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June 2015 The Pharmacologist
t h ePharmacologist
A Publication by The American Society for Pharmacology and
Experimental Therapeutics
Inside:Farewell Message from the President
EB 2015 in Review
Call for Award Nominations
Vol. 57 Number 2 June 2015
How the Humble Horseshoe Crab Saves Lives
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The Pharmacologist June 2015
Message from the President
EB 2015 in Review
EB 2015 Program Highlights
Fun Stats from EB 2015
Call for Award Nominations
Feature Story: Blue Bloods: How the Humble Horseshoe Crab Helps
Save Human Lives
Science Policy News
Education News
Journal News
New Membership
In Sympathy Dr. William Fleming
Members in the News
Division News
Meetings & Congresses
Contents...717278828588
97106110114116118119130
The Pharmacologist is published and distributed by the American
Society for Pharmacology and Experimental Therapeutics. THE
PHARMACOLOGIST
PRODUCTION TEAM
Prateeksha NagarSuzie Thompson Rich DodenhoffJudith A. Siuciak,
PhD
COUNCIL
President Annette E. Fleckenstein, PhD
President-Elect Kenneth E. Thummel, PhD
Past President Richard R. Neubig, MD, PhD
Secretary/Treasurer Paul A. Insel, MD
Secretary/Treasurer-Elect Dennis C. Marshall, PhD
Past Secretary/Treasurer Sandra P. Welch, PhD
Councilors Charles P. France, PhD John D. Schuetz, PhD Margaret
E. Gnegy, PhD
Chair, Board of Publications Trustees Mary E. Vore, PhD
Chair, Program Committee Scott Waldman, MD, PhD
FASEB Board Representative Brian M. Cox, PhD
Executive Officer Judith A. Siuciak, PhD
The Pharmacologist (ISSN 0031-7004) is published quarterly in
March, June, September, and December by the American Society for
Pharmacology and Experimental Therapeutics, 9650 Rockville Pike,
Bethesda, MD 20814-3995. Annual subscription rates: $20.00 for
ASPET members; $45.00 for U.S. nonmembers and institutions; $70.00
for nonmembers and institutions outside the U.S. Single copy:
$20.00. Copyright 2015 by the American Society for Pharmacology and
Experimental Therapeutics Inc. All rights reserved. Periodicals
postage paid at Bethesda, MD. GST number for Canadian subscribers:
BN:13489 2330 RT.
ASPET assumes no responsibility for the statements and opinions
advanced by contributors to The Pharmacologist.
Postmaster: Send address changes to: The Pharmacologist, ASPET
9650 Rockville Pike, Bethesda, MD 20814-3995.
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June 2015 The Pharmacologist
Message from
The PresidentMy Fellow Pharmacologists:
It has been my privilege to serve as president of the American
Society for Pharmacology and Experimental
Therapeutics (ASPET). It has been a year highlighted by many
positive changes that are directly attributable
to the outstanding efforts of those in the ASPET office. These
consist of important improvements in
communications with ASPET members, including a fresh new look
for both The Pharmacologist and the
ASPET website. Cooperative initiatives with the International
Union of Basic and Clinical Pharmacology
continue. A Division for Cancer Pharmacology was initiated.
Important new journal features were initiated,
including the introduction of visual abstracts,
CrossCheck/iThenticate, and revised instructions to authors
that proactively and enthusiastically support the
NIH-Nature-Science/AAAS workshop-initiated Principles and
Guidelines for Reporting Preclinical Research.
Legislative outreach continues, including visits to Capitol Hill
in efforts to advocate for sustained funding
from the National Institutes of Health (NIH) and other agencies
so as to ensure a bright future for biomedical
research. Interaction with the NIH and other important thought
leaders continues, as evidenced in part by the
inaugural Presidential Symposium at EB 2015.
Both the David Lehr Research Award and the Reynold Spector Award
in Clinical Pharmacology were
presented for the first time. ASPET continues to be very
grateful to Mrs. Lehr and to Dr. and Mrs. Spector for
their very generous support of the Society.
During the past year, ASPET has invested substantial resources
toward supporting young scientists.
As one example, three inaugural BIG IDEAS initiatives were
launched, with particular emphases on
undergraduate education, mentoring and diversity, and partnering
with industry. As another example, the
Mentoring and Career Development Committee provided exceptional
programming for young scientists at the
recent Experimental Biology meeting. Young scientists are key to
the future of the discipline of pharmacology,
and their support must continue to be a key priority for the
Society.
I sincerely thank both the ASPET staff and the ASPET Council for
their tremendous efforts this year. I
also thank the Division leadership, who have met regularly this
year and have cooperated to initiate many
improvements for the membership. Finally, I express gratitude to
the ASPET membership for the opportunity
to serve the Society as its president.
Sincerely,
Annette E. Fleckenstein
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The Pharmacologist June 2015
ASPET members filled the room on Saturday, March
28 to attend this years business meeting. President
Annette Fleckenstein updated members on the
Societys current activities, programs, and initiatives.
Highlights included the 2015 ASPET election results,
an announcement about our new Division