed Unit's Organizational Structure
Internal Structure of the Center
The Director of the Center will be Professor Asrar Malik, a leading figure in lung and
vascular biology research (curriculum vitae is Attachment #6). He has been among the top 10 funded
researcher at UIC for last five years, million plus dollars annually. He has developed and has had
successfully funded multi-investigator and inter-disciplinary research and training programs. He sits
on many national committees such at NIH where he was a member of the Program Project Review
Committee of the National Heart, Lung and Blood Institute and the Pulmonary Disease Advisory
Committee. He is currently the chairman of the American Heart Association Vessel Wall Biology
Study Section. Dr. Malik has been the recipient of the NIH MERIT Award from 1987-1997. He is
currently the editor-in-chief of the leading lung biology journal, The American Journal of
Physiology: Lung Cell and Molecular Physiology. An Associate Director will be selected to assist
the Director in the management of the Center.
The Center faculty will be chosen by the Standing Committee (described below) which will
advise the Center Director on all appointments following recommendations made by the Center
Director and department heads. The Center will appoint selectively and only specific faculty
members. These individuals will be selected on the basis of the following criteria: (i) sophistication
of research expertise in specific areas relevant to the Center’s thrust in lung and vascular biology, (ii)
research discipline that adds to the Center’s inter-disciplinary focus, (iii) excellence in research as
demonstrated by important publications in first-tier journals and extramural funding, and (iv) ability
to interact and work in an inter-disciplinary setting.
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The Center Director will report to the Dean of the College of Medicine. The Center Director
will be responsible for the day-to-day management, financial issues relevant to the Center, and annual
progress reports. An organizational chart showing the reporting lines is indicated in Attachment #7 .
External and Internal Advisory Boards
The Center will have an External Advisory Committee consisting of nationally prominent
individuals to be invited to review its progress. The individuals to be selected are Dr. Joe G. N.
Garcia, David Marine Professor of Medicine at Johns Hopkins University School of Medicine and
Director of Division of Pulmonary and Critical Care Medicine, Johns Hopkins Hospital, Dr. David
Pinsky, Professor Medicine and Head of the Division of Cardiology at the University of Michigan,
School of Medicine, Ann Arbor, and Dr. Iasha Sznajder, Professor of Medicine and Chief of
Pulmonary Division, Northwestern School of Medicine, Chicago.
The Center will also have an Internal Advisory Committee consisting of the new Chief of
Pulmonary and Critical Care Medicine, Dr. John Christman and Dr. Lester Lau, Professor of
Biochemistry and Molecular Genetics, University of Illinois at Chicago, College of Medicine.
The External and Internal Advisory Committees will annually evaluate the Center's
performance and provide input to the Center Director.
The Center will have a Standing Committee of the following individuals: Dr. Richard Ye,
Professor of Pharmacology; Dr. Tatyana Voyno-Yasenetskaya, Associate Professor of Pharmacology;
Dr. Robert Costa, Professor of Biochemistry and Molecular Genetics; Dr. C. Tiruppathi, Associate
Professor of Pharmacology, and Dr. Richard Minshall, Assistant Professor of Anesthesiology and
Pharmacology.
The Standing Committee will meet more regularly to advise the Center Director on
appointments following the recommendations made by the Center Director.
7.2 Organization of the Center
To achieve the Center’s goals and objectives (i.e., to develop a synergistic and inter-disciplinary
center of excellence in the thematic area of lung and vascular biology, increase the programmatic
research funding in this area, provide an intellectually-charged training environment for doctoral
level students and post-doctoral fellows, and foster clinical interactions with hope of improving
patient outcomes), the Center will be organized in a manner that fosters interactions and dialog,
provides appropriate resources to the newly recruited faculty, supports a nurturing environments in
which ideas can take wing, and encourages and rewards innovative thinking, entrepreneurship, and
team approach to science and solving difficult problems. The example and leadership of the Center
Director are among the factors essential for this task. Thus, Center Director working in concert with
other individuals listed in the organizational chart is an absolute requirement for the Center to meets
its outlined goals and objectives.
The Center Director will be responsible for day-to-day management and progress reports to
the Dean of the College of Medicine.
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8. UNIT OUTCOMES
8.1 Targets for Assessment of Center's Success in Achieving Objectives
Gauge of success of the Center:
1.
2.
3.
4.
Level of extramural funding (chiefly from NIH). A bench-mark in this regard is
the ratio of external-to-internal funding of 5:1 to 10:1 in 5 years. Thus, the Center's
success will be assessed by the level of extramural funding generated.
Quality tenure-track faculty appointments to the Center and the ultimate success of
the faculty as defined by research publications, level of independent research
funding, and research contributions to the scientific vitality of the Center.
Discoveries, inventions, and patents developed in the Center.
Quality and excellence of trainees recruited into the Center receiving advanced
training with Center-designated investigators and their ultimate success as funded
and independent scientists.
9. RESOURCES
9.1 Number of Faculty and Trainees (PhD students and Postdoctoral fellows) and Served by the
Center
At its maximum strength, the Center will provide partial salary support for 5-8 tenure-track
faculty members with remaining amount provided by individual departments on a cost-sharing basis.
An equal number of existing faculty will have an appointment in the Center without salary support at
the tenured or tenure-track level. The Center will also have at any given point approximately 10 PhD
students and an equal number of postdoctoral (M.D. and/or Ph.D.) level trainees.
Other Constituencies Served by the Center:
In terms of drug discovery and drug delivery systems, the Center will collaborate with
scientists in the pharmaceutical and biotechnology industries. This will be essential for the practical
applications of the technologies to be developed by the Center faculty.
Total Resource Requirements:
Line 1: indicates the total amount (ranging from $300,000-$400,000) needed for the
recruitment and salary support of tenure-track faculty at the Assistant Professor level per year. The
additional amount in Line 1 in the out years reflects the Center’s contribution to faculty salaries with
addition of each new faculty member (approx $40,000).
Line 2: approximate amount of ICR/year. This estimate is based on 20% recovery on a NIH
with total/year direct costs of approximately $250,000 and IDC of $125,000.
.
Line 4: this is amount of state support committed by the Dean of COM
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Line 5: reallocations obtained by the Center Director from Department of Pharmacology and
other participating departments for recruitment packages.
Note: In this scheme, at the end of 5 years, if it is assumed each of the 5 new faculty generate
$375,000 in DC and IDC, the total amount will be $1,875,000. The institutional investment of state
dollars will be $200,000. Thus, this represents an outstanding return on the investment.
Library Resources:
The University Library estimates that it currently spends $50 thousand per year for resources that
support work in the area of lung and vascular biology. Lung and vascular biology faculty and
Library faculty find that current resources are adequate, so that no additional University Library
funding will be necessary.
10. QUALITY ASSURANCE PROCESSES
In terms of demonstrating ultimately the quality of the Center for Lung and Vascular Biology,
there will be three evaluative quality control measures. This assessment can be made on an annual
basis under the following categories: 1) evidence that the Center investigators are recipients of NIH
support and support from foundations and organizations such as the American Heart Association and
American Lung Association (the dollar amount); 2) demonstration of effective recruitment of
outstanding junior faculty who are NIH funded or have the potential of NIH funding; 3) publications
of appointees to the Center. Each of these criteria can be quantified and the results can be tabulated
on an annual basis. Thus, these measures will be used to improve the Center's effectiveness in
fulfilling its goals.
Quality Assurance.
1. Center's support of university mission and statewide goals:
The Center's mission in developing a nationally recognized and intellectually and financially
profitable center of excellence is in full accord in the institutional mission in supporting research and
scientific activity for the sake of knowledge as well as for the societal good. The development of the
Center also serves the state mission in that the Center promises to be a driving engine for new ideas
and technologies, recruitment of talented and energetic faculty and technical staff, and partnering
with biotechnology companies and other businesses. To measure the quality of the "product", the
Center will be reviewed on a yearly basis by advisory committees as described above. The review
will address the Center's progress in fulfilling its objectives. An annual report will be generated
detailing the funding of the Center's faculty, publications, and other measures of their academic
productivity such as awards, patents filed and awarded, grants submitted, trainees mentored, etc. The
Center will also be reviewed every eight years as part of the IBHE's review of degree programs and
research/public service centers and institutes.
2. Determination of organizational effectiveness:
The organizational effectiveness of the Center and its administration will also be evaluated by
the aforementioned committees and report provided to the Dean of COM.
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3. Faculty qualifications and reward structures:
The qualifications of the Center faculty will be annually reviewed by the Standing Committee
and the Center Director on the basis of their commitment to the Center's goals and objectives and
participation in the Center's activities. Faculty will be recommended for promotion and salary
increments to the respective department heads on the basis of standard academic criteria including
success in extramural funding, publications in prestigious journals, and service and commitment to
the Center.
4. Results from evaluation to improve the Center's effectiveness:
The constructive feedback obtained from these reviews as well as the input from the Dean of
COM and Center's faculty will be used to make the Center a more effective entity and to drive it
forward. In this regard, the Center will strive to continually "raise the bar" and excel in its scientific
endeavors, the essential elements of the Center’s success.
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Table II
TOTAL RESOURCE REQUIREMENTS FOR THE NEW UNIT
1
2
Total Resource Requirements
Resources Available from Federal
Sources
3
4
5
6
Current
Budget
2nd
3rd
4th
Year
Year
Year
Year
Year
365.0
405.0
445.0
485.0
525.0
25.0
(375.0)
25.0
(375.0)
25.0
(375.0)
25.0
(375.0)
25.0
(375.0)
340.0
380.0
420.0
460.0
340.0
40.0
40.0
40.0
40.0
1
2
3
4
5
'
Resources Available from Other
Non-State Resources
Existing State Resources
Resources Available through Internal
Reallocation
New State Resources Required
Breakdown: New State Resources
Required
7
FTE Staff 5
8
Personal Services
9
Equipment and Instructional Needs
10 Library
11 Other Support Services6
1. This line reflects total funds to be provided to each new tenure-track Assistant Professor recruit from all sources (state, ICR, and
cost-sharing commitments made by departments) in any given year. This total amount represents the 9 month salary component and
start-up costs for the junior faculty member.
2. This amount ($25,000) is the expected ICR calculated on the basis of the Center receiving 20% of the total ICR for each faculty
member. For example, if the Assistant Professor is to receive a grant of $250,000 per year (a rather typical amount for an NIH grant),
the ICR would be $125,000, and the Center would receive 20% of this amount or $25,000. The amount in brackets indicates the total
budget of a typical NIH grant with direct amount of $250,000/yr plus indirect cost amount of $125,000. This calculation of $25,000 is
based on the assumption that each Assistant Professor recruited into the Center will be successful in generating this support amount on
an annual basis.
3. The commitment of state support ($40,000) made by the Dean of COM for the recruitment of one position per year at the Assistant
Professor rank for 5 years.
5. This is the amount ($300,000) to be reallocated from other sources such as departmental cost-sharing for the recruitment; i.e., one
time $300,000 reallocation in each of the five years.
7. Represents the recruitment of one tenure-track Assistant Professor faculty per year over 5 years, for a cumulative total of 5 recruits;
this number of recruits may increase with overall success of the Cent
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ATTACHMENT #1
UNIVERSITY OF ILLINOIS AT CHICAGO
Focus Statement
Located in the nation’s third largest metropolitan area, the University of Illinois at Chicago offers
instruction at the baccalaureate, master’s, first professional and doctoral levels. The University
conducts research and public service in a variety of fields and ranks among the top universities
nationally in attracting external support for these activities. A significant proportion of the campus’
undergraduate student body commutes, is older than traditional college age, attends part time, and has
transferred from other institutions. In addition to pursuing statewide goals and priorities, the
University of Illinois at Chicago:

strengthens the economic and social vitality of the Chicago metropolitan area through its
urban land grant mission that emphasizes business and industrial development, health care,
school improvement, and enhanced opportunities for minority groups;

offers instruction, research and public service in traditional fields such as engineering and the
arts and sciences complemented and enhanced by a focus on health and medical sciences and
services;

provides off-campus programs in community college districts in the Chicago metropolitan
area; and

has a statewide mission to provide off-campus programs in health sciences and in selected
other areas not generally available through other colleges and universities in the state.
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ATTACHMENT #2
RESEARCH TRAINING PROGRAM PLAN
1.
Background
A.
Introduction to “Training Program in Lung Biology and Pathobiology”
(T32HL07829)
Our preeminent goal has been and remains as outlined in this renewal application: to
provide state-of-the-art research training for predoctoral and postdoctoral trainees in areas of
pulmonary biology and pathobiology. The training approach is a multidisciplinary enterprise with
the training faculty having their primary appointments in six academic departments at The University
of Illinois (Departments of Pharmacology, Medicine, Molecular Genetics, Biochemistry and
Molecular Biology, Physiology and Biophysics, and Microbiology and Immunology) and one at
Northwestern University Medical School (Department of Pharmacology and Molecular Biology).
The training program is geared to provide research expertise in specific areas relevant to lung
biology and pathobiology. The program goes beyond simply the reductionistic level in its approach
to research training. The trainees also have opportunities to address specific research questions at the
integrative and systems level as we have available in the program expertise ranging from molecular
biology to the study of intact lungs. The research expertise of the faculty impacts on important
questions in lung biology and pathobiology as indicated in Table 2. The training faculty (listed in
Table 1) is exceptionally talented as evident by their research competence and accomplishments.
Thus, the training program has fostered the development of an intellectually stimulating environment
that provides the critical underpinnings for the training of individuals planning research careers.
The interdisciplinary training faculty has been assembled on the basis of their research areas,
productivity, competence, and general relevance to research themes in pulmonary biology and
pathobiology. All training faculty members have established credentials as independent
investigators. Although some members of the faculty are not strictly pulmonary researchers, their
specific areas of expertise and disciplines have implications in addressing fundamental research
questions in lung biology and pathophysiology of lung injury (Table 2). Therefore, these faculty
members complement the pulmonary research strengths of the program and contribute to the unique
multidisciplinary training environment, the hallmark of the program. Given the multi-disciplinary
nature of today’s research, we believe that the environment fostered by the diverse backgrounds of
our faculty provides a unique approach to training in lung research. This “cross-fertilization” of
research interests and “cross-training” of fellows enhances the quality of the program and provides
the trainees with the up-to-date approaches needed to address their research questions.
It is important to note that the multidisciplinary training faculty, although from different
academic departments, is a cohesive and experienced group of investigators who know each other
well and whose research interests are relevant to the overall theme and thrust of the training program.
They have been brought into the training program on the basis of their abilities to offer training in
areas that are directly related to the fundamental questions and themes in lung research.
B. Rationale for the Lung Research Training Program
We seek continuation of support for the following reasons. First, an outstanding
training faculty providing in-depth and comprehensive research training has been integrated into the
overall theme of the program. Thus, the program is clearly an on-going and vital institutional entity
with significant commitment as outlined in Resources Section (page 68). The training faculty
members have also been thoroughly integrated into the program on the basis of their strong interests
in either lung research or in areas that strongly impact on lung biology. As evident by the detailed
descriptions of the research interests, the training program provides a breadth of opportunities for the
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prospective fellows. All of the training faculty members also have a strong commitment to training
predoctoral and postdoctoral fellows as evident by their individual records as preceptors (Table 7).
Many of the individuals have had well over 10 years of experience as preceptors. Thus, a committed
and dedicated faculty has been assembled under the aegis of the training program. Their commitment
to providing training in lung research is well recognized and represents a major rationale for this
program.
Second, we seek continuation because the program has been a success in attracting an
outstanding and highly competitive group of trainees and providing them with state-of-art training
even in the short period of four years since the program’s inception. We are confident in saying that
the objective of providing research training in specific areas in lung biology and pathobiology of
acute lung injury and inflammation is being realized.
Third, we believe it is important that the program serves an important function in providing
training to predoctoral fellows in aspects of lung biology, an area in which a relatively few Ph.D.s.
are trained nationally. Because of the training that they receive at the doctoral level, we believe that
these individuals will bring their unique basic science perspectives to research questions that will
contribute to future advancements in lung research. We also believe that individuals trained at both
predoctoral and postdoctoral levels in an interdisciplinary setting will contribute in tangible ways to
the future development of diagnostic tools and therapies. Their skills are highly sought in clinical
and basic departments of academic medical centers as well as the burgeoning biotechnology industry
where a great deal of high quality research is being carried out. Thus, the training program from
these perspectives serves an important purpose and need.
Finally, the program adheres to the underlying premise of providing training that will enable
trainees to adapt to the multidisciplinary approach of today’s research environment as they progress
in their research careers. Thus, the training program stimulates and fosters interdisciplinary thinking,
a requisite for a productive career in research. The program provides research expertise in areas
ranging from molecular biology to integrative physiology as they impact on lung biology and
pathobiology. Although the reductionist approach is useful and valid, we as a training faculty believe
it is also important for the trainees to be aware of the systems biology and to integrate approaches and
methodologies from multiple disciplines and apply them to addressing important questions in lung
research.
The program is able to provide a multidisciplinary approach to research problems because the
training faculty has been integrated into the program on the basis of the research areas that are
fundamental to lung biology and pathobiology. The areas of research in which fellows can receive
training include: (1) pulmonary vascular endothelial injury, (2) determinants of pulmonary
endothelial barrier function and control of endothelial permeability by second messengers, (3)
molecular mechanisms of endothelial-leukocyte interaction, (4) signaling of phagocytic cell
activation, (5) mechanisms of activation of interferon receptors and the Jak-Stat pathway, (6)
thromboxane receptor activation mechanisms, (7) integrin-regulated signaling, (8) pulmonary
vascular processing of peptides via angiotensin converting enzyme and other carboxypeptidases, (9)
regulation of NO production in endothelial cells, (10) seven-transmembrane receptor activation
mechanisms, (11) regulation of K+ channels, (12) G-protein regulation of cytoskeleton, (13)
mechanisms of endothelial cell proliferation and angiogenesis, (14) differentiation of epithelial cells,
(15) caspases and regulation of apoptosis, (16) collagen production and its role in fibrosis, (17)
alveolar clearance mechanisms regulated by alveolar epithelial cells, (18) pathology of pulmonary
hypertension, (19) regulation of thrombin activation and inactivation, (20) molecular determinants in
cell proliferation, and (21) oxidant-activated signaling in endothelial cells. As evident by the detailed
description of the faculty research interests (Table 2), we can provide a wealth of opportunities for
research training in basic lung biology and pathobiology of acute lung injury and inflammation.
C.
Relationship of Lung Research Training Program to Current Training
Activities
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This program integrates 29 investigators into the activities of the training program. To
fulfill its objective of providing multidisciplinary training, the program has breached departmental
barriers. We as a training faculty are committed to continuing the dialogue and cross-fertilization
fostered by the training program so as to enhance the training experience of our trainees. Since the
inception of the program, the multiple faculty interactions developed under the umbrella of this
program have made it into an institutional effort, and have led to even greater research collaborations
among the training faculty members (as described in detail in Table 6).
In terms of relationship of the current training program to research activities, it is evident that
all participating departments have strong faculties and training activities. Thus, this training program
is embedded in a strong academic and research environment of the institution. The Department of
Pharmacology at the University of Illinois has 18 faculty (10 professors, 4 associate professors, and 4
assistant professors) with 28 predoctoral fellows and 20 postdoctoral fellows. The Department of
Biochemistry has 13 faculty (7 professors, 5 associate professors, and 1 assistant professor) with 24
predoctoral fellows and 21 postdoctoral fellows. The Department of Physiology and Biophysics has
22 faculty (12 professors, 8 associate professors, and 2 assistant professors) with 25 predoctoral
students and 27 postdoctoral fellows. The Department of Molecular Genetics has 15 faculty (4
professors, 4 associate professors, and 3 assistant professors) with 49 predoctoral fellows and 21
postdoctoral fellows. The Department of Microbiology and Immunology has 17 faculty (7
professors, 7 associate professors, and 3 assistant professors) with 23 predoctoral fellows and 13
postdoctoral fellows. The Department of Medicine has 124 faculty (24 professors, 45 associate
professors, and 55 assistant professors) and 81 postdoctoral fellows.
Table 3 lists the participating departments, the number of faculty participating in this training
grant, and trainees working specifically in the laboratories of the participating faculty. This list
demonstrates that the training faculty members provide a fertile training environment in their
laboratories that attracts a variety of trainees.
Table 4 lists the current and pending training support available to the faculty in the various
departments. The other institutional training programs in which some of our training faculty
members participate center on the theme of signal transduction mechanisms in the cardiovascular
system and the general area in cellular signaling (listed in Table 4). This program in lung biology
and pathobiology constitutes the only lung research training program in the institution.
5.
Program Plan
A. Program Direction
(a) Program Director
The Program Director, Dr. Asrar B. Malik, is professor and head of the Department of
Pharmacology at The University of Illinois. He will devote 15% of his time to administration of the
training program and has a minimum of 50% effort directed towards his research activities. The
Program Director will direct and coordinate the activities of the research training faculty and the
program as a whole. Among his activities will be to organize weekly seminars and serve as the point
of contact within the pulmonary research group. Other primary functions will be to facilitate faculty
interactions and promote an active and continuous dialogue between trainees and faculty by holding
frequent meetings and group discussions. The director is also responsible for ensuring the proper
functioning of the training quality control mechanisms as outlined in a later section of the proposal.
The research of the Program Director, Dr. A.B. Malik, centers on the biology of the
pulmonary vascular endothelial cell, a field in which he has worked for 25 years and a research area
in which he has been continuously funded by the NIH. He will continue to focus most of his effort to
research- and training- related activities. In addition to administration of this training program, his
previous experiences include directing a training program in lung research entitled “Mechanisms and
Treatment of Pulmonary Insufficiency” initiated by him as Professor of Physiology and Cell Biology
at The Albany Medical College. He served as its Program Director for 12 years (3 rd NIH grant cycle)
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prior to his move to Chicago. Dr. Malik’s research interests cut across the area of pulmonary
vascular biology from molecular aspects to analysis of the integrated control of lung fluid balance.
He addresses research questions from a multidisciplinary viewpoint (the hallmark of the training
program). Dr. Malik’s research focuses on three specific but related areas in pulmonary vascular
biology. The first is the study of the mechanisms that regulate endothelial permeability, both the
paracellular and transcellular pathways. This work involves addressing questions related to the
regulation of paracellular endothelial permeability via cadherins junctions and their associated
proteins, catenins, and the determinants of transcellular pathways as regulated by formation of
vesicles and their vectorial transport across the endothelial barrier. Another research question being
addressed relates to the mechanisms of expression of endothelial adhesion molecules, ICAM-1 and
E-selectin. Studies address the intracellular signals that lead to activation of specific transcription
factors mediating gene expression and regulate expression of these molecules. The third research
area related to the above areas is in defining mechanisms of acute lung injury, in particular, the role
of the neutrophil in the mechanism of vascular and alveolar epithelial injury. These studies address
the factors and pathways that determine migration of neutrophils across the vascular barrier into the
airspace and novel strategies of preventing inappropriate neutrophil adhesion and airspace migration.
(b) Experience in Research Training
Dr. Malik has been involved in institutional research training programs throughout his
academic career. He first served as a training faculty member of the long-standing and successful
General Medical Sciences training program in Shock and Trauma Research directed by Dr. Thomas
M. Saba at The Albany Medical College. He subsequently conceived and directed a NHLBIsupported lung research training program entitled “Mechanisms and Treatment of Pulmonary
Insufficiency” for 12 years at the same institution. The individuals trained in Dr. Malik’s laboratory
in the last 10 years are listed in Table 7. Dr. Malik has trained 8 predoctoral students and 38
postdoctoral fellows. Virtually all of his trainees have remained in research-related careers in
academia or the pharmaceutical industry. Some of the individuals who have trained in his laboratory
include Dr. J. G. N. Garcia, Professor and Head of Pulmonary and Critical Care Division, Johns
Hopkins Medical Center, Dr. Philip Barie, Professor of Surgery, Cornell Medical College, and Dr.
Arnold Johnson, Research Career Investigator, Albany Veterans Administration Hospital and The
Albany Medical College. Currently, there are 2 postdoctoral fellows in his laboratory. Dr. Malik
also currently sponsors through the Minority Investigator Research Supplement awards program
(NIH grants HL45638-10 and HL27016-19), Mr. Raudel Sandoval (a predoctoral fellow) and Dr.
Theresa John (a postdoctoral fellow), respectively. This funding provides Ph.D. training and
postdoctoral training to support these individuals engaged in lung research. Both of them are integral
members of the training program.
(c) Leadership Positions
Dr. Malik has been the head of the Department of Pharmacology at The University of
Illinois College of Medicine in Chicago for 4 years. During this period, he has recruited 6 tenured or
tenure-track faculty members to the department. These individuals (Drs. Du, Colamonici, Ye,
Tiruppathi, Kozasa, and Voyno-Yasenetskaya) have been incorporated into the training activities of
the program because of their backgrounds and the outstanding research skills they bring to the
training activities of the program.
He serves on several key committees of the University including the University Senate and
the Dean’s Executive Committee. He has been a member of several study sections including
Cardiovascular Sciences, Respiratory and Applied Physiology, Lung Biology and Pathobiology, and
Program Project Review Committee A. He has also served as a member of the Pulmonary Disease
Advisory Committee of NHLBI. His other experience on NIH panels includes reviews of Clinical
Sciences Awards, regular ad hoc membership of Surgery, Anesthesiology, and Trauma Study
Section, NIH panels reviewing NHLBI awards for support of research development at minority
institutions, and the Parent Committee reviewing Specialized Centers of Lung Research.
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Dr. Malik has also recently taken on the editorship of American Journal of Physiology (Lung
Cellular and Molecular Physiology). In addition, he serves on the editorial boards of several journals
(e.g., Circulation Research).
(d) Training Grant Administrative Structure
The Internal Training Program Committee consists of Dr. Richard Green, Professor of
Pharmacology Director of Graduate Training, Department of Pharmacology at University of Illinois,
Dr. Heidi Hamm, Department of Pharmacology and Molecular Biology at Northwestern University,
Dr. Lester Lau, Professor of Molecular Genetics and Director of Graduate Program in Molecular
Genetics at University of Illinois, and Dr. Peter Gettins, Professor of Biochemistry and Molecular
Biology and Director of Graduate Training, Department of Biochemistry and Molecular Biology, at
University of Illinois. Thus, this training program interacts closely with the graduate committees of
basic sciences departments involved in the program. This is of fundamental importance in the
recruitment of predoctoral trainees who apply to individual departments represented in the program
(as described below).
The Internal Training Program Committee working with the Program Director is responsible
for the final selection of the predoctoral and postdoctoral fellows admitted into the program as well as
monitoring their progress in the program. As the members of this committee are also on the training
faculty, they interact with the Program Director on a regular basis both at the weekly seminars as well
as regularly scheduled meetings. In addition, the committee meets monthly to discuss the specific
applicants who have been recommended for admission into the training program by the various
Graduate Committees of the departments participating in the program. The administrative aspects of
managing the program are directed by Ms. Barbara Frese, an Administrator of the Department of
Pharmacology at the University of Illinois.
As indicated above, The graduate students entering the training program are recommended by
the Graduate Committee Directors of the departments participating in the program, three of whom,
Drs. Green, Gettins, and Lau, are also members of the Internal Training Program Committee. The
Graduate Committees of the participating departments to which the students apply for formal entry
into the Graduate College act as the initial screening committees for all candidates. At the time of
their application to the Ph.D. program, the Graduate Program Directors of each department
participating in the program advise the applicant about the training program. Specific candidates who
are admitted into the Ph.D. program and express interest in specifically joining the lung research
training program are then recommended for admission into the training program. This
recommendation is made to the Internal Training Program Committee which makes the final decision
regarding admission. All prospective candidates are interviewed by several members of the training
faculty as well as the Program Director prior to being admitted into the program. Thus, there is an
initial review and recommendation by Departmental Graduate Committees (which approves the
admission into the Ph.D. program), and this is followed by screening for admission into the training
program by the Internal Training Program Committee. In contrast, the postdoctoral candidates are
recruited directly into the program through contact with colleagues at other institutions and letters of
interest from prospective candidates.
At least three letters of reference are requested for both predoctoral and postdoctoral
candidates and all final candidates are invited for an interview with members of the training program.
Postdoctoral candidates are also asked to present their research findings in a seminar to which all
members of the program training faculty are invited. Trainees are inducted into the program fully
cognizant of their responsibilities and made aware that this is a specific and differentiated fellowship
program. They are also made aware of their own responsibilities and the need for their participation
in lung research seminars and visits by external consultants and professors sponsored by the training
program.
In addition to the above selection criteria, the training director of two other programs at the
University of Illinois College of Medicine (Dr. Peter Gettins who directs the Training Program in
Signal Transduction and Dr. John Solaro who directs the Training Program in Cellular Signaling in
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the Cardiovascular System) meet frequently to discuss candidates applying to their specific programs
so that the candidates can be directed to the appropriate program. Thus, there is free communication
among the other training Program Directors so as to provide the applying candidates the necessary
information concerning the various available training opportunities.
B.
Program Faculty
(a) List of Program Faculty (Table 1)
(i) General Faculty Attributes
The training faculty consists of 26 individuals with 23 full professors and
associate professors (who are all tenured) with 3 advanced level tenure-track assistant professors.
There are also 3 faculty members who comprise the Resource Faculty. The faculty members have
research strengths in the areas of cell and molecular biology, cell physiology, immunology, and
systems physiology (their research areas are detailed in Table 2).
It is evident from the faculty list that the training program is truly an interdisciplinary one.
There is also significant overlap of the faculty research interests as reflected in the collaborative
research carried out by the training faculty members (indicated in Table 6). Moreover, all faculty
members are active participants in the training program activities such as research seminars and
sharing research core facilities and major instrumentation. All of the faculty members also have
ongoing research support (Table 5) and they are active in research as evident by their recent
publication records (Biographical Sketches).
Three junior faculty members are engaged in research relevant to lung biology, but they are as
yet in the early stages of their career development and are relatively inexperienced in training
predoctoral and postdoctoral fellows. These faculty members will serve as Resource Faculty for the
trainees. Their research areas are detailed in Table 2a and their extramural funding is listed in Table
5a; their collaborations with other members of the training faculty are listed in Table 6. We believe
that the addition of these junior faculty members to the program adds strength and vitality to the
training environment. Since the last submission, junior faculty members who were originally listed
as Resource Faculty have been incorporated as regular faculty upon taking on more training
responsibilities and obtaining additional or independent research funding. Thus, with the
development of their research programs and increased training responsibilities, members of the
Resource Faculty have the possibility of joining the program as regular training faculty members.
All faculty members, except one, have their primary appointments in the College of Medicine
at The University of Illinois; one faculty member, Dr. Heidi Hamm, is a senior professor at
Northwestern University Medical School (a 10 minute drive). Prior to this appointment, she was
affiliated with the Department of Physiology and Biophysics at the University of Illinois and still
maintains daily research collaborations with several members of the training faculty including the
Program Director. She also has a cross-appointment at The University of Illinois. All of the research
ties amongst the members of the training faculty are ongoing, strong, and in many cases interdependent. The training program has been essential in maintaining and fostering these interactions.
The extent of their collaborations is highlighted below and described in detail in Table 6.
From the point of view of institutional geography, the various training faculty members are
close to one another (i.e., in the same buildings or in buildings connected with walkways). This helps
in the frequent exchange that occurs amongst the faculty members on a daily basis. Thus, the
location of their laboratories does not pose any hindrance to frequent communications and exchanges.
(b) Research Opportunities Available for Trainees
Table 2 details the specific research opportunities available in the laboratories of the
Training Faculty; Table 2a indicates research interests of the Resource Faculty. These tables
highlight the specific relevance of faculty research areas to providing research training in areas in
lung biology and pathobiology.
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(c) Thematic Areas of Lung Research Training
The training program can be subdivided into three areas that have been incorporated
into the overall theme of the program. These areas are: (1) Vascular Injury and Lung Repair, (2)
Cellular and Humoral Basis of Lung Injury, and (3) Cellular Signaling and Regulation of Lung
Function.
(i) Vascular Injury and Lung Repair
Research training under this theme is provided in the laboratories of Drs.
Malik, Du, Colamonici, Erdös, Skidgel, Tiruppathi, Lau, Ucker, Schraufnagel, Sznajder, and Varga.
The underlying focus of the research in these laboratories has direct relevance to mechanisms of
vascular injury and lung repair. This group of training faculty members provides training
opportunities in the general area of vascular injury and lung repair from an interdisciplinary
perspective.
Dr. Malik investigates mechanisms of cellular interactions occurring at the level of the
endothelial cell and mechanisms of leukocyte migration across the endothelial barrier. These
interactions have a direct bearing on the mechanisms of vascular endothelial injury. Drs. Erdös and
Skidgel investigate the role of endothelial-specific carboxypeptidases in modulating endothelial
function. Their underlying premise is that these peptidases cleave arginine-containing peptides,
thereby releasing arginine and providing it as a substrate for nitric oxide synthetase; thus, the
induction of carboxypeptidases and their role in NO production has implications in modulating
endothelial injury. Dr. Tiruppathi investigates the mechanisms of loss of endothelial barrier
function by studying how thrombin increases endothelial permeability following activation of
specific signaling pathways. Dr. Colamonici addresses cytokines such as inteferons implicated as
key mediators of endothelial injury, and may under certain instances be involved in repair of lung
tissue. Dr. Du investigates apoptosis in endothelial cells, a factor resulting in detachment of
endothelial cells from the underlying matrix and denudation of vascular lining cells, and which may
also be important in removal of dead endothelial cells after vascular injury. Using similar
approaches, Dr. Ucker investigates role of caspases in the induction of apoptosis; his work on
apoptosis has similar implications in vascular injury as Dr. Du’s studies. Dr. Sznajder investigates
mechanisms of clearance of fluid from the alveolar space by addressing the function of alveolar type
II epithelial cell Na+/K+ ATPases. Dr. Varga investigates the deposition of extracellular matrix
proteins such as collagen in response to proinflammatory cytokines. He addresses its role in the
reorganization of extracellular matrix after lung injury. Dr. Lau investigates role of matrix proteins
in endothelial proliferation and induction of angiogenesis. Dr. Tyner investigates the basis of
epithelial cell differentiation, which is important in understanding the process of epithelial cell
proliferation after lung injury.
(ii) Cellular and Humoral Basis of Lung Injury
Research training under this theme is provided in the laboratories of Drs. Le Breton,
Lam, Ye, Malik, Tiruppathi, Sznajder, Erdös, Skidgel, Gettins, Olson, and Lau. This group of
investigators carries out research addressing the role of cellular and humoral mechanisms of lung
injury. They study the specific cellular and humoral pathways believed to be critical in the
mechanism of lung injury. Each faculty member is involved in the program because of the particular
expertise they bring to the understanding of cellular and humoral basis of lung injury.
Drs. Le Breton and Lam carry out studies in their laboratories concerned with platelet
activation mechanisms; in particular, the regulation of thromboxane receptor and mechanisms and
consequences of integrin signaling in platelets, respectively. Dr. Malik carries out research in his
laboratory on the mechanisms of loss of endothelial barrier function. Dr. Ye studies the mechanisms
of phagocytic cell activation and effects of modulating agents such as NO. Dr. Tiruppathi addresses
mechanisms of endothelial cell activation, a critical cell involved in orchestrating lung’s
inflammatory response. Dr. Sznajder investigates type II alveolar epithelial cell function during
lung injury and the failure of Na+/K+ ATPase-regulated alveolar fluid clearance mechanisms. Dr.
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Erdös and Skidgel investigate the function of carboxypeptidases found on the surface of endothelial
cells and phagocytic cells in modulating in endothelial and phagocytic cell functions. Drs. Gettins
and Olson carry out studies in their laboratories dealing with the mechanisms of activation and
inactivation of thrombin and other serpins. These studies shed light on how thrombin function can be
modified, thereby affecting how it mediates cell injury. Dr. Lau’s laboratory investigates using
transgenic approaches the role of novel matrix proteins released by endothelial cells and other cell
types in promoting angiogenesis.
(iii) Cellular Signaling and Regulation of Lung Function
Research training under this theme is provided in the laboratories of Drs. Ucker,
Raychaudhuri, Hamm, Ye, Kozasa, Rasenick, Malik, Benya, Tiruppathi, Green, and Colamonici.
The third area deals with signaling pathways and how they bear on the mechanism of lung injury.
These investigators provide opportunities for the in-depth analysis of signal transduction pathways,
involving G-proteins, second messengers, and the cytoskeleton in the regulation of function of
specific lung cells, and role of dysregulation of signaling pathways in mediating cellular impairment.
Drs. Du and Ucker study in their laboratories the role of pathways activated by calpain and
caspases in the induction of apoptosis. In contrast Dr. Raychaudhuri investigates the signaling
pathways that lead to activation of retino-blastoma (Rb) factor and induction of cellular proliferation,
an important consequence of repair after lung injury. Drs. Hamm, Ye, Kozasa, and Rasenick
investigate from their perspectives the coupling of receptors with heterotrimeric and monomeric Gproteins and how these interactions mediate activation of cells. This is important for understanding
not only physiological response of cells, but also the basis of dysregulation of cellular function. Dr.
Hamm’s laboratory also investigates interactions of receptors with specific G-proteins with the
objective of selectively interfering with the receptor’s binding to particular G-protein without
affecting the receptor’s interaction with other G-proteins. Dr. Malik studies in his laboratory how
oxidants activate signaling pathways that lead to increased transcription and expression of proteins
such as ICAM-1 in endothelial cells. Drs. Green, Colamonici, Ye, Tiruppathi, and Benya each
study a specific receptor on a surface of cells (adenosine, α-inteferon, FMLP, thrombin, and GRP
receptors, respectively). Their objective is to define the basis of activation of these receptors and
how dysregulation of function of these receptors and the altered signals generated by these receptors
can lead to impaired cell function.
(d) Extent and Evidence of Faculty Collaborations and Interactions
The training program provides a strong interactive environment conducive to the
development of research trainees as independent and creative thinkers and investigators. Although
the interests of some faculty are not primarily in lung research, their participation reflects the
intellectual vitality of the program in being able to provide expertise in important research areas that
impact lung biology and pathobiology. Thus, the interdisciplinary diversity of training faculty
reflects a key strength of the program. The detailed interactions that exist among members of the
training faculty are outlined in Table 6.
The on-going interactions and collaborations of the training faculty is a distinguishing
characteristic of the training environment. We believe that these interactions are a requisite for the
program, and are necessary for the development of young scientists for a variety of reasons. The
cohesiveness of the faculty has enabled the trainees to have available to them “on-call” research
expertise that may be needed to address specific research question. Thus, it is quite common for the
trainees in the program to consult informally with members of the training faculty not only to receive
critical evaluation of the work, but also to seek out other experimental approaches to help them
address a particular research question. Thus, the existing faculty interactions have facilitated an
intensive “trainee-faculty dialogue”, the hallmark of an outstanding program. In addition, the faculty
interactions and collaborations have contributed to the heralded success of the Lung Research
Seminar. These seminars of the lung research group held on Friday mornings and are an anticipated
part of the week’s academic calendar. They provide an exciting and formalized venue for the
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exchange of information and presentation of data by the trainees. This opportunity is important in
allowing the progress of the trainees to be evaluated by members of the training faculty, who fully
participate in these seminars. Other formalized faculty interactions occur at the level of the regular
meetings of Ph.D. Advisory Committees of the predoctoral fellows supported by the training
program. As members of these Ph.D. Advisory Committees are also members of the training faculty,
this enables members of the training faculty to interact and exchange ideas with each other and the
trainees.
(e) Past and Current Training Record of Faculty (detailed in Table 7)
All of the regular faculty members have served as preceptors, and thus are experienced
at taking on this responsibility. Even though Table 7 lists the training record for the past 10 years, it
is apparent that most former trainees are occupied in productive research careers. Table 7a lists the
honors and awards received by many of the recent trainees working in the laboratories of the training
faculty members. It is evident that the recent and current trainees working under the various
members of the training faculty have been successful during their research training phase. This
portends well for their future careers.
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Proposed Training
(a) Overall Aspects of the Program
We are seeking continuation of this program that trains highly qualified predoctoral
and postdoctoral fellows in specific research areas of pulmonary biology and pathobiology as related
to acute lung injury and inflammation. The training program emphasizes an in-depth and intensive
research experience at the multidisciplinary level. The training faculty members have been integrated
into the training program on the basis of the underlying philosophy of providing multidisciplinary
training. The training encompasses all aspects of the research enterprise from formulation of the
hypothesis, obtaining preliminary data in support of the hypothesis, the design and conduct of further
experiments, and ultimately to submission of the research manuscript and its publications. Advanced
level graduate courses in areas relevant to lung biology such as in receptors and signaling and lung
and vascular biology are provided by the participating basic science departments, which include the
Departments of Physiology and Biophysics, Biochemistry and Molecular Biology, Molecular
Genetics, Pharmacology, and Microbiology and Immunology. The Ph.D. degree requirements for the
trainees in the program are listed in Table 8. The advanced level required courses and those courses
relevant to research being carried out by trainees supported by this program are described in Table
8a. The specific course requirements for the trainees in the program are also described below.
Postdoctoral fellows also have the opportunity to take these courses, but only on an audit basis.
All trainees also participate in three weekly scheduled seminars (two scheduled at noon on
Wednesdays and Fridays and one (Lung Research Seminar) scheduled at 8:30 A.M. on Friday to
minimize interference with their research schedules). These are (i) Lung Research Seminar on Friday
at 8:30 A.M., attended by lung research trainees and the training faculty; (ii) Graduate Student
Seminar (Wednesdays at noon), which are formal seminars presented by all graduate students; and
(iii) Visiting Professor Seminar given by distinguished experts are held at noon on Fridays (listed in
Table 8c). These three weekly sessions are an important component of the training program and
attendance is a requirement for all trainees.
(i) Differentiation of Trainees in the Program
It is important to note that the cohort of predoctoral and postdoctoral trainees are
treated as a distinct group. The first distinction is that they share common experiences in terms of
seminars, presentations, and meetings with visiting scientist organized and supported by the program.
Second, the trainees share common resources such as the Cell Culture Core and the Imaging Core
(described in the Resources and Environment Section on pages 68-70). In addition, the trainees
supported by the program are differentiated by the fact that they are addressing research questions in
areas with specific relevance to lung biology and pathobiology. The trainees also have available to
them a lounge with study carrels and computers. To enhance program cohesiveness, in the last 2
years, we have had a one and half day retreat for the trainees and faculty members (at Lake Geneva,
Wisconsin) where posters are presented and talks are given by both trainees and faculty. Finally, all
faculty and trainees are connected by an e-mail server list which provides direct linkage to all
participants in the program. The specific course requirements for the trainees in the program are
described below, which is a key element of differentiating the program. Finally, the trainees are
distinct from other trainees who are not a part of the program because the intent is to provide a true
sense of fellowship amongst the trainees and faculty.
(ii) Number of Trainees
In the last cycle, we were approved for 4 predoctoral and 4 postdoctoral fellows per
year since this was a number that could be accommodated by the faculty; however, due to NIH
budget constraints, the number of positions was reduced to 3 predoctoral and 3 postdoctoral fellows.
Although the reduction has not affected the quality of the program, it has prevented us from having
an institutionally broad-based program as we initially envisioned. Nevertheless, the program has
thrived and the quality and size of the faculty have grown. Thus, we believe that we can easily
accommodate 5 predoctoral and 5 postdoctoral fellows per year on a basis of the 26 training faculty
members and 3 resource faculty members. Moreover, we have more than enough highly qualified
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predoctoral and postdoctoral fellowship candidates to fill 5 predoctoral and 5 postdoctoral positions.
The number of applications made to participating units of the training program are listed in Table 9.
Tables 9b and 9c lists the number of candidates expressing interest in admission into the training
program since the beginning of the program. In 1998-1999, we had 29 Ph.D. predoctoral and 65
postdoctoral applicants with superb credentials.
The proposed size of the faculty matches well the number of outstanding candidates applying
to the program as well as the size of the faculty. The ratio of training faculty-to-predoctoral fellows
and-postdoctoral fellows is 5 in each case. It is clear that the trainees will have enough flexibility in
choosing preceptors and their specific research projects. Thus, we are requesting an increase in the
number of predoctoral and postdoctoral trainees by 1 each over the previously recommended number
of 4 in each category because of not only the training faculty size and strength but also the cadre of
strong applicants to the program.
(iii) Duration of Research Training
The duration of training will depend on factors such as the trainee’s career goal and
unquantifiable issues such as the difficulty of the research questions under investigation. Predoctoral
fellows will be supported by the training program for 3 years. Beyond the 3 years, it is expected that
the support be derived from funds of the preceptor.
Postdoctoral trainees will be supported for 2 years. Our general experience has been that this
represents an optimal period for support of postdoctoral trainees in the program. In the event that the
training period needs to be extended to the third year because of a specific research area, there will be
a contingency for this. Each case will be evaluated by the Internal Training Program Committee.
The postdoctoral trainees will be encouraged in specific cases to apply for outside support such as
Parker B. Francis Foundation Fellowship. One of the 6 postdoctoral trainees recruited into the
program has received a fellowship by this mechanism. However, in most instances we have felt that
it is necessary to have a continuity of fellowship support by the training program mechanism for at
least 2 years so that the trainees are committed to the program and its approach, and are able to
receive training under the aegis of the program.
(iv) Trainee Participation in Research
The intent of the program is to provide “hands-on” intensive research training; thus,
the research training component is the quintessential element of the program. Courses (listed in
Table 8a) are available for postdoctoral fellows on an audit basis to remedy weaknesses in specific
areas as may be identified by the trainee or Internal Training Program Committee.
Although research is the key element of Ph.D. training, the doctoral students have to meet the
set Graduate College requirements (Table 8). The first year the Ph.D. trainee has a number of
courses, but this is also the beginning of the research phase of training. The second and third years
have fewer course requirements for trainees and a significantly greater amount of research.
Predoctoral fellows, thereafter, engage in full-time research. The usual experience has been that in
the first year 50% of the time is devoted to research training which increases to 75% in year 02, 90%
in year 03, and thereafter 100%. Postdoctoral trainees are involved in full-time research from the
outset.
(v) Availability of Graduate Courses and Course Requirements
Graduate level courses are available for the predoctoral fellows so that they can meet
the Ph.D. degree requirements. The predoctoral fellows are required to take Core courses which
include physiology and biophysics, biochemistry, and a course in pharmacology for pharmacology
graduate students or a course in immunology for immunology and microbiology graduate students.
The specific Ph.D. degree requirements are listed in Table 8.
The specific course requirements for Ph.D. trainees in this program are the 500 level courses
in Molecular Biology offered by the Department of Molecular Genetics (BIOS 524) and the Lung and
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Vascular Biology (PCOL 530) offered by the Department of Pharmacology. In addition, the students
are required to take 3 other 500 level courses listed in Table 8a (i.e., courses that would be most
pertinent to their research area). This is to be done in consultation with their advisor and approval of
the Internal Training Program Committee. Finally, all trainees (predoctoral and postdoctoral) are
required to take the course in the responsible conduct of research (Table 8b). We feel that these
courses for Ph.D. students are needed to build the basic foundations of a research career.
In contrast to the predoctoral trainees, the laboratory phase for the postdoctoral fellows is
upon entry into the program. The postdoctoral trainee may have the option of auditing advanced
level courses in areas relevant to the research project of the trainee (the relevant courses are listed in
Table 8). This will be decided in consultation with the advisor, Program Director, and Internal
Training Program Committee. A course can only be audited if it does not interfere with laboratory
research of the postdoctoral trainee. We generally will not encourage course work after the first year
since postdoctoral fellows are expected to concentrate full-time on the research projects during this
period.
(vi) Field in Which Trainees Qualify Upon Completion of Training
A common career path for the Ph.D. postdoctoral trainee is an academic position in a
basic science or clinical department, a position in the biotechnology industry, or a government
research laboratory such as NIH. Our recent experience (albeit of only 4 years) has been that the
biotechnology industry offers very competitive and intellectually stimulating research positions with
outstanding starting packages and with opportunities for independent research; thus, these have
become highly desirable for some recent trainees. The general experience of the former trainees
employed in biotechnology firms is that they provide a challenging research environment and a
considerable degree of independence along the lines of company’s research focus. Thus, in the
rapidly changing market for postdoctoral trainees, it is quite apparent that research positions in
private industry have become a desirable career objective.
Other individuals, however, gravitate towards academia with positions in either basic science
or clinical departments. Positions in clinical departments have become particularly attractive for
postdoctoral trainees because they present opportunities for interactions with physician-scientists;
moreover, for the qualified individuals, these are independent tenure-track positions. This program
because of its multidisciplinary and integrated approach to training benefits those individuals
planning independent research careers in academic departments or government and research institute
laboratories, as well as those entrepreneurial individuals choosing the biotechnology industry. For
the doctoral candidate completing the Ph.D. degree, the primary option is postdoctoral training in a
recognized laboratory in either an academic institution, NIH, or a research institute.
Although this program is relatively new, the postdoctoral trainees have all found challenging
positions in academia (both basic and clinical departments) and in pharmaceutic/biotechnology
industry. The predoctoral trainees have also found outstanding postdoctoral positions. This
information is provided in Table 13 and discussed in the Progress Report on page 93. The
Publication records and the various achievements of former trainees in the program are provided in
Tables 14 and 14a, respectively.
(b) Program Integration and Coordination
(i) Program Director’s Role
The major responsibilities of the Program Director are in guiding the program and
providing the overall integration to the training activities of the program. The integration and
coordination require regular and frequent meetings with the trainees, training faculty, and the
Advisory Committees. The Program Director chairs the Friday morning Lung Research Seminar and
is responsible (in consultation with the training faculty) for organizing visits of professors and
consultants for the noon seminars.
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A key mechanism by which the Program Director integrates the training program activities is
by bringing together the faculty at seminars and other regularly scheduled events. These group
meetings are a vital element of the training process since they provide opportunities for the faculty to
gauge trainees’ progress in research and they help to build an esprit de corps amongst both trainees
and faculty members. Feedback and evaluations are provided by e-mailing an evaluation form to the
faculty for their comments directed to the trainees. In addition, informal feedback is provided after
the session by the Program Director as well as the advisor, and, often, by individual faculty members.
The Program Director also deals with training-related issues in consultation with the Internal
Training Program Committee. A part-time administrative secretary facilitates all of the coordination
of the program by scheduling visiting professors/consultants, informal research meetings among
members of training faculty, filling out NIH and institutional appointment forms, dealing with
administrative matters relating to trainees (e.g. health insurance), assisting in organization of
scheduled weekly research meetings, and typing of progress reports.
Another critical aspect of the Program Director’s role in coordination of the program is his
active and direct participation in the recruitment of trainees (described below). This involves
working with the training faculty to establish outreach programs to various regional undergraduate
institutions (e.g., Knox College, Oberlin, etc) as well as minority institutions in order to attract
trainees into the program.
(ii) Role of Training Faculty
The intellectual vitality of the training program, and its ultimate success, rest in large
measure on the quality and dedication of the faculty. We feel the program is well balanced in this
regard because a talented and committed training faculty has been assembled under this umbrella of
this program.
The faculty has been selected on the basis of several quantifiable criteria: (i) experience as
mentors (as outlined in Table 7), (ii) areas of research and disciplines with relevance to pulmonary
biology and pathobiology (as outlined in Table 2), (iii) commitment to work with one another (as
outlined in Table 6), (iv) a resolute commitment to provide the multidisciplinary underpinnings to the
research training program, and (v) independent research support (as outlined in Table 5).
For the purposes of coordination and integration of the program, the faculty meets weekly at
research seminars organized by the training program: the Lung Research Seminar on Friday
mornings and the Visiting Professor Seminar at noon. In addition, the faculty meets in small group
sessions when research findings of fellows are discussed and critique is provided (such as the
frequent meetings of the Thesis Advisory Committee, for the predoctoral fellows supported by the
training program). All faculty members are active participants in these weekly seminars and at the
various research sessions such as Ph.D. Student Advisory Committees. These meetings are essential
in providing input concerning each trainee’s research and assessing their general progress in the
development as an investigator. The faculty also has the opportunity of influencing the program
through the Internal Training Advisory Committee as described below. Finally, the faculty members
also meet once a year with the trainees at the retreat to discuss research and to coordinate the various
training issues.
(iii) Roles of Advisory Committees
(I) Internal Training Program Committee
The Internal Training Program Committee functions to (1) screen finalist Ph.D. and
postdoctoral trainees for admission into program (2) evaluate progress of fellows, (3) discuss any
changes in direction of program, and (4) discuss issues relating to organization and management of
training program. The members of this committee are Drs. Richard Green, Heidi Hamm, Lester Lau,
Peter Gettins, and Asrar Malik
(II) External Advisory Committee
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The External Advisory Committee will consist of Dr. Michael Shasby, Professor of
Medicine, University of Iowa, Dr. Michael Matthay, Professor Medicine, University of California at
San Francisco, and Dr. Joe (Skip) Garcia, Professor of Medicine, and Head Pulmonary and Critical
Care Division, Johns Hopkins University. This committee will provide a regular review of activities
of the training program, deal with concerns of the training faculty, and help to identify potential
trainees. They will also provide a measure of the trainees in this program relative to the research
trainees at their home institutions. Other issues to be dealt with by this committee in conjunction
with Program Director include trainee performance as carried out by annual evaluation of the
progress of trainees in the program. Each of these individuals has visited the training program and
consulted with the Program Director in the previous cycle.
(iv) Basic Science and Clinical Integrations
An important strength of the program is the strong linkage between the clinical and
basic science training faculty members. These basic science and clinical interactions are an integral
component of the training program. They are facilitated by having as members of the training
faculty, individuals whose primary appointments are in clinical departments: Drs. Dean
Schraufnagel, Richard Benya, Donovan Yeates, Jacob Sznajder, and John Varga. Moreover, both Dr.
Benya and Dr. Sznajder have cross-appointments in the Department of Pharmacology. In addition,
Dr. Benya directs the Confocal Core facility at the Veterans Administration Hospital available to
training program fellows. There are also several members in the basic science faculty with either
M.D./Ph.D. or M.D. degrees: Drs. Oscar Colamonici, Ervin Erdös, Xiaoping Du, Tatyana VoynoYasenetskaya, and Shigehiro Nakajima. Thus, there are ample opportunities for basic scienceclinical integration through the daily interactions of physician-scientists and basic science training
faculty members. These individuals not only serve as role models, but also provide a clinical
prospective to the basic science trainees. As the trainees are exposed to training faculty members
with an integrated perspective of their research areas, this further points to the training approach as
being truly multidisciplinary.
We believe that these clinical interactions are of fundamental benefit to the predoctoral and
postdoctoral trainees. For example, trainees may not easily recognize the relevance of a particular
research question; however, because they are exposed to issues first-hand through their contacts and
discussions with the physician-scientist members of the training faculty, the trainees are able to
integrate their bench research observations to the physiological and clinical relevance of their
research question. This continuous dialogue has been an integral aspect of the training process.
Although the research questions of the trainees are focused and targeted, these interactions are
essential in providing the trainees with a broader and well-rounded perspective of the problem; thus
they go beyond viewing the question only at the reductionist level.
(v) Coordination in Selection of Research Projects
(I) For Postdoctoral Fellows
Postdoctoral fellows inducted into the program apply to a particular individual in the training
program, or are attracted to the program itself, and thus they have preconceived notions of the
specific research area they intend to pursue. However, if the selection of the research advisor has not
been made during the first two months in the program, a committee consisting of the Program
Director and Internal Training Program Committee assists in this process. The intent is to match
optimally the research training experience desired by the fellow to the research interests of the
faculty.
Also during the first two months, all postdoctoral trainees are required to present their
proposed research program under headings of specific aims, background, rationale, and methods of
their proposed research. They are guided in this exercise by their selected advisor and other training
program faculty convened on the basis of the closeness of their research areas to the research interests
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of the trainee. This allows for a more focused definition of the hypothesis and experimental design.
In addition, we ask all our incoming postdoctoral trainees, within a 2 to 3 month period of having
selected an advisor, to present at the Lung Research Seminar the ideas that will form the basis of their
work and preliminary supporting data. The intent of this exercise is to outline the hypothesis and
present preliminary data in support of the hypothesis. Our experience indicates that this period of
“acclimatization” assists the trainees in developing a more focused and directed approach to their
future studies. During this initial period, the fellows are advised about the planned experimental
approaches and methodologies available to them within the program as well as the necessary
equipment and other resources needed to conduct their studies.
(II) For Predoctoral Fellows
After their selection into the training program, the Ph.D. students meet with the Internal
Training Program Committee to discuss research areas and interests. The committee advises them
about the particular training faculty members with matching interests. The predoctoral students then
rotate through the laboratories (usually 4-5 laboratories) of these training faculty for 6 months after
admission into the program. They make their choice of an advisor at the end of this period on the
basis of research interests and the particular interactions that they have had during the laboratory
rotation period. The selection of the research project and preceptor is made with the approval of the
Internal Training Program Committee. Following this selection period, the Thesis Advisory
Committee members are chosen by the Ph.D. student in consultation with the Ph.D. advisor. The
Ph.D. Advisory Committee then meets at intervals of 2-3 months during the entire training period of
the Ph.D. student. A year to 18 months prior to the anticipated graduation date, the Ph.D. student is
required to submit the Preliminary Ph.D. Thesis which outlines the hypothesis, presents the detailed
supporting data, and outlines the series of experiments to be carried out for completion of the thesis,
and subsequently for the defense of the thesis in an in viva examination.
(vi) Coordination in Selection of Preceptors
Most prospective postdoctoral trainees entering the program are aware of the research
areas that they wish to pursue, and thus have already directed their attention to their primary advisor.
In those instances where a postdoctoral trainee has not made a choice, a period of laboratory rotations
(usually to several laboratories) is allowed to obtain a better sense of the available training
opportunities. Usually, 2 months are allowed for rotation; thereafter, it is expected that the advisor be
identified.
In some instances, the postdoctoral trainee has the option of selecting more than one
preceptor. This has been a valuable modus operandi in this program because it allows more than one
way of viewing a particular research problem and provides opportunities for the trainees to address
research questions with wider array of experimental strategies and methodologies than would be
otherwise possible. This flexibility enhances the quality of the program because it enables the
fellows to cross disciplines and to receive cross-training on the basis of research questions being
addressed rather than there being departmental or laboratory barriers to training. This goal of
interdisciplinary research training is realized within this program because of the many close
collaborations amongst the training faculty members and the cooperative attitudes fostered within the
program.
The selection of the preceptor for the Ph.D. student varies from that of the postdoctoral
trainee. The Ph.D. students have a distinct and well-defined 6-month period of laboratory rotations
after which they are required to select their primary research advisor. The Internal Training Program
Committee along with the Program Director approves the advisor and the laboratory as the primary
site of Ph.D. training. The Ph.D. student also has the opportunity to interact with other members of
the training faculty through the Ph.D. Advisory Committee; within 6 months of selecting a primary
advisor, the Ph.D. student selects the Ph.D. Advisory Committee which incorporates members of the
training faculty into the overall training structure of the Ph.D. student. Members of the Ph.D.
Advisory Committee of the student (who are also members of the training faculty) have a critical role
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in advising the Ph.D. student along with the primary advisor during the student’s entire training
period. Individuals comprising the Ph.D. Advisory Committee are selected on the basis of their
research interests, which enables them to participate effectively in the research training of the Ph.D.
student.
(vii) Research Performance and Evaluation: Quality Control Measures
(I) Research Presentations at Weekly Meetings
The Lung Research Seminar on Fridays at 8:30 A.M. in which trainees present their
findings and which are interspersed with seminars given by training faculty and visiting professors
provide an important opportunity to evaluate trainee progress. A trainee is afforded typically
opportunities for three seminars during the academic year; thus, this enables an ongoing and
continuous review of the research progress being made by the trainee.
In addition to these research-in-progress seminars, there are the formal noon seminars in
which leaders in the field are invited to the institution (Table 8c list the individuals who have
participated as visiting professors and consultants).
(II) External Consultants/Visiting Professors
The training program organizes a series of Visiting Professorships and Consultant
sessions during the academic year (Table 8c). These individuals are invited to review the research
and serve in an advisory capacity to the Program Director and program. The consultants’ visits are
usually for one and a half days during which the fellows present their work often on a one-to-one
basis. Sufficient time is allocated for the fellows’ presentations and for feedback and critique.
Important benefits accrue to both trainees and we believe to the training faculty from these
visits. The consultants/ visiting professors are important in enhancing the training process as they
critically and objectively review the research of the trainees and provide insightful feedback from an
objective perspective. Their evaluations are also relayed to the Program Director who then shares
them with trainees and the Internal Training Program Committee.
(III) Presentation of Other Weekly Seminars
In addition to the weekly Lung Research Seminar and the regularly scheduled visits by
external consultants/visiting professors, there are also weekly Trainee Research Colloquia held in the
basic science departments participating in the program. Ph.D. students and post-doctoral fellows
present their research at these meetings. These are more generally attended, and thus provide a
broader exposure of the trainees’ research findings. Although these seminars are more structured
than Lung Research Seminar, they serve as important means for monitoring progress of the trainees
in the program.
(IV) Presentations at National Meetings and Preparation of Manuscripts
An important goal of the training program is to guide trainees in presenting their
research findings at national meetings (e.g., Experimental Biology Meetings, American Thoracic
Society Meetings, or Gordon Conference, etc.). This involves members of the training faculty
providing critiques of oral presentations and poster presentations for content and style (e.g., quality of
graphics). This is done at the Friday morning Lung Research Seminar one to two weeks before the
national meeting. Although the primary responsibility for publication and critiquing lies with the
preceptors, other members of the training faculty, with particular interests in the research project also
participate in the process. The various faculty members are also often asked to review manuscripts
by trainees. It is our experience that these critiques invariably lead to improvements in the trainees’
communication and writing skills.
(V) Presentations at Annual Retreat
All trainees are also asked to present their research at the annual retreat held at Lake
Geneva, Wisconsin in mid-September, near the beginning of the academic year. This provides an
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opportunity for the trainees to be evaluated and also for the trainees to learn about the research
activities of the faculty.
(VI) Role of Advisory Committees
The Internal Training Program Committee is significantly involved in monitoring
trainee performance during the various presentations given by the trainees at the research seminars
described above. In addition, the committee is kept apprised of the trainee’s progress by regular
contacts with the trainee and by the frequent meetings organized by the Program Director. The basic
question addressed by the Internal Training Program Committee with respect to trainee evaluation is
whether the fellows are achieving their goals under the guidance of their advisors. Feedback by this
committee is provided to the Program Director during the meetings held with the Internal Training
Program Committee.
Any decision to terminate a fellow is made by the Internal Training Program Committee and
Program Director. Specific and persistent concerns will result in such an issue being discussed and a
letter being written by the Program Director to the concerned individual. Although this policy is in
effect, there have been no such issues raised about any of our trainees.
(VII) Role of Program Director
The Program Director has a critical role in evaluating the research performance of
trainees during the multiple opportunities available to him. These include the Friday morning Lung
Research Seminar and the graduate student colloquia, held on Wednesdays at noon, as well as
evaluations by external consultants/visiting professors. The Program Director also directly consults
with the preceptors concerning developments being made by the trainees in the program.
The Program Director provides the feedback whenever appropriate and deemed necessary.
The close working relationships between members of the training faculty and Program Director are
key to the director playing an essential role in evaluation of the trainees’ progress.
D. Trainee Candidates
(a) Overall Qualifications of Prospective Trainees
It is the policy of the program to recruit those candidates with high academic
performance and significant potential for a research career. The predoctoral trainees are required to
have undergraduate degrees in biology, chemistry and biophysics, molecular biology, or related
disciplines. All postdoctoral candidates are required to present a seminar of their previous research
work (this does not apply to the M.D. candidates without prior research experience).
Academic grades from graduate school and medical school are requested from all candidates.
The prospective Ph.D. trainees are required to submit scores from their college and the Graduate
Record Examination (GRE). All predoctoral and postdoctoral candidates are asked to submit letters
of reference or provide a list of references.
(b) Specific Criteria and Procedures for Selection of Trainees
The training positions are advertised in widely distributed scientific journals.
Postdoctoral candidates are also identified at annual meetings of scientific societies such as
Experimental Biology and American Society of Cell Biology. Postdoctoral candidates are also
considered on the basis of recommendations from colleagues at other institutions. Predoctoral
candidates apply to The University of Illinois and to the basic departments participating in the
training program. They are informed about the program, if they are not already aware of it. They are
recommended for admission by the Graduate Committee of the participating departments on the basis
of the criteria established by the departments. The names of those candidates expressing an interest
in the program are then forwarded to the Internal Training Program Committee of the program, which
has 3 Graduate Program Directors (Drs. Lau, Gettins, and Green) as well as Drs. Hamm and Malik.
This committee then does the final screening in admitting the Ph.D. candidate into this program. The
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decision to admit is based on the candidates research interests and aptitude, scholarly activity,
recommendations, and interview with members of the training faculty.
Table 9 lists the predoctoral trainee recruitment data of the basic science departments
participating in the training program. As is evident from this list, all departments connected with the
program have a large applicant pool, and only a small fraction of the outstanding applicants are
considered for admission into the department Ph.D. programs, and after further screening the number
entering the Ph.D. programs is yet smaller. Thus, the Ph.D. programs of all departments involved
with the training program are highly selective.
Table 9a provides further details about the applicants who were eligible for the program.
These individuals received their undergraduate education from a spectrum of institutions reflecting
the national visibility of the predoctoral training programs of the University of Illinois College of
Medicine. The criteria for admission into the program are described below. They are based not only
on grades and GRE scores but also on personal interviews, letters of recommendation, previous
research experience, future goals, and aptitude for research.
We have been successful in recruiting a highly qualified group of candidates into the training
program without regard to race, color, creed, or sex. Our experience to date is that we have a
substantially larger pool of both qualified predoctoral and postdoctoral candidates (Table 9b and 9c)
than the available positions. Tables 9b and 9c list the pre-doctoral and post-doctoral candidates
expressing interest in the lung research training program. Regrettably, many of the interested and
potentially acceptable outstanding candidates cannot be accepted.
In 1998-1999, there were 29 finalist predoctoral Ph.D. trainee candidates; that is, those
individuals with expressed interest in lung research training who were recommended by the
Departmental Graduate Committees for possible admission into the training program (Table 9b). In
1998-1999, there were 65 postdoctoral candidates (Table 9c) for the available positions in the
training program. The Internal Training Program Committee further narrowed down the list of
postdoctoral candidates to the 16 individuals listed in Table 9d.
Prior to inviting predoctoral or postdoctoral candidates for interview, members of the Internal
Training Program Committee as well as the Program Director screen all applicants and select those to
be invited. All travel and accommodation expenses for the short-listed candidates are covered. The
selection criteria are: (1) strong academic background reflected in grades and previous research
experience, if the candidate has a Ph.D. or M.D. degree; (2) strong motivation for and commitment to
a research career; (3) strong references from individuals knowledgeable about the candidate (e.g.,
departmental chairperson, previous mentor, and other faculty members from the candidate’s
institution); and (4) future goals and commitment to devote full-time effort to research training in this
program.
If any of the prospective trainees have had previous research experience, they are asked to
present their research findings at a seminar. The interviews are scheduled so that the candidates have
an opportunity to meet many members of the training faculty, establish a sense of our expectations,
and for the faculty to evaluate the candidates at first-hand prior to admission into the program.
(c) Sources of Availability of Trainees
The training faculty as a group has a wide network of colleagues, an essential
component of a successful recruitment process. The training program also publicizes itself by
advertisements in scientific journals as well as through placement services of scientific organizations
such as Experimental Biology and American Society of Cell Biology. Postdoctoral trainees are often
attracted to the program because of the research being carried by a faculty member, the quality and
reputation of the training faculty members as a whole, and the growing visibility of the training
program itself. The Ph.D. students are attracted to the program because of the training faculty and
the visibility of the graduate programs of the University of Illinois academic departments represented
in the training program.
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In the coming year, we will further address this issue by establishing outreach programs to
various outstanding undergraduate institutions in the Great Lakes area and the upper Mid-west. The
Program Director and members of the training faculty will make presentations in Departments of
Biology of these institutions with hope of further increasing our applicant pool, and attracting some
of these students into the program.
3. Recruitment of Under-Represented Minorities
A. Goals and Programs
The training program remains actively committed to providing opportunities for
training of candidates of under-represented minorities. We actively participate in programs designed
by The University of Illinois Graduate College to attract undergraduate minority students to graduate
programs. Members of the Graduate College and the Office of Vice Chancellor for Research have
established direct contacts with a number of minority institutions. These include Xavier University,
Morgan State University, Jackson State University, and Prairie View University all of which have
excellent academic programs. In addition, senior members of the faculty at The University of Illinois
College of Medicine such as Dr. Richard Davidson, Professor and Head of Molecular Genetics, has
visited Howard University, Morgan State University, and Xavier University to establish good
working relationships that we believe will be important in recruiting their best students. A summer
internship program has also been established to provide research opportunities for undergraduate
students from Howard University and Morgan State University. This ten-week summer program has
allowed 14 students to work in various laboratories at The University of Illinois College of Medicine.
The Committee on Institutional Cooperation (CIC), the academic consortium of the Big Ten
Universities and The University of Chicago, has designed a Summer Research Opportunity Program
(SROP). The goal of SROP is to introduce talented minority sophomores and juniors, early in their
undergraduate education, to research and rewards of graduate study. Working with faculty members,
some of whom also are on the training faculty, we hope to attract minority trainees on this basis. The
goal of this project is to identify potential graduate school applicants within the consortium and share
their names with member institutions, and ultimately influence their eventual graduate and academic
career choices.
Another program involves students from community colleges. The Graduate College of the
University of Illinois has received funding from the U.S. Department of Education through a grant
application to the Minority Participation in Graduate Education Programs to provide research
experience for talented students from community colleges. These students are selected to participate
in an 8-week program under the supervision of University of Illinois faculty members the summer
prior to transferring from their associate degree program at the community college to the
baccalaureate program at The University of Illinois. The intent of this program is to enable some of
the top students to apply eventually to graduate programs leading to the doctorate degree.
The institution also participates in an outreach program to bring minority high school students
into the laboratory with the hope that they become interested in a research career following their
graduation. An example of such a program for high school students is that from the Illinois
Mathematics and Science Academy (IMSA), a public residential high school for talented science and
mathematics students. Students from institutions such as IMSA and others in the area have
demonstrated high academic abilities and potential. Students under this mentorship program
participate in research during the summer months with investigators in the training program. These
students are assigned mentors during the summer and they participate in seminars and research
discussions of the lung biology training program. Although no grade is assigned, students receive an
appropriate note in their transcripts indicating their participation in this research program. Many of
these students have returned to their mentor’s laboratory in successive summers prior to enrolling in
an undergraduate degree-granting institution. We hope in the future to be able recruit back some of
these outstanding students into the training programs under the auspices of this training grant.
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In addition to the strategies indicated above, in the future, the program intends to play a more
active role in advertising at specific minority undergraduate institutions (as described below under the
heading “Direct Involvement of Program Director in Minority Recruitment”). We will also continue
to advertise this program on the GRE Minority Placement Service, Minority Access to Research
Careers training programs, and Deans of Honors Programs in the National Collegiate Council.
We have been successful in recruiting two minority trainees in lung research through contacts
with colleagues at other institutions and within the institution. Two trainees, Dr. Theresa John and
Mr. Raudel Sandoval, are receiving training in lung research as other the other trainees in the in the
program. Because of the cutbacks in the original recommended budget of the program from 4
postdoctoral fellows and 4 predoctoral trainees to 3 in each category, Dr. John and Mr. Sandoval are
being supported by supplements to ongoing NIH grants in lung research (these trainees are described
below.)
B. Fellowship Opportunities Supported by The University of Illinois
The Graduate College of The University of Illinois is committed to increasing the racial
diversity of the graduate student population. We are well aware of the dramatic decrease in the
number of African-American and Hispanic graduate science students (greater than 20% drop between
1996 and 1997 according to a report released by the American Association for Advancement of
Science, “Losing Ground: Science and Engineering Graduate Education of Black and Hispanic
Americans”, AAAS publications, Washington D.C.). The University of Illinois by its University
Fellowship Stipend program provides stipends as well as waives tuition for many eligible underrepresented minority students entering the Ph.D. program.
C. Retention of Minority Trainees
It is the intent of the training program, and indeed that of the institution, to improve
the retention of minority students. The Graduate College along with relevant academic departments
regularly monitors the academic progress of all students. The minority students experiencing
academic difficulty are invited to meet the Graduate College staff to review and assess their progress,
and subsequent referrals are made to appropriate support services. Continuation of fellowship awards
is contingent upon the fellow’s academic progress towards the completion of the degree. The
Graduate College regularly verifies the satisfactory academic progress of externally supported
fellows and formulates reports to the appropriate funding agencies. These reviews are held in concert
with the student’s academic unit.
A minority graduate student organization at The University of Illinois, Helping Other People
Excel (HOPE), also provides academic and non-academic support for minority graduate students.
HOPE focuses on the promotion of intellectual growth and fellowship among students, faculty, and
university administrators. To this end, HOPE sponsors regular events, such as orientation and brown
bag lunches where speakers discuss topics important to professional and academic development, and
social events are held for potential minority graduate students. In addition, the Urban Health
Program at the University of Illinois at Chicago was established to increase the number of minorities
in health professions. Although a main goal of this program is recruitment, the program also
emphasizes retention, financial aid assistance, academic and personal counseling, and early outreach.
The program functions in seven different colleges of the University, including the College of
Medicine.
D. Direct Involvement of Program Director in Minority Recruitment
The Program Director will be directly involved in minority student recruitment. He
will organize meetings with (i) minority students majoring in biology, chemistry, and physics in the
Colleges of Liberal Arts and Sciences at both at the Chicago and Urbana campuses, (ii) minority
students enrolled in medicine at the Chicago campus (approximately 15% of our medical school class
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in the first year enrolled under the auspices of the Urban Health Program are minority students), and
(iii) minority students in biomedical engineering programs at both the Chicago and Urbana campuses.
Any student expressing an interest in the program with be invited for meetings and interviews with
members of the training faculty as well as trainees in the program. The Program Director will also
seek out and visit undergraduate advisors and students at the traditionally African-American
institutions of higher learning (such as the ones mentioned above) as well as institutions with large
Hispanic undergraduate populations. It is our hope both of the above strategies to be spear-headed by
the Program Director as well as the other aforementioned will increase the minority applicant pool.
Table 10 list the minority predoctoral and postdoctoral fellowship recruitment data for the
Department of Pharmacology; this table reflects the general experience of all other academic
departments connected with the training program. Our experience has been that all minority
candidates accepted to the various predoctoral and postdoctoral positions are outstanding and they are
eligible to receive support from via a variety of mechanisms including University Fellowships, and
NIH grants. We hope to increase the number of applicants using the strategies described above and
to enroll them into this training program.
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E. Successes in Recruitment of Under-Represented Minority Trainees in Lung
Research
We have been successful thus far in recruiting a minority graduate student and a postdoctoral fellow in lung research, Mr. Raudel Sandoval and Dr. Theresa John, respectively. Both are
outstanding trainees and are full and integral members of the training program. They participate in
all training activities as described in this proposal, although they derive their stipend support from
supplement awards from ongoing R01s because of the reduction in the number of trainee positions at
the time of funding by 1 predoctoral and 1 post-doctoral position.
Raudel Sandoval is a Ph.D. student. He received his undergraduate degree at The University
of Illinois at Chicago. His work deals with the endothelial contractile protein, myosin light chain
kinase, and regulation of endothelial barrier function. He works in Dr. Tiruppathi’s laboratory and
collaborates with Dr. Malik. Mr. Sandoval is in the fourth year of his Ph.D. program. He has
published 2 papers as a co-author (Time Course of Recovery of Endothelial Cell Surface Thrombin
Receptor (PAR-1) Expression, C.A. Ellis, C. Tiruppathi, R. Sandoval, W. D. Niles and A. B. Malik,
Am. J. Physiol., 276:C38-C49, 1999) and (Thrombin Induces Proteinase-activated Receptor-1 Gene
Expression in Endothelial Cells via Activation of Gi-linked Ras/Mitogen-activated Protein Kinase
Pathway, C.A. Ellis, A.B. Malik, A. Gilchrist, H. Hamm, R. Sandoval, T. Voyno-Yasenetskaya, and
C. Tiruppathi, J. Biol. Chem. 274:13718-13727, 1999. He has also just submitted a paper as first
author (Intracellular Calcium Stores Regulate Thrombin-Induced Increase in Endothelial
Permeability, R. Sandoval, A. B. Malik, C. A. Ellis, L. Kiedrowsky, and C. Tiruppathi, Am J.
Physiol.)
Dr. Theresa John is a post-doctoral fellow in lung research. She is an U.S. citizen, who
received her doctorate at the University of Lagos in Nigeria, and subsequently received research
training at the University of Manchester in England. She came to Dr. Malik’s laboratory in the
summer of 1998 to study mechanisms of clearance of albumin across alveolar epithelial cells. She
has recently presented a paper (Faseb J. 13(A), 219.8, 1999) at the 1999 Experimental Biology
Meetings (John, et. al., Transalveolar Albumin Transport: Evidence for a Saturable Albumin Binding
Site).
Dr. Cordus Easington is a medical student enrolled in The University of Illinois College of
Medicine. Through the effort of the program, he receives an institutional stipend because of his
future interest in entering lung research training program. He completed his B.Sc. at Georgetown
University in Washington, D.C. and Ph.D. in Pharmacology at Rush-Presbyterian-St. Luke’s Medical
Center, and has subsequently enrolled as a medical student at the University of Illinois College of
Medicine. Although Dr. Easington is not a research trainee in the program as yet, his intent is to
pursue advanced training in lung research after having completed his medical degree. Thus, he
spends all of his available time in research with the presumption that he will enter the training
program upon completion of his medical training. We have developed this novel approach to nurture
Dr. Easington’s interests in lung research during his medical program.
F. Recruitment of Women in the Training Program
Table 11 lists the number of women in the training program from its inception. It is clear that
we have been committed to recruiting women at both the predoctoral and postdoctoral levels.
4. Responsible Conduct of Research
All trainees are required to take the course entitled “Scientific Integrity and Responsible
Conduct” given by Professor Robert Kelly (outline is in Table 8b). This is a lecture-based course
interspersed with in-depth discussions and case studies of the key topics in the outline. The specific
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details of the course, its contents, involvement of the faculty and trainees, and how the course is
given is described in Table 8b.
5. Progress Report (7-1-95 to 5-1-99)
A. General Attributes of the Program and its Developments
There have been several important accomplishments of the Lung Biology and Pathobiology
Training Program in its initial cycle. The first is that the training program has now been systematized
and integrated along interdisciplinary lines at The University of Illinois College of Medicine.
Multiple productive linkages based on research interests and the common goal of providing
multidisciplinary lung research training have developed amongst the training faculty members to the
ultimate benefit of the trainees. In addition, the training program has become an integral component
of the institution as evident by the well-attended weekly research seminars in lung biology (Lung
Research Seminar) that are supplemented by presentations from visiting scientists and consultants. In
other words, the training program has now become a distinct institutional entity.
Because of the recruitment of the specialized and outstanding training faculty, which is the
“heart and soul” of the program, the trainees have access to a variety of research opportunities and
strategies ranging from molecular approaches to lung physiology. The training program has been
essential in bringing together such a talented and diverse faculty along a thematic line. The trainees
themselves have also aided in building linkages amongst the training faculty because of the strengths
such as the inquisitive spirit that they have brought to the program. A major aspect of the program is
that it has enabled these trainees access to the various training faculty members solely on the basis of
the particular research questions being addressed and their own intellectual curiosity. This level of
integration could not have occurred without the program. The Ph.D. students supported by the
training program (who were uninitiated in research when they joined the program) have benefitted
from the many layers of research interactions and different ways of addressing questions as well as
the availability of specialized courses (e.g., Lung and Vascular Biology) that cross departmental
lines. Moreover, all trainees have benefitted from the intellectually stimulating environment created
and fostered by the program. An important spin-off of the program has been the improvement in the
overall quality of the training efforts of several components of the College of Medicine.
Another major accomplishment has been our ability even in a short time since the program’s
inception to compete successfully for outstanding Ph.D. students and postdoctoral fellows for
admission into the program. We believe that the interdisciplinary research training environment has
been a major reason for our successes in recruitment. Table 12 lists the positions awarded and
committed since the inception of the program. Table 13 identifies these individuals, their research
areas, current positions, and other training support received by them. Tables 14 and 14a list the
trainees’ publications and their achievements.
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Seven postdoctoral and six predoctoral trainees have been recruited into the program since its
inception (Table 12). All were recruited on the basis of intense competition and their outstanding
potential. Because this is our first funding period, these individuals are still early in their career
development (Table 13). Nevertheless of the four postdoctoral fellows who completed their
training, Dr. David Beno is an Assistant Professor in the Department of Pediatrics at RushPresbyterian-St. Luke’s Medical Center in Chicago, IL., Dr. Walter Niles is a Group
Leader/Principal Investigator at Aurora Biosciences in La Jolla, CA, Dr. Karen Buchkovich-Sass is a
Senior Scientist at Cephalon Laboratories in Philadelphia, PA, and Dr. Annette Gilchrist has
accepted a position as Assistant Professor in the Department of Pharmacology and Molecular
Biology at Northwestern University School of Medicine. Of the predoctoral trainees who have
completed their training, Dr. Chad Ellis is in postdoctoral training with Dr. Geoffrey L. Clark at
National Institutes of Health; Dr. Andrea True has accepted a postdoctoral training appointment at
the Department of Medicine, University of Michigan under Dr. Elizabeth G. Nabel. Dr. True will
move with Dr. Nabel to NIH upon completion of her laboratories. One of the trainees (Dr.
Buchkovich-Sass) was the recipient of the competitive Parker B. Francis Award, thus freeing up a
position for another trainee to be appointed. Three out of the six postdoctoral trainees and one out of
the six predoctoral trainees have been women. In addition, one Hispanic-American (Raudel
Sandoval) pre-doctoral trainee and one African-American post-doctoral trainee (Dr. Theresa John)
are both integral members of the lung research training program as described above. Thus, it is
evident that our trainees are on a successful track, and future evidence of their successes will
accumulate as they establish their research careers. Trainee publications, an indication of their
accomplishments and their other achievements, are listed in Table 14 and Table 14a, respectively.
Another significant accomplishment has been the development of a graduate level course in
Lung and Vascular Biology. This course is directed by Drs. R. Skidgel and C. Tiruppathi (both
members of the training faculty) and has significant involvement from all members of the training
faculty, including the Program Director. The establishment of this specialized required course as
well as other graduate level courses directed by other training faculty members (such as the course
on Platelet Activation Mechanisms directed by Drs. LeBreton and Lam and the course on Receptors
and Signaling developed by Drs. Mark Rasenick and Richard Green) greatly benefit the trainees not
only in the program but also a variety of other trainees at The University of Illinois.
In summary, although we are nearing the end of the first cycle (Year 04), the training
program has matured progressively during this period. It has created a productive and intellectually
stimulating training environment by assembling a group of highly qualified interdisciplinary faculty
members who provide research training opportunities for predoctoral students and postdoctoral
fellows. The trainees recruited on the basis of their credentials, research interests, and potential have
been outstanding and the training environment created under the umbrella of the training program
has clearly benefitted them. Even at the early stages of their development, it is apparent that our
postdoctoral trainees have accepted fulfilling research and academic positions and the graduate
students who have completed their Ph.D. training have accepted outstanding postdoctoral
fellowships.
B. Research Conducted by Trainees Supported by the Program
The specific research carried out by the trainees supported by the program is indicated in
Table 14b. This table provides the abstracts of their research studies.
6. HUMAN SUBJECTS
See Table 15
7. VERTEBRATE ANIMALS
See Table 16
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ATTACHMENT #3
PCOL 530
PHARMACOLOGY AND VASCULAR BIOLOGY
Fridays, 2:00 – 4:00 PM, 419 CMW
Course Directors: Dr. Randal A. Skidgel, 412 CSN; Phone: 996-9179; Email: rskidgel@uic.edu
Dr. Chinnaswamy Tiruppathi, 1006A CME; Phone: 355-0249; Email:tiruc@uic.edu
Date
Topic
Jan 16
Introduction to the Course
Overview of Vascular Biology
Jan 23 Endothelial cell cytoskeleton, signaling and barrier function
Jan 30 The plasminogen system in cardiovascular disease
Feb 6
Endothelial cell adhesion molecules
Feb13 Endothelial cell adhesion and leukocyte trafficking
Feb 20 Vascular Inflammation, oxidant signaling and gene expression
Feb 27 Nitric Oxide and the regulation of nitric oxide synthases
Mar 5 Novel functions of myeloperoxidase
Smooth muscle cell proliferation
Mar 12 Cadherins in cellular adhesion and signaling
Mar 19 Thrombin receptor activation and signaling in endothelial cells
Mar 26 Spring Break, No Class
Apr 2
Angiotensin converting enzyme, angiotensin and bradykinin
Apr 9
Hypertension
Measurement of barrier function in vivo
Apr 16 Transcellular permeability pathway in endothelial cells
Apr 23 Student Presentations
Apr 30 Angiogenesis
May 7
Final Exam
Grading:
Grades will be based on the following formula:
Attendance: 10%
Student Presentation: 15%
Student Paper: 25%
Final Exam: 50%
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Instructor
Randal Skidgel
D. Mehta
Randal Skidgel
Xiaoping Du
Richard Ye
Asrar Malik
Randal Skidgel
C. Tiruppathi
Richard Minshall
Michael Broman
C. Tiruppathi
Ervin Erdös
Steve Vogel
Richard Minshall
Karen Snapp
Principal Investigator/Program Director (Last, first, middle):
Malik, Asrar B.
ATTACHMENT #4
(2
United States Patent
Sutton , et al.
of
3)
6,204,054
March 20, 2001
Transcytosis vehicles and enchancers for drug delivery
Abstract
Transcytosis of a physiologically-active agent that exerts its action following passage across
endothelia, epithelia or mesothelia containing the GP60 receptor is enhanced by formulation with or
conjugation to a transcytosis enhancer or vehicle selected from albumin and fragments thereof, antiGP60 antibody and fragments thereof, GP60 peptide fragments, and PDI (protein disulphide
isomerase) and fragments thereof.
Inventors:
Sutton; Andrew Derek (Grantham, GB); Malik; Asrar Bari (Chicago, IL);
Tiruppathi; Chinnaswamy (Chicago, IL); Johnson; Richard Alan (Nottingham,
GB)
Assignee:
Andaris Limited (GB)
Appl. No.:
043412
Filed:
June 25, 1998
PCT Filed:
September 20, 1996
PCT NO:
PCT/GB96/02326
371 Date:
June 25, 1998
102(e) Date:
June 25, 1998
PCT PUB.NO.: WO97/10850
PCT PUB. Date: March 27, 1997
Foreign Application Priority Data
Mar 26, 1996[GB]
Current U.S. Class:
9606315
435/334; 424/143.1; 424/158.1; 424/447; 435/371
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Intern'l Class:
Field of Search:
Malik, Asrar B.
C12N 005/06; C12N 005/08; A61K 039/395; A61L 015/16
435/334,371 424/447,143.1,158.1
References Cited [Referenced By]
5254342
WO 88/00834
WO 93/20834
U.S. Patent Documents
Oct., 1993
Shen et al.
Foreign Patent Documents
Feb., 1988
WO.
Oct., 1993
WO.
Other References
Ghitescu, L. et al., "Specific Binding Sites for Albumin Restricted to Plasmalemmal
Vesicles of Continuous Capillary Endothelium: Receptor-mediated Transcytosis," J. Cell.
Biol. 102:1304-1311 (1986).
Schnitzer, J. E., "gp60 is an albumin-binding glycoprotein expressed by continuous
endothelium involved in albumin transcytosis," Am. J. Physiol. 262 (Heart Circ. Physiol.
31):H246-H254 (1992).
Schnitzer, J. E. and Oh, P., "Antibodies to SPARC inhibit albumin binding to SPARC,
gp60, and microvascular endothelium," Am. J. Physiol. 263 (Heart Circ. Physiol.
32):H1872-H1879 (1992).
Schnitzer, J. E. and Oh, P., "Albondin-mediated capillary permeability to albumin.
Differential role of receptors in endothelial transcytosis and endocytosis of native and
modified albumins,"J. Biol. Chem. 269:6072-6082 (Feb. 1994). Database Medline,
Accession No. 94164970 (Abstract).
Primary Examiner: Park; Hankyel T.
Attorney, Agent or Firm: Saliwanchik, Lloyd & Saliwanchik
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a U.S. national phase application corresponding to International Patent
Application No. PCT/GB96/02326, filed Sep. 20, 1996 (pending).
This application claim benefit to provisional application Ser. No. 60/004,097 Sep. 21, 1995.
Claims
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Principal Investigator/Program Director (Last, first, middle):
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What is claimed is:
1. A composition comprising, or conjugate of, a physiologically-active agent that exerts its action
following passage across endothelia, epithelia or mesothelia containing the GP60 receptor, and a
transcytosis enhancer or vehicle selected from albumin and fragments thereof, anti-GP60 antibody
and fragments thereof, GP60 peptide fragments, and PDI (protein disulphide isomerase) and
fragments thereof; wherein said composition or conjugate is a dry powder suitable for inhalation.
2. The composition or conjugate according to claim 1, wherein the transcytosis enhancer or vehicle
includes the CGMC motif.
3. The composition or conjugate according to claim 1, wherein the transcytosis enhancer or vehicle
comprises albumin or an albumin fragment.
4. The composition or conjugate according to claim 1, wherein the transcytosis enhancer or vehicle
comprises anti-GP60 antibody or an anti-GP60 antibody fragment.
5. The composition or conjugate according to claim 1, wherein the transcytosis enhancer or vehicle
comprises a GP60 peptide fragment.
6. The composition or conjugate according to claim 1, wherein the transcytosis vehicle comprises
albumin or an albumin fragment in combination with a GP60 peptide fragment.
7. The composition or conjugate according to claim 5 or claim 6, wherein the GP60 peptide fragment
is or comprises SEQ ID No. 1.
8. The composition or conjugate according to claim 1, wherein the physiologically-active agent is
selected from the group consisting of Luteinizing hormone (LH), chorionic gonadotropin, atrial
peptides, interferon, lymphokines I, lymphokine II, lymphokine III, lymphokine IV, lymphokine V,
lymphokine VI, lymphokine VII, a colony-stimulating factor, growth hormone-releasing factor,
corticotropin-releasing factor, luteinizing hormone-releasing hormone (LHRH), somatostatin,
calcitonin, thyrotropin-releasing hormone, calcitonin gene-related peptide (CGRP), transferases,
hydrolases, isomerases, proteases, ligases, oxidoreductases, esterases, phosphatases, nerve growth
factor (NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glialderived neurotrophic factor (GDNF), epidermal growth factor (EGF), fibroblast growth factor
(FGF), insulin-like growth factor, tumour necrosis factor (TNF), transforming growth factor (TGF),
encephalins, endorphins, gonadoliberin, melanostatin, melonoliberin, somatostatin, thyroliberin,
substance P, neurotensin, corticotropin, lipotropin, melanotropin, lutropin, thyrotropin, prolactin,
somatotropin, neurohypophyseal hormones, parathyrin, calcitonin, thymosin, thymopoietin,
circulating thymic factor, thymic humoral factor, insulin, glucagon, somatostatin, gastrin,
cholecystokinin, secretin, gastric inhibitory polypeptide, vasointestinal peptide, motillin, relaxin,
angiotensin, bradykinin, somatomedins, epidermal growth factors, urogastrone, deferoxamine,
buserelin, deslorelin, gonadorelin, goserelin, histrelin, leuprorelin, nafarelin, and triptorelin.
9. In a method for administering a physiologically-active agent to a mammal, the improvement
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comprising:
administering said active agent either conjugated to or in admixture with a transcytosis vehicle or
enhancer as defined in claim 1, wherein said transcytosis vehicle or enhancer delivers or enhances
passage of said physiologically-active agent across epithelia, endothelia, or mesothelia containing
the GP60 receptor.
10. The method of claim 9, wherein said physiologically-active agent is selected from the group
consisting of Luteinizing hormone (LH), chorionic gonadotropin, atrial peptides, interferon,
lymphokine I, lymphokine II, lymphokine III, lymphokine IV, lymphokine V, lymphokine VI,
lymphokine VII, a colony-stimulating factor, growth hormone-releasing factor, corticotropinreleasing factor, luteinizing hormone-releasing factor (LHRH), somatostatin, calcitonin, thyrotropinreleasing hormone, calcitonin gene-related peptide (CGRP), transferases, hydrolases, isomerases,
proteases, ligases, oxidoreductases, esterases, phosphatases, nerve growth factor (NGF), ciliary
neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic
factor (GDNF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth
factor, tumor necrosis factor (TNF), transforming growth factor (TGF), encephalins, endorphins,
gonadoliberin, melanostatin, melonoliberin, somatostatin, thyroliberin, substance P, neurostensin,
corticotropin, lipotropin, melanotropin, lutropin, thyrotropin, prolactin, somatotropin,
neurohypophyseal hormones, parathyrin, calcitonin, thymosin, thymopoietin, circulating thymic
factor, thymic humoral factor, insulin, glucagon, somatostatin, gastrin, cholecystokinin, secretin,
gastric inhibitory polypeptide, vasointestinal peptide, motillin, relaxin, angiotensin, bradykinin,
somatomedins, epidermal growth factors, urogastrone, deferoxamine, buserelin, deslorelin,
gonadorelin, goserelin, histrelin leuprorelin, nafarelin, and triptorelin.
11. The method of claim 9, wherein said physiologically-active agent is conjugated to said
transcytosis vehicle by a method selected from the group consisting of glutaraldehyde conjugation
using Schiff base formation, carbodiimide reaction between proteins and carboxylic acids, acid
anhydride activation of amine containing drugs followed by carbodiimide linkage, activation of
primary amine containing drugs with 3-(2-pyridyldithio)proprionate-N-succinimidyl anhydride
followed by coupling to cysteine groups of proteins, coupling of sugar alcohols to proteins utilizing
cyanuric chloride, and conjugation between amines and hydroxyl groups via bisperoxidation.
12. The method of claim 9, wherein said mammal is human.
13. The composition or conjugate, according to claim 1, which further comprises an excipient.
14. The composition or conjugate, according to claim 1, which comprises microparticles of the
active agent which are from 2 to 5 .mu.m in size.
15. An inhaler device which comprises a composition comprising, or conjugate of, a physiologicallyactive agent that exerts its action following passage across endothelia, epithelia or mesothelia
containing the GP60 receptor, and a transcytosis enhancer or vehicle selected from albumin and
fragments thereof, anti-GP60 antibody and fragments thereof, GP60 peptide fragments, and PDI
(protein disulphide isomerase) and fragments thereof; wherein said composition or conjugate is a dry
powder suitable for inhalation.
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Description
FIELD OF THE INVENTION
The invention relates to drug delivery. In particular, the invention relates to transcytosis vehicles and
enhancers capable of delivering and enhancing passage of drugs across endothelia, epithelia and
mesothelia containing the GP60 receptor.
BACKGROUND OF THE INVENTION
For most therapeutic drugs administered by intra-arterial or intravenous routes the intended site of
molecular activity lies outside the vasculature. For drugs administered via the airways, the intended
site of activity normally is beyond the first cellular barrier of alveolar, bronchiolar or tracheal
epithelia. In both cases, there is an endothelial or epithelial barrier which must be crossed before the
drug can mediate its effect.
For small lipophilic drugs, there appears to be a paracellular route between the tight junctions of the
barrier cells. However, for hydrophilic drugs and larger macromolecular active agents, such as
peptides, proteins, genes or anti-sense nucleotides, the only route across the barrier is through the
cells. This poses a particular problem for drugs administered intravenously which have exceedingly
short half-lives due to rapid degradation or first pass clearance by the liver. In order to maintain
therapeutic levels in balance with such excretion and degradation, large doses or infusions are often
necessary. Thus, there is clearly a need in the art for more rapid mechanisms for delivering drugs
across cellular barriers.
There have been numerous reports of specific receptors which mediate endocytotic events, where a
ligand binds to the receptor and is then internalized, complexed to the receptor, by a process similar
to pinocytosis. This involves invagination of the cell membrane in the region of the ligand receptor
complex and then release of the ligand into the cell by a process which is not fully understood.
Numerous endocytotic receptor systems have been reported including LDL, insulin, epidermal
growth factor, insulin-like growth factor and tPA-PAI-I (hybrid molecule).
Transcytosis entails invagination and vesicle formation around a ligand receptor complex, followed
by transcytotic passage with release by a reverse invagination process at the basolateral membrane.
Monoclonal antibodies to the transferrin receptor have been conjugated with toxins, so that they can
undergo transcytosis, across blood-brain endothelia. However, there is a continuing need in the art
for agents capable of delivering or enhancing passage of drugs by receptor-mediated transcytosis
across cellular barriers other than blood-brain endothelia, such as endothelia of the vasculature,
alveolar epithelia, and peritoneal mesothelia.
The GP60 receptor, also referred to as albondin, is one of several albumin-binding proteins reported
in the literature (Schnitzer and Oh, J. Biol. Chem. 269(8):6072-6082 (1994)). Others include SPARC
(serum protein, acidic, rich in cysteine), oesteonectin or basement membrane protein 40, GP30,
GP18 and GP60. SPARC and oesteonectin are extra-cellular proteins. GP60 shares some homology
with SPARC as determined using anti-SPARC antibodies (Schnitzer and Oh, Am. J. Physiol.
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263:H1872-H1879 (1992)).
GP18 and GP30 are membrane glycoproteins found in a variety of cell types but are particularly
prevalent in the macrophage (Schnitzer et al, J. Biol. Chem. 267: 24544-24553 (1992)). GP18 and
GP30 are the so-called "scavenger receptors" responsible for mediating removal of oxidized,
glycated or adduced forms of albumin by endocytosis and are thus believed to play a role in albumin
catabolism for a wide variety of organs (Schnitzer and Bravo, J. Biol. Chem. 268(10):7562-7570
(1993)).
In contrast to GP18 and GP30, the GP60 receptor has found to be expressed exclusively in
continuous endothelia of the vasculature (Schnitzer, Am. J. Physiol. 262:H246-H254 (1992)), in
alveolar epithelia (Kim et al, Am. J. Resp. and Crit. Care Med. 151:A190, (1994) and inferentially in
peritoneal mesothelia (Gotloib and Shostak, Kidney International. 47:1274-1284 (1995)). GP60 is
particularly abundant in the microvessel endothelia and is, interestingly, absent from the blood-brain
barrier, where little albumin flux is observed (Rousseaux et al, Methods in Enzymology 121:163
(1986)). It has been shown that polyclonal antibodies to endothelial GP60 also bind alveolar
epithelial GP60 (Kim et al, supra). The GP60 receptor has been implicated in receptor-mediated
transcytosis of albumin across epithelia and endothelial cell barriers (Kim et al, supra; Tirrupathi et
al, Molecular Biology of the Cell 4 (Supp):338a, Abstract No. 1964 (1993)).
The GP60 amino acid sequence is known in the art (Yamauchi et al, Biochem. Biophys. Res. Comm.
146:1485 (1987)).
SUMMARY OF THE INVENTION
The present invention provides transcytosis vehicles and enhancers capable of transporting
physiologically-active agents across epithelia, endothelia and mesothelia containing the GP60
receptor. The GP60 receptor has been implicated in receptor-mediated transcytosis of albumin across
cell barriers. By means of the invention, GP60 receptor-mediated transcytosis can be exploited for
the transport of not only albumin, but also physiologically-active agents which do not naturally pass
through epithelia, endothelia and mesothelia via the GP60 system.
Transcytosis vehicles and enhancers of the invention include albumin, albumin fragments, anti-GP60
polyclonal and monoclonal antibodies, anti-GP60 polyclonal and monoclonal antibody fragments,
and GP60 peptide fragments. Further, they include PDI (protein disulphide isomerase) and fragments
thereof (any subsequent reference to GP60 fragments may be interpreted as referring also to PDI
fragments). A common factor may be a CGMC motif found in PDI and at least the T.sub.1-44
fragment of GP60. If the transcytosis vehicle or enhancer is a GP60 peptide fragment, it is preferably
co-administered with other transcytosis vehicles or enhancers of the present invention such as
albumin or an albumin fragment. Suitable albumin fragments of 14, 20 and 32 kDa can be generated
by cleavage at methionine residues using cyanogen bromide and can be further reduced in size by
reduction of disulfide bridges. Anti-GP60 polyclonal and monoclonal antibody fragments useful as
transcytosis vehicles and enhancers according to the present invention include Fab, Fab',
F(ab').sub.2, and Fv fragments. Preferred GP60 peptide fragments include the T3118 peptide which
corresponds to the N-terminal 18 amino acids of the GP60 protein.
In accordance with the invention, when the above compounds are conjugated to a physiologicallyPHS 398 (Rev. 5/95)
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active agent, they are referred to herein as "transcytosis vehicles". When co-administered with but
not conjugated to a physiologically-active agent, the above compounds are referred to herein as
"transcytosis enhancers". In preferred embodiments, the transcytosis vehicles and enhancers of the
present invention are useful for delivering or enhancing passage of physiologically-active agents
across endothelia of the vasculature, alveolar epithelia and peritoneal mesothelia.
DETAILED DESCRIPTION OF THE INVENTION
As its name indicates, the GP60 protein has been reported in the art as having a molecular weight of
about 60 kDa. After a more careful analysis, it has been discovered that the "true" molecular weight
for this protein is more probably about 57 kDa. This discrepancy in molecular weight is thought to
be due to differences in protein preparation and gel conditions. However, to be consistent with the
art, this protein is referred to herein (with the exception of Example 1 below) as the GP60 receptor.
It has been discovered that GP60 receptor-mediated transcytosis can be exploited for the transport of
not only albumin, but also for a vast number of therapeutically-important physiologically-active
agents which do not naturally pass through epithelia, endothelia and mesothelia via the GP60
system. Thus, the present invention provides an improved method for transporting physiologicallyactive e.g. those having relatively high molecular weights, e.g. 50, 100, 150 kDa or more, across the
cellular barriers of the endothelia of the vasculature, alveolar, bronchiolar, and tracheal epithelia, and
the peritoneal mesothelia. Transcytosis vehicles and enhancers capable of delivering or enhancing
passage of physiologically-active agents across GP60-containing endothelia, epithelia and
mesothelia include albumin, albumin fragments, anti-GP60 polyclonal and monoclonal antibodies,
anti-GP60 polyclonal and monoclonal antibody fragments, and GP60 peptide fragments. If the
transcytosis vehicle or enhancer is a GP60 peptide fragment, it will preferably be co-administered
with other transcytosis vehicles or enhancers of the present invention such as albumin or an albumin
fragment.
Mammalian albumin is well known in the art and readily available. Preferably, the albumin used will
be from the same mammalian species as the patient. For example, if the patient is human, human
serum albumin will preferably be used as the transcytosis vehicle or enhancer. Similarly, if the
patient is equine or bovine, equine or bovine serum albumin is preferably used, respectively.
Methods for generating albumin fragments are well known in the art. For example, cleavage of
albumin at methionine residues by cyanogen bromide yields three particularly suitable peptides of
14, 20 and 32 kDa which can be further reduced in size by reduction of the disulfide bridges, to
peptides ranging in size from 3.3-20 kDa. Alternatively, protease digestion can be used to generate
albumin peptide fragments.
Whether any particular albumin fragment is useful as a transcytosis vehicle or enhancer according to
the present invention can be determined according to the routine screening assay described below.
As indicated in the Examples below, it has now been demonstrated that both bovine and human
serum albumin, acting as transcytosis enhancers, stimulate uptake of a physiologically-active agent
2.5-4 fold over the control.
Anti-GP60 polyclonal and monoclonal antibodies can be generated from the GP60 receptor purified
from endothelia, epithelia or mesothelia. As discussed above, endothelial, epithelial and mesothelial
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cells which express the GP60 receptor include endothelia of the vasculature (including capillary
endothelia (Ghinea et al, J. Cell Biol. 107:231-239 (1988)); arterial endothelia (Silflinger-Birnboim
et al, J. Cellular Physiology 149:575-584 (1991); aortic and vein endothelia (Schnitzer and Oh, Am.
J. Physiol. (1992), supra); epithelia of alveolar tissue (Kim et al, supra); and mesothelia of the
peritoneum (Gotloib and Shostak, supra). GP60 can be purified from epithelia, endothelia and
mesothelia according to art-known methods (see, for example, Schnitzer and Oh, J. Biol. Chem.
(1994), supra) and as described in Example 1 below.
Producing polyclonal antibodies against purified GP60 or a GP60 peptide fragment (such as the
T3118 peptide discussed below) can occur in mice, rabbits, or goats according to art-known
techniques. In Example 1 below, the GP60 receptor was eluted from preparative SDS-PAGE to
immunize rabbits. Approximately 50 .mu.g protein per rabbit was injected intramuscularly after
mixing with equal volume of Freund's complete adjuvant. A second injection was given after two
weeks. Rabbits were bled at 4 to 6 weeks after the second injection, and the immune response was
tested. The antiserum IgG was then purified using a Protein A-Sepharose column.
Monoclonal antibody preparation can also occur according to known techniques (Goding, J.
Immunol. Methods 39:285 (1980); Oi and Herzenberg, Selected Methods in Cellular Immunology,
p. 352, Freeman, San Francisco, 1979)). For example, Balb/c mice are injected intraperitoneally with
50-150 .mu.g of GP60 or a GP60 peptide fragment. Three to five days before the fusion, positive
mice receive a booster injection of antigen (50-150 .mu.g of GP60 or GP60 fragment), and then 10
.mu.g (intravenous and intraperitoneal route) every day until spleen removal. The spleen cells are
fused with Sp2/0-Ag14 myeloma cells essentially according to St. Groth et al, J. Immunology
Methods 35:1-21 (1980). Culture supernatants are screened by ELISA using unconjugated GP60 or
GP60 fragment as antigen. Positive cultures are then tested by immunofluorescence and Western
blotting on cDNA-transfected COS-1 cells as described in Lutz et al, Experimental Cell Research
175:109-124 (1988). Hybridomas secreting specific antibodies are cloned twice on soft agar. Each
hybridoma can be adapted in serum-free medium SFRI-4. For ascites fluid production,
approximately 2.times.106 cells are injected in pristine-primed Balb/c mice. Class and subclass
determination is performed using an Isotyping Kit. Both SFRI culture supernatants and ascites fluids
can be used as monoclonal antibody sources.
As discussed, the anti-GP60 polyclonal and monoclonal antibodies and antibody fragments of the
present invention are useful as transcytosis vehicles and enhancers capable of delivering or
enhancing passage of physiologically-active agents across endothelia, epithelia and mesothelia
containing the GP60 receptor. Anti-GP60 antibody fragments useful as transcytosis vehicles or
enhancers of the present invention include fragments containing single (Fab) antigen binding
domains produced by papain digestion; or F(ab').sub.2 fragments produced by limited pepsin
digestion (Olsson and Kaplan, Methods in Enzymology 92:3 (1983)). Other suitable fragments
include Fab' and Fv. Whether any particular antibody fragment is useful as a transcytosis vehicle or
enhancer can be determined according to the routine screening assay described below. In Example 3
below, it is demonstrated that administering anti-GP60 polyclonal antibodies at 37.degree. C. results
in a 1.6-2 fold increase in uptake of a physiologically-active agent over the level of a pre-immune
serum control.
According to the invention, anti-GP60 antibodies raised in animals other than humans such as mice
and rats are suitable for short-term administration only (i.e., non-chronic dosage) due to the wellPHS 398 (Rev. 5/95)
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known adverse immune response to foreign antibodies. However, art-described methods can be used
to produce human monoclonal antibodies to the GP60 receptor, to overcome the problems of
administering murine monoclonals to humans (Olsson and Kaplan supra), thereby rendering the
antibodies suitable for long-term or chronic administration. Moreover, the murine antibodies of the
present invention can be "humanized" by chimeric or CDR grafting. The recognition region of the
murine antibody is grafted into the appropriate region of a human antibody, in order to avoid or limit
an adverse immune response in a patient.
GP60 peptide fragments are also useful as transcytosis vehicles and enhancers according to the
present invention. Particularly suitable GP60 peptide fragments include the first 18 amino acids from
the N-terminus of GP60; it has been discovered that this is at least 80% homologous to a stretch of
the bovine, membrane-bound thyroid hormone (T3) binding protein. Such GP60 peptide fragments
can be produced according to any known enzymatic or physical technique, including proteolytic
degradation. Alternatively, GP60 peptide fragments can be produced synthetically. As indicated in
Example 5 below, a synthetic N-terminal peptide (T3118) of GP60 corresponding to the first 18
residues may be produced by solid-phase synthesis. This peptide, acting as an agonist of
transcytosis, stimulated uptake of human albumin 5-fold over the control.
Methods for conjugating the transcytosis vehicles of the present invention to a physiologically-active
agent will be readily apparent to the skilled artisan and include, but are not limited to, glutaraldehyde
conjugation involving Schiff base formation; carbodiimide reaction between proteins and carboxylic
acids; acid anhydride activation of amine-containing drugs followed by carbodiimide linkage;
activation of primary amine-containing drugs with 3-(2-pyridyldithio)propionate-N-succinimidyl
anhydride followed by coupling to cysteine groups of proteins; coupling of sugar alcohols to proteins
utilizing cyanuric chloride; and conjugation between amines and hydroxyl groups via
bisperoxidation.
For example, the amino sugar moiety of a physiologically-active agent can be oxidized by sodium
periodate treatment and directly attached to lysine residues on a transcytosis vehicle of the present
invention via Schiff base formation according to the method described in Hurwitz et al, Cancer Res.
35:1175-1181 (1975). Alternatively, a physiologically-active agent can be linked to a transcytosis
vehicle of the present invention through carbodiimide-mediated linkage of the amino group of the
active to carbonyl groups on the vehicle or to an aminoalkyl group according to the method
described in Hurwitz et al, Int. J. Cancer 21:747-755 (1978). The physiologically-active agent can
also be linked to a transcytosis vehicle of the present invention by cross-linking the amino sugar of
the active and amino groups of the vehicle with glutaraldehyde according to the method described in
Belles-Isles et al, Br. J. Cancer 41:841-842 (1980).
Other suitable conjugation sites for conjugating physiologically-active agents to one of the
transcytosis vehicles of the present invention can be routinely determined empirically. For example,
a transcytosis vehicle of the present invention can be labelled with fluorescein or .sup.125 I either
before or after conjugation to a physiologically-active agent such as insulin. After conjugation and
labelling, a screening assay such as that described in the Examples below can be used to determine
the endothelial cell uptake, the epithelial cell flux, or the mesothelial cell flux of any candidate
vehicle/active conjugate. Such a routine screening assay allows the skilled artisan to determine
which transcytosis vehicles of the present invention retain the ability to undergo transcytosis after
being conjugated at a particular site to a physiologically-active agent. Such an assay is also useful
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for routine screening of candidate albumin fragments, anti-GP60 antibody fragments and GP60
peptide fragments to determine which are suitable (for use as transcytosis vehicles and enhancers
according to the present invention.
The conjugation of physiologically-active agents to a transcytosis vehicle of the present invention is
particularly suited for intravenous delivery of low molecular weight drugs which otherwise have
exceedingly short serum half-lives, or of peptide drugs that are rapidly degraded in the blood stream
or removed by first pass excretion in the liver. Of course, where the physiologically-active agent is
covalently conjugated to one of the transcytosis vehicles of the present invention, the residual
activity of the therapeutic agent must be assessed after conjugation. Techniques for assaying a
therapeutic agent's activity are well established in the art, and many therapeutics have successfully
been conjugated and retained substantial activity. For example, the literature describes conjugates
between receptor ligands, or fragments thereof, and drugs to promote transcytosis across the blood
brain barriers. Fukta et al, Pharm. Research 11(12):1681 (1994), describe conjugation of horse radish
peroxidase (HRP) to insulin which enabled HRP to cross the blood, brain barrier. The investigators
went on to produce fragments of insulin which were screened for their ability to bind to the insulin
receptor on bovine brain microvessel endothelial cells in culture. Similarly, other transcytosis
systems allow the passage of antibodies linked with active drugs including, among others, antibodymethotrexate targeted to the transferrin receptor (Friden et al, Proc. Natl. Acad. Sci. USA 88:4771
(1991)), and antibody-polylysine targeted to the epidermal growth factor receptor (Chen et al, FEBS
Lett. 338:167 (1994)).
By contrast to the transcytosis vehicles, transcytosis enhancers of the invention are not conjugated to
the physiologically-active agent. It has been discovered that co-residence on epithelia, endothelia
and mesothelia containing the GP60 receptor of one of the transcytosis enhancers of the present
invention and a physiologically-active agent is sufficient to enhance uptake and passage of the agent
across the cell barrier. Without wishing to be bound by theory, the transcytosis enhancers of the
present invention apparently "trigger" the GP60-mediated transcytosis mechanism, thereby
stimulating the enhanced uptake of co-resident macromolecules, including therapeutic agents.
Uptake or passage of physiologically-active agents by or across epithelia, endothelia and mesothelia
can be induced or enhanced with any of the transcytosis enhancers of the present invention either
alone or in combination. For example, the experiments below demonstrate that, acting as an agonist
of transcytosis, the GP60 peptide T3118 enhanced human albumin uptake 5-fold over the control. In
a further embodiment of the present invention, delivery of active agents can be achieved when one
of the transcytosis vehicle conjugates discussed above is administered together with one or more of
the transcytosis enhancers of the present invention.
The transcytosis vehicle conjugates and the transcytosis enhancer compositions (including an active
agent) of the present invention can be administered with a pharmaceutically-acceptable carrier or
excipient, i.e., pharmaceutically-acceptable organic or inorganic substances suitable for application
which do not deleteriously react with the conjugate or composition. Suitable pharmaceuticallyacceptable substances include but are not limited to water, salt solutions, alcohol, vegetable oils,
polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters,
hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be
sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers,
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wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colourings, flavouring
and/or aromatic substances, which do not deleteriously react with the conjugates. For parenteral
application, particularly suitable preparations are solutions, preferably oily or aqueous solutions, as
well as suspensions, emulsions, or implants, including suppositories. Ampoules are convenient unit
dosages. For enteral application, particularly suitable preparations are tablets, dragees or capsules
having a carrier binder such as talc and/or a carbohydrate, the carrier preferably being lactose and/or
corn starch and/or potato starch. A syrup, elixir or the like can be used wherein a sweetened vehicle
is employed. Sustained release compositions can be formulated including those wherein the active
component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple
coatings, etc.
Administration of a conjugate or composition comprising one or more physiologically-active agents
and one or more of the transcytosis vehicles or enhancers of the present invention can occur
according to any art-known technique including injection or via the pulmonary airways. Injection is
particularly suitable for administration to the vasculature and the peritoneum, whereas the
pulmonary airways are particularly suitable for administration to the alveoli. Suitable formulations
for pulmonary administration include one or more of the transcytosis enhancers of the present
invention admixed with a physiologically-active agent. Alternative suitable formulations for
pulmonary administration include a transcytosis vehicle conjugated to the agent. For example,
formulations may be made from a nebulizer device such as an Acorn or DeVilbiss jet nebulizer,
wherein the agent and transcytosis enhancer or vehicle are presented as an aqueous solution in the
nebulizer reservoir. Alternatively, in a preferred embodiment for pulmonary administration, the
formulation is discharged from a dry powder inhaler (DPI) device. DPI devices are described by
Sutton et al in U.S. patent application Ser. No. 08/487,420 and in WO-9609814. They require spraydrying the formulation into microparticles of 2-5 .mu.m which are preferred for alveolar penetration.
In particular, a transcytosis enhancer or vehicle of the present invention or a mixture thereof,
preferably at a concentration of about 20% w/v, is used for spray-drying. The preparation to be
sprayed may contain substances other than the transcytosis enhancers or vehicles and solvent or
carrier liquid. For example, the aqueous phase may contain 1-20% by weight of water-soluble
hydrophilic compounds such as sugars and polymers as stabilizers, e.g., polyvinyl alcohol (PVA),
polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), gelatin, polyglutamic acid and
polysaccharides such as starch, dextran, agar, xanthin and the like. Similar aqueous phases can be
used as the carrier liquid in which the final microsphere product is suspended before use. Emulsifiers
may be used (0.1-5% by weight), including most physiologically-acceptable emulsifiers, for instance
egg lecithin or soya bean lecithin, or synthetic lecithins such as saturated synthetic lecithins, for
example, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, or distearoyl
phosphatidylcholine or unsaturated synthetic lecithins, such as dioleyl phosphatidylcholine or
dilinoleyl phosphatidylcholine. Emulsifiers also include surfactants such as free fatty acids, esters of
fatty acids with polyoxyalkylene compounds, e.g. polyoxypropylene glycol and polyoxyethylene
glycol; ethers of fatty alcohols with polyoxyalkylene glycols; esters of fatty acids with
polyoxyalkylated sorbitan; soaps; glycerol-polyoxyethylene ricinoleate; homo-and copolymers of
polyalkylene glycols; polyethoxylated soya-oil and castor oil as well as hydrogenated derivative;
ethers and esters of sucrose or other carbohydrates with fatty acids, fatty alcohols, these being
optionally polyoxyalkylated; mono-, di- and triglycerides of saturated or unsaturated fatty acids,
glycerides or soya-oil and sucrose.
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Additives can be incorporated into the wall of the microspheres to modify the physical properties
such as dispersibility, elasticity and water permeability. Among the useful additives include
compounds which can "hydrophobize" the wall in order to decrease water permeability, such as fats,
waxes and high molecular weight hydrocarbons. Additives which improve dispersibility of the
microspheres in the injectable liquid-carrier are amphipathic compounds such as phospholipids; they
also increase water permeability and rate of biodegradability. Additives which increase wall
elasticity include plasticizers such as isopropyl myristate and the like. The quantity of additives to be
incorporated in the wall is extremely variable and depends on the needs. In some applications, no
additive is used at all; in other cases, amounts of additives which may reach about 20% by weight of
the wall are possible.
A solution containing one or more transcytosis enhancers or vehicles of the present invention and
additive, if any, is atomized and spray-dried by any suitable technique which results in discrete
microspheres or microcapsules of 2 to 5 .mu.m as discussed above. As used herein, "microcapsules"
refers to hollow particles enclosing a space, which space is filled with a gas or vapour but not with
any solid materials.
The atomization forms an aerosol of the transcytosis vehicle or enhancer formulation, for example
by forcing the formulation through at least one orifice under pressure, or by using a centrifugal
atomizer in a chamber of warm air or other inert gas. The chamber should be big enough for the
largest ejected drops not to strike the walls before drying. The gas or vapour in the chamber is clean
(preferably sterile and pyrogen-free) and non-toxic when administered to the bloodstream in
amounts concomitant with administration of the microcapsules in use. The rate of evaporation of the
liquid from the preparation should be sufficiently high to form hollow microcapsules but not so high
as to burst the microcapsules. The rate of evaporation may be controlled by varying the gas flow
rate, concentration of transcytosis vehicle or enhancer in the formulation, nature of liquid carrier,
feed rate of the solution and, more importantly, the temperature of the gas encountered by the
aerosol. For example, an albumin or albumin fragment concentration of 15-25% in water, and an
inlet gas temperature of at least about 100.degree. C., preferably at least 110.degree. C., is sufficient
to ensure hollowness and the temperature may be as high as 250.degree. C. without the capsule
bursting. About 180-240.degree. C., preferably about 210-230.degree. C. and most preferably about
220.degree. C., is optimal. Since the temperature of the gas encountered by the aerosol will depend
also on the rate at which the aerosol is delivered and on the liquid content of the preparation, the
outlet temperature may be monitored to ensure an adequate temperature in the chamber. An outlet
temperature of 40-150.degree. C. is suitable. Controlling the flow rate is useful in controlling other
parameters such as the number of intact hollow particles.
The microparticles may comprise at least 50%, more preferably 70% or 80%, and most preferably
90%, by weight transcytosis enhancer. For use in an inhaler device, the microparticles may be
formulated with a conventional excipient such as lactose or glucose. The amount of the
physiologically-active agent will be chosen with regard to its nature and activity, to the mode of
administration and other factors known to those of skill in the art. By way of example, the number of
particles administered may be such as to deliver 100 mg/day .alpha.-1 anti-trypsin, or 0.1 mg/day of
an active agent such as beclomethasone. Other possible physiologically-active agents that can be
administered via microparticles are given below.
A further embodiment of the present invention is the co-spray-drying of the physiologically-active
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agent with the transcytosis enhancer in order to facilitate stabilization of the active agent during
formulation, packing, and most importantly, during residence on the alveolar lining. In this
environment, there can be intense proteolytic activity. In this or another embodiment, the active
agent may be covalently linked to the transcytosis vehicle via cleavable linkages prior to spraydrying. This embodiment represents a method of carrying the active agent all the way from the
device to the bloodstream, and possibly to targets within the body. The formation of particles with
optimal aerodynamic size means that the "physical" vehicle delivers the active agent to the site of
absorption. Once deposited upon the alveoli, the "molecular" vehicle then protects and facilitates
passage into the bloodstream via the GP60-mediated transcytosis system and, once in the
bloodstream, can further enhance circulatory half-life and even direct the active agent to certain sites
which are found to contain the GP60 receptor. Suitable linking technologies are discussed above;
further, WO-A-9317713 describes esterase-sensitive polyhydroxy acid linkers. Such technology,
used in the derivatization of the transcytosis vehicle prior to spray-drying, enables the production of
a covalent carrier system for delivery of active agents to the systemic vasculature. This utilizes the
potential of the transcytosis vehicles to cross the alveoli and to carry active agents over a prolonged
period while protecting potentially unstable entities.
Although the physiologically-active agent used in the present invention my be imbibed into or
otherwise associated with the microparticles after their formulation, it is preferably formulated with
the transcytosis vehicle or enhancer. The microparticles may be at least partly coated with a
hydrophobic or water-insoluble material such as a fatty acid, in order to delay their rate of
dissolution and to protect against hydroscopic growth.
Methods and equipment for spray-drying and generating the microparticles, e.g. for use in a dry
powder inhaler device are described in more detail in WO-A-9609814 and in U.S. patent application
Ser. No. 08/487,420, the contents of which are incorporated herein by reference.
The optimal proportions of drug to transcytosis enhancer in a formulation for pulmonary delivery
can be determined according to any suitable method. An in vitro optimization of the formulation
entails using epithelial monolayers of primary human or immortalized human epithelial cells grown
as monolayers on porous filters, as described in the Examples below. Combinations of drug and
enhancer may then be applied to the upper chamber of a transwell flux system also as described
below. Using either labelled tracer or an immunoassay, flux rates of the drug or gene to the lower
layer are determined. The optimal formulation is defined as the one showing maximal rate and extent
of passage through the restrictive monolayer.
An alternative way of optimizing the formulation entails performing an in vivo determination of lung
to blood passage of the drug under investigation. There are well-reported studies in rat, pig and
sheep (Patton et al, Journal of Controlled Release 28:79 (1994), Folkesson et al, Acta. Physiol.
Scand. 147:73 (1993); Schreier et al, Pharm. Res. 11:1056 (1994)); these studies describe methods of
instilling or aerosolizing drug formulations into the trachea and bronchioles and assessing the
appearance in blood of the drug by immunoassay or pharmacological activity. Optimization would
entail a series of animal preparations using differing proportions of the drug and enhancer, the
optimal formulation being defined by the most beneficial area under the curve that matched the
desired pharmacological profile for the drug. For instance, the drug may simply be required to show
the maximal bioavailability or alternatively to show a protracted or sustained release profile. For
each case, it is likely that there would be differing requirements for the level of enhancer
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incorporated in the formulation. For drugs requiring maximal availability, it would be desirable to
utilize the maximal level of enhancer and/or the enhancer showing the highest activating effect upon
the GP60 receptor. For drugs requiring a longer period of presentation across the lung, it would be
desirable to utilize lower levels of enhancer and/or enhancers showing lower activation potential on
the transcytosis GP60 receptor.
The "strength" of the enhancer or vehicle can be defined, by the extent to which transcytosis of a
given tracer can be enhanced, by the presence of the GP60 receptor-binding ligand, antibody or
mimetic, over the level of transcytosis in the absence of the ligand. The "strength" of the enhancing
agent may be somewhat drug-dependent also. Enhancement of marker uptake can vary dependent
upon the nature of the marker and the transcytosis enhancer. Tabulated below is a synopsis of the
markers, enhancers, cell system and extent of enhancement over the control achieved for differing
markers cell systems and experimental type.
Abbreviations used:
.sup.125 I-BSA
bovine albumin
.sup.125 I-IgG
HSA
BSA
FITC-Insulin
GP60 Ab
T3118
.sup.125 Iodine-labelled
Marker
Enhancer
.sup.125 I-BSA GP60 Ab
.sup.125 I-BSA GP60 Ab
anti-BSA
BSA
.sup.125 I-IgG
FITCHSA
Insulin
FITCBSA
Insulin
.sup.125 I-BSA BSA/T3118
.sup.125 Iodine-labelled Immunoglobulin G
Human albumin
Bovine albumin
fluorescein-labelled insulin
Anti-GP60 polyclonal antibody
Synthetic peptide derived from N
terminal 18 residues of GP60
Fold
Cell Type
Enhancement
Bovine/Endothelia/ 1.6
flux
Bovine/Endothelia/ 2.0
flux
Bovine/Endothelia/ 1.5
flux
Human
2.5
Endothelia/flux
Rat Epithelia/flux 4
Bovine
Endothelia/uptake
5
By "physiologically-active agent" is intended drugs which include nucleic acid molecules and
medicinal peptides and proteins. "Physiologically-active agent" is used interchangeably herein with
"drug", "active", "active agent" and "therapeutic". Drugs that would benefit from a more rapid
transcytosis across the endothelia and epithelia include Luteinizing hormone (LH), chorionic
gonadotropin, atrial peptides, interferon, the various lymphokines such as the interleukins (I, II, III,
IV, V, VI, and VII), and colony-stimulating factors.
Other drugs suitable for use in the present invention include: Growth hormone-releasing factor,
corticotropin-releasing factor, luteinizing hormone-releasing hormone (LHRH), somatostatin,
calcitonin, thyrotropin-releasing hormone, calcitonin gene-related peptide (CGRP), proteins such as
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enzymes, including transferases, hydrolases, isomerases, proteases, ligases, oxidoreductases,
esterases and phosphatases, and various growth and neurotrophic factors, such as somatomedins,
epidermal growth factors, urogastrone, nerve growth factor (NGF), ciliary neurotrophic factor
(CNTF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF),
epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor, tumour
necrosis factor (TNF) and transforming growth factor (TGF). Further drugs include endogenous
opioid agonists, such as encephalins and endorphins; hypothalamic hormones, such as gonadoliberin,
melanostatin, melonoliberin, somatostatin, thyroliberin, substance P, and neurotensin;
adenohypophyseal hormones, such as corticotropin, lipotropin, melanotropin, lutropin, thyrotropin,
prolactin, and somatotropin; neurohypophyseal hormones; calcitrapic (thyroid) hormones, such as
parathyrin and calcitonin; thymic factors, such as thymosin, thymopoietin, circulating thymic factor,
and thymic humoral factor; pancreatic hormones, such as insulin, glucagon and somatostatin;
gastrointestinal hormones, such as gastrin, cholecystokinin, secretin, gastric inhibitory polypeptide,
vasointestinal peptide, and motillin; ovarian hormones, such as relaxin; vasoactive tissue hormones,
such as angiotensin and bradykinin; and artificial or pseudo peptides, such as deferoxamine; and
LHRH analogs such as buserelin, deslorelin, gonadorelin, goserelin, histrelin, leuprorelin, nafarelin,
or triptorelin.
Having generally described the invention, the same will be more readily understood through
reference to the following Examples which are provided by way of illustration but are not intended
to be limiting.
EXAMPLE 1
Growth of Endothelial and Epithelial Monolayers
Bovine pulmonary microvessel endothelial cells (BPMVEC) and (BPAEC) bovine pulmonary artery
endothelial cells were isolated and cultured according to described methods (Del Vecchio et al, In
Vitro. Cell. Dev. Biol. 28A:711-715 (1992)). Endothelial cells were routinely cultured with DMEM
containing 20% FBS. For isolating plasma membranes, the endothelial cells were cultured in 850
cm.sup.3 roller bottles. To each roller bottle, 75 ml culture medium was added. An air-CO.sub.2
mixture was introduced. The cells were then transferred to a roller bottle incubator at 37.degree. C.,
and were allowed to grow for 10-12 days.
Primary rat alveolar epithelial cells (AEC) were isolated by methods described in Uhal et al, Am. J.
Physiol. 257:C528-C536 (1989). Cells were cultured in DMEM containing 10% FBS for either 2 or
4 days, at which times they exhibited a type II or type I cell-like phenotype respectively. Phenotype
was verified by methods described by Uhal et al, Am. J. Physiol. Suppl. 261:110-117 (1991).
Endothelial Cell Membrane Isolation
Endothelial cells grown in roller bottles were washed 2.times. with phosphate buffered saline. The
cells were scraped from roller bottles and suspended in Buffer-A (20 mM HEPES/Tris, 0.15 M
NaCl, 0.1 mM PMSF at pH 7.4) and washed 2.times. by centrifuging at 700.times.g for 10 minutes.
The cells obtained from 6-8 roller bottles were suspended in 75 ml of buffer-A and homogenized
using a Polytron homogenizer for 1 minute at full speed. The homogenate was centrifuged at
3000.times.g for 10 minutes. The supernatant was collected and centrifuged at 40,000.times.g for 60
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minutes. The pellet obtained was then suspended in buffer-A and recentrifuged at 40,000.times.g for
60 minutes. The final membrane pellet was suspended in a small volume of buffer-A containing 0.2
mM EDTA and the protein concentration was determined (Lowry et al, J. Biol. Chem. 193:265-275
(1951)). The plasma membrane marker enzyme activities were determined and the sample stored at 70.degree. C. until further use.
Ligand Blotting
Endothelial cell membranes were preincubated with 1 mM PMSF and 0.5 mM EDTA for 20 minutes
at 22.degree. C., and then solubilized by mixing with 1.5 volume of solubilizing buffer (9M urea, 2%
SDS, 2% .beta.-mercaptoethanol, 0.1 M Tris, 0.02% bromophenol blue pH 6.8). The mixture was
incubated at 22.degree. C. for 30 minutes. The solubilized proteins were separated by SDS-PAGE
(Laemmli, Nature (London) 227:680-685 (1970)) using a slab-gel electrophoretic system with 3%
acrylamide in the stacking gel and 10% acrylamide in the separating gel. After electrophoresis, the
proteins were transferred to either PVDF or nitrocellulose membrane. The transfer was carried out
for 2 hours at 150 volts using 25 mM Tris, 192 mM glycine, and 20% methanol as transfer buffer.
The non-specific binding was blocked by incubating the membrane with 5 mM CaCl.sub.2 in TBS
(20 mM Tris, 0.5 M NaCl at pH 7.5) for 10 minutes and then with 0.5% Tween-20 in TBS overnight.
After this step, the membrane was washed and cut into two strips. One strip was incubated with 0.6
mg/ml globulin-free BSA in TBS containing 1.5% gelatin for 2 hours and the other strip was
incubated without BSA. The strips were washed and incubated with anti-bovine BSA for 60 minutes
in TBS containing 1.5% gelatin. The membranes were then washed 2.times. and incubated with
second antibody (goat anti-rabbit IgG) conjugated with alkaline phosphatase. The protein bands
were localized after adding 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium salt.
Protein Purification
BPMVEC membranes were used to isolate a 57 kDa albumin-binding protein. The ligand blotting
was carried out to assess the presence of this protein in each step. BPMVEC membranes (100 mg)
were preincubated with 1 mM PMSF and 0.5 mM EDTA for 30 minutes at 22.degree. C. The
membranes were solubilized using a final concentration of 2.5% sodium cholate and 4 M urea, at
4.degree. C. for 3 hours, with gentle stirring. The protein concentration was adjusted to 4 mg/ml
during solubilization. After this treatment, the suspension was centrifuged at 100,000.times.g for 60
minutes. The supernatant was collected and dialyzed against 5 mM HEPES/Tris (pH 7.2). More than
80% of membrane proteins were recovered in the supernatant. The dialysed suspension was
concentrated by 60% ethanol precipitation at 4.degree. C. The ethanol precipitate was collected by
centrifugation at 10,000.times.g for 30 minutes at 4.degree. C. and suspended in Buffer-A. This
precipitate was solubilized with 2.5% Triton X-100 overnight at 4.degree. C. with gentle stirring.
The suspension was centrifuged at 100,000.times.g for 60 minutes. The supernatant was collected
and dialysed against 4 l of 50 mM Tris-HCl, 0.2 mM EDTA, 0.15% Triton X-100 and 0.1 mM
PMSF, pH 8.0 (Buffer-B). The dialysed extract was applied on a DEAE-52 column (10.times.13
cm). The column was previously equilibrated with Buffer-B. The column was washed with 50 ml of
Buffer-B after applying the sample. The bound proteins were eluted from the column with 80 ml of
0-500 mM linear NaCl gradient in Buffer-B at a flow rate of 15 ml/hr. The fractions from individual
peaks were pooled separately and concentrated by 50% acetone precipitation. The acetone
precipitate was used for ligand blotting. Only peak-I showed albumin-binding activity. The proteins
present in peak-I were further separated by using preparative SDS-PAGE (16 cm.times.16 cm, 3 mm
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thick slab-gel), and a 57 kDa protein eluted from the gel was used for further studies.
Antibody Production and Purification
The 57 kDa albumin-binding protein eluted from preparative SDS-PAGE was used to immunize
rabbits. Approximately 50 .mu.g protein (per rabbit) was injected intramuscularly after mixing with
equal volume of Freund's complete adjuvant. A second injection was given after two weeks. Rabbits
were bled at 4 to 6 weeks after the second injection and the immune response was checked. The
preimmune serum IgG and the antiserum IgG were purified using protein A-sepharose column.
Immunoblotting
Endothelial cell membranes were subjected to SDS-PAGE (Laemmli, supra) , and
electrophoretically transferred to nitrocellulose or PVDF membrane. Non-specific binding was
blocked with 3% gelatin in TBS for 5 hours at 22.degree. C. The membrane was washed 2.times.
with 0.5% Tween-20 in TBS and incubated with antiserum diluted in TBS containing 1% gelatin.
The incubation was carried out for 4-6 hours, washed 2.times., and then incubated for 60 minutes
with the second antibody (goat anti-rabbit IgG coupled to alkaline phosphatase). After incubation,
the membranes were washed 2.times. and the protein bands were localized as described under
"Ligand Blotting". Molecular weights of the proteins were determined using known marker proteins.
Monolayer Binding Studies
BPMVEC were seeded (3.times.105 cells/well) in six well Corning tissue culture plates and grown
to confluence. The monolayers were washed 2.times. with serum-free medium (20 mM
HEPE/DMEM pH 7.4) and incubated with serum-free medium for 15-20 hours in a tissue culture
incubator. After this incubation, the monolayers were washed 2.times. with binding buffer (20 mM
HEPES/Tris HBSS pH 7.4) and the binding was initiated by adding 1 ml of 1 .mu.M .sup.125 I-BSA
in binding buffer. The incubation was carried out at 4.degree. C. for 60 minutes. The binding was
terminated by washing the monolayer 3.times. with the binding buffer. The radioactivity associated
with the monolayer was determined after lysing the cells with 1 N NaOH (Tiruppathi et al, Am. J.
Physiol. (Lung. Cell. Mol. Physiol.) L595-L601 (1992)). Non-specific binding was determined by
the inclusion of unlabelled BSA (40 mg/ml) during the binding procedure. The test components,
preimmune serum-IgG and the anti-57 kDa-IgG were preincubated for 30 minutes with the
monolayer prior to the addition of .sup.125 I-BSA.
Trans-cellular Flux Experiments
Transendothelial .sup.125 I-albumin flux rates in cultured endothelial monoloyers were used to
assess transendothelial albumin transport. The system used for this study has previously been
described (Cooper et al, J. Appl. Physiol. 62:1076-1083 (1987); Garcia, et al, J. Cell. Physiol.
128:96-104 (1986); Del Vecchio, et al, Vitro. Cell. Dev. Biol. 28A:711-715 (1992) and SiflingerBirnboirn et al, J. Cell. Physiol. 132:111-117 (1987)). The system measures the transendothelial
movement of tracer macromolecules in the absence of hydrostatic and oncotic pressure gradients. It
consists of luminal and albuminal compartments separated compartments separated by a
polycarbonate microporous filter (0.8 .mu.m pore diameter). BPMVEC were seeded at 105
cells/filter and grown for 3-4 days to attain confluency. Both compartments contained the same
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medium (20 mM HEPES-DMEM, pH 7.4) at volumes of 600 ml and 25 ml, respectively. The
luminal compartment was fitted with a Styrofoam outer ring, and "floated" in the abluminal medium
so that fluid levels remained equal after repeated samplings from the abluminal compartment. The
abluminal compartment was stirred continuously and the entire system was kept at 37.degree. C. by
a thermostatically regulated water bath. Transendothelial clearance of .sup.125 I-albumin was
determined as the volume of luminal chamber radioactivity cleared into the abluminal chamber. The
change in volume over time provided the .sup.125 I-albumin clearance rate in .mu.l/min as
determined by weighted least-squares non-linear regression analysis (BMDP Statistical Software,
Berkeley, Calif.).
At the beginning of the experiment, the luminal compartment was floated in the abluminal medium,
and filled with medium containing about 6 .mu.Ci/ml .sup.125 I-albumin. Albuminal samples, 400
.mu.l, were collected at 10 minute intervals for up to 60 minutes and the radioactivity was measured
using a gamma counter. At the end of the experiment, free .sup.125 I in the luminal and abluminal
compartments was determined using 12% TCA precipitation and the transendothelial .sup.125 Ialbumin flux rates were corrected for free .sup.125 I.
The day before the experiment, the BPMVEC monolayers were washed 2.times. with 20 mM
HEPES-DMEM pH 7.4 (serum-free medium) and incubated at 37.degree. C. in cell culture incubator
with serum-free medium for 12-15 hours. After this incubation period, the test components
(preimmune serum-IgG and the anti-57 kDA-IgG) were diluted in serum-free medium and incubated
with the monolayers for the desired periods. These monolayers were then used for transendothelial
albumin transport measurement.
Trans-epithelial flux rates were measured with slight modification to the method described for
endothelial cells. Flux rates were determined on primary AEC or the A549 human lung carcinoma
cell line cultured as described on Transwell filters (Costar) (Evans et al, Exper. Cell Res. 18:375-387
(1989)). Monolayer integrity is defined by transepithelial electrical resistance being greater than 500
ohms/cm.sub.2. Filters with intact monolayers were placed in a 24 well culture plate containing 1 ml
serum-free DMEM per well (abluminal chamber). The luminal chamber was filled with 200 .mu.l
serum-free DMEM containing the tracer molecule of interest (FITC-Insulin). The fluid levels in the
two compartments were the same, eliminating hydrostatic pressure. The filter system was
preincubated (30 mins) and then maintained at 37.degree. C. in a CO.sub.2 incubator throughout the
flux experiment. At one and two hours, 300 .mu.l samples were withdrawn from the abluminal
chamber and immediately replaced with serum-free DMEM. The fluorescence of the transcytosed
material was recorded on a plate reader, and the ratio of bound vs. free FITC determined by gel
filtration chromatography of the abluminal samples.
Actin Filament Distribution
The actin filament distribution and cytoskeletal changes in endothelial monolayers grown on the
filters were studied under the conditions identical to those used for the measurement of .sup.125 Ialbumin clearance rates. After the required pretreatment period with the test components, the
monolayers on the filter were fixed in 10% buffered formalin (Pallescences Inc., Warrington, Pa.),
permeabilized with 1% Nonidet P40 (Sigma), and stained with rhodamine phalloidin (Molecular
Probes, Inc., Eugene, Oreg.) as described by Phillips and Tsan, J. Histochem. Cytochem. 36:551-554
(1988). The intact filters containing the monolayers were removed from the wells and mounted on
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coverslips, covered with a 1:1 solution of glycerine in phosphate-buffered saline, and then covered
with a round coverslip and sealed. The slides were analyzed using a Nikon Lab Diaphot fluorescent
microscope (NiKon Inc., Melville, N.Y.) and photographed using TRI X Pan 400 ASA Kodak film).
Identification of Albumin-Binding Proteins
Plasma membranes were first isolated from BPMVEC by differential centrifugation and the
albumin-binding proteins present in this membrane fraction were identified using ligand blotting (see
above). A simple method was developed, to identify native albumin-binding proteins in endothelial
cell membranes. The membrane proteins were separated using SDS-PAGE and then transferred to
PVDF or nitrocellulose. Non-specific binding was blocked by incubating the membrane strips with
Tween-20, and then treated with globulin-free monomeric native BSA. The BSA-binding regions
were identified using polyclonal antibody raised against native BSA. In the absence of exposure of
the membrane strip to native BSA, the anti-BSA recognized only a 67 kDa polypeptide, indicating
the presence of a significant amount of BSA bound to endothelial cell membranes. However, when
the strip was treated with BSA, the anti-BSA antibody reacted with 3 additional polypeptides (110
kDa, 57 kDa and 18 kDa). Of these polypeptides, the antibody reacted most intensely with 57 kDa,
indicating the 57 kDa polypeptide to be the major native albumin-binding protein. Total endothelial
cell membrane fractions (100,000.times.g particulate fraction from BPMVEC and BPAEC) were
also prepared and used for ligand blotting. These particulate fractions also showed a primary
interaction of BSA with the 57 kDa polypeptide.
Isolation of the 57 kDa Albumin-Binding Protein
Since binding of native albumin was seen primarily with the 57 kDa protein, a method was
developed for the isolation of this protein from BPMVEC membranes. Ligand blotting was
employed to assess the presence of this protein during purification. BPMVEC membranes were
initially solubilized with 2.5% sodium cholate and 4M urea, and the extract was dialyzed and
concentrated by 60% ethanol precipitation. This precipitate was re-extracted with Triton x-100 (see
above). The Triton x-100 solubilized extract was chromatographed on the DEAE column, and the
bound proteins were eluted with linear gradient (0-500 mM NaCl). The proteins were eluted as 3
peaks. The fractions from each peak were pooled and screened for albumin-binding using the ligand
blotting assay. Only one peak (I) showed albumin-binding with the 57 kDa protein region.
SDS electrophoresis was conducted, using proteins from native BPMVEC membrane and DEAE
column peak I after staining with Coomassie brilliant blue R-250. The presence of 57 kDa protein
corresponding to albumin-binding was observed with ligand blotting in both native membranes as
well as in DEAE peak I. SDS-PAGE was also performed under non-reducing conditions (in absence
of .beta.ME), and the albumin-binding was observed only with 57 kDa region, suggesting the
absence of sulfide link in this protein. This protein was further purified using preparative SDSPAGE, and the protein eluted from gel was used for the antibody preparation.
Immunoblotting
BPMVEC and BPAEC membrane proteins were separated by using SDS-PAGE and transferred to
nitrocellulose strips. The strips were immunoblotted with the 57 kDa antiserum. The preimmune
serum did not recognize any proteins from BPMVEC and BPAEC membranes. The antiserum
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recognized two major proteins (57 kDa and 36 kDa) and one minor protein (43 kDa) in both
membrane preparations. The particulate fractions from BPMVEC and BPAEC were also used for
immunoblotting. The antibody recognized only these three proteins in the particulate fractions. This
suggests that the albumin-binding protein was purified to an apparent homogeneity.
To study the proposed structural relationship between the endothelial membrane-associated and
secreted (SPARC) albumin-binding proteins, immunoblotting of BPMVEC membranes was carried
out with the antibodies raised against purified bovine SPARC. The antiserum raised against purified
bovine SPARC recognized 67 kDa, 61 kDa, 57 kDa, 43 kDa and 36 kDa polypeptides in BPMVEC
membranes. The anti-SPARC-NH2 terminal peptide antiserum reacted strongly with a 36 kDa
polypeptide and weakly with a 43 kDa polypeptide. This suggests that scavenger receptors are quite
different from native albumin receptors.
Effect of Anti-57 kDa-IgG on Binding of .sup.125 I-BSA to BPMVEC Monolayers
Preimmune serum-IgG and the anti-57 kDa-IgG were affinity-purified using Protein-A Sepharose
column. The influence of IgG fractions on binding of .sup.125 I-BSA to BPMVEC monolayers at
4.degree. C. was investigated: non-specific binding ranged from 40-50%. The preimmune serumIgG did not significantly affect the specific binding of .sup.125 I-BSA to the BPMVEC monolayers.
In contrast, the anti-57 kDa-IgG reduced the specific binding of .sup.125 I-BSA to BPMVEC
monolayers in a dose-dependent manner. The reduction was maximum (40-50%) at 200 .mu.g/ml
concentration in anti-57 kDa-IgG, and remained unchanged up to 1000 .mu.g/ml.
These results demonstrate that the antibody developed against the 57 kDa protein does not fully
recognize the albumin-binding domain in the receptor, or that the native albumin may interact with
other binding sites on endothelial cell surface.
Activation of Transendothelial Albumin Flux by Anti-57 kDa-IRG in the Absence of Endothelial
Cell Shape Change
To study the effects of the anti-57 kDa-IgG on transendothelial transport of albumin, the
transendothelial .sup.125 I-BSA clearance rates in BPMVEC monolayers was measured. The
monolayers were preincubated with preimmune serum-IgG and anti-57 kDa-IgG for 15 minutes, 30
minutes and 60 minutes, and then the transendothelial .sup.125 I-BSA clearance rates were measured
up to 60 minutes. The anti-57 kDa-IgG-induced increase in permeability was time-dependent. A 30minute period of preincubation of anti-57 kDa-IgG resulted in a 2-fold increase in .sup.125 I-BSA
clearance rate over preimmune IgG. No significant increase in permeability was seen with 15 min.
preincubation, and a 40-50% change was noticed when anti-57 kDa-IgG was pre-incubated with the
monolayer up to 60 min. The preimmune serum-IgG had no influence on transendothelial albumin
transport at all preincubation periods tested. The anti-57 kDa-IgG effect on the permeability of
.sup.125 I-albumin reverted at 4.degree. C.
The shape change of endothelial cells after treating with preimmune serum-IgG and anti-57 kDa-IgG
was studied, using a technique described previously (Phillips and Tsan, supra; Siflinger-Birnboim et
al, Lab Invest. 67:24-30 (1992)). BPMVEC grown on nucleopore filters were preincubated with
preimmune serum-IgG and anti-57 kDa IgG for 30 min., and the monolayers were stained with
rhodamine phalloidin (see above). No cell "rounding" or formation of interendothelial gaps was
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Principal Investigator/Program Director (Last, first, middle):
Malik, Asrar B.
observed in either case.
These results suggest that anti-57kD albumin-binding protein antibody activates albumin transport.
There is another possibility, i.e. that this antibody may non-specifically increase the pericellular
transport of albumin, by widening the interendothelial junctional gaps. To delineate this, the effect of
anti-receptor IgG and preimmune serum IgG on endothelial cell morphology was studied.
Pretreatment of BPMVEC monolayers with either preimmune serum-IgG or anti-receptor-IgG had
no influence on interendothelial junctional gaps. This antibody to the 57 kDa albumin-binding
protein may activate the transcytosis of albumin. The permeability increasing effect of this antibody
did not occur at 4.degree. C., supporting the conclusion that the antibody activated albumin
transcytosis via formation of vesicles, which have been shown to be temperature-sensitive (Lo et al,
J. Cell. Physiol. 151:63-70 (1992)).
EXAMPLE 2
Antibodies Raised Against GP60
Antibody raised against GP60 from endothelial cells was used to probe epithelial membrane extracts
as described in Example 1. The anti-GP60 antibodies recognized a 60 kDa protein found in the
epithelial extracts. This clearly shows that an immunologically-related protein is present in this
system.
Epithelial and endothelial cells were grown as monolayers, as described in Example 1, to produce
confluent monolayers showing the appropriate reactivity to solute flux. Anti-GP60 antibody (200500 .mu.g/ml) was incubated with the monolayers at 4.degree. C. to bind antibody to the receptor, in
the absence of metabolic activity that might result in internalization of the GP60. Binding of antiGP60 antibody under these conditions resulted in a 80-90% decrease in .sup.125 I-BSA binding by
the endothelial monolayers. The epithelial monolayers were further incubated with a second
antibody to the primary rabbit anti-GP60 antibody, to cross-link the receptors. Both monolayers
were washed and then incubated with .sup.125 I-BSA for the epithelial cells or .sup.125 I anti-BSA
immunoglobulin for the endothelial monolayers at 37.degree. C., to allow internalization of the
receptor-antibody complex and co-transcytosis of the .sup.125 I-labelled tracer. Incubation with antiGP60 antibody resulted in a 1.6-2 fold increase in uptake over the level of a pre-immune serum
control. Thus, binding the GP60 receptor by an anti-GP60 antibody results in activation of the
transcytosis mechanism, thereby enhancing uptake of a macromolecule in the vicinity of the
invaginating membrane.
EXAMPLE 3
Use of Albumin with Macromolecules
Endothelial monolayers were incubated at 4.degree. C. in the presence of BSA, to initiate the
binding of BSA to GP60 but to prevent the internalization of the ligand receptor complex. After
extensive washing to remove unbound BSA, the cells were incubated with .sup.125 I-labelled antiBSA immunoglobulin at 37.degree. C., as the macromolecular tracer. Pre-treatment with BSA
enhanced transcytosis of the immunoglobulin tracer by 1.5 fold over the control cells pre-incubated
with unlabelled anti-BSA immunoglobulin. Further, when the cells incubated at 37.degree. C. were
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Principal Investigator/Program Director (Last, first, middle):
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washed and immediately taken through the same protocol, no macromolecular flux was observed;
this shows that, once internalized, the GP60 receptor is unavailable for ligand binding. Thus, large
(150 kDa) molecules can be co-transcytosed in concert with HSA using the GP60 system.
EXAMPLE 4
Use of Albumin with Peptides
Human and rat epithelial monolayers were grown to confluence, as described in Example 1. The
cells were then incubated with FITC-insulin (1 mg/ml) or FITC-insulin and BSA (each 1 mg/ml) at
37.degree. C. in the transcellular flux system described above. For human and rat epithelial
monolayers, there was a 2.5 or 4 fold increase in FITC-insulin flux over the control of FITC-insulin
alone. Thus, albumin also stimulates co-transcytosis of small molecular weight peptides across
epithelial cells containing the GP60 receptor.
EXAMPLE 5
Use of N-Terminal Peptide 1-18 of GP60
A synthetic N-terminal peptide (T3118) of GP60 corresponding to the first 18 residues was produced
by solid-phase peptide synthesis. The sequence (SEQ ID No. 1) shows at least 80% homology with
the bovine, membrane-bound thyroid hormone (T3)-binding protein (Yamauchi et al, Biochem.
Biophys. Res. Comm. 146:1485 (1987)). It has 97% homology with PDI.
Antibodies were raised in rabbits against T3118, and used to probe endothelial membrane extracts,
to determine cross-reactivity with proteins recognized by anti-GP60 antibodies as described below.
BPMVEC membrane proteins (100 .mu.g) were separated on SDS-PAGE and transferred to
nitroulose membrane strips. Non-specific binding was blocked with 5% non-fat dry milk in Trisbuffered saline. The antisera were diluted in blocking solution, incubated for 4-5 hrs at 4.degree. C.,
washed and treated with goat-anti-rabbit-IgG conjugated with alkaline phosphatase. The protein
bands were identified using known molecular weight marker proteins. The anti-T3118 antibodies
showed only reactivity towards the GP60 protein and not towards the SPARC peptides recognized
by the anti-GP60 antibody.
The T3118 peptide was then used in an endothelial uptake experiment to determine if it would act as
an antagonist of albumin recognition and uptake. Endothelial monolayers were incubated at
4.degree. C. in the presence of .sup.125 I-BSA or .sup.125 I-BSA plus the T3118 peptide. After
incubation, the cells were washed extensively, lysed and counted for tracer uptake. Surprisingly,
rather than acting as an antagonist, the T3118 peptide actually stimulated uptake of albumin 5-fold
over the albumin alone control. The enhancement was saturable at a concentration of 500 .mu.m of
T3118 peptide. These data suggest that the T3118 peptide, acting as an agonist, may induce a
conformational change in albumin, which enhances recognition by GP60, or is the signal for uptake
by the endothelial cells.
It will be appreciated by those skilled in the art that the invention can be performed within a wide
range of equivalent parameters of composition, concentrations, modes of administration, and
conditions without departing from the spirit or scope of the invention or any embodiment thereof.
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Principal Investigator/Program Director (Last, first, middle):
*****
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Principal Investigator/Program Director (Last, first, middle):
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Reporting Structure Attachment #7
Dean COM
Standing
Committee
Associate
Director
Center Director
Center Faculty
Trainees
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Internal and
External
Advisory
Committee
Department
Heads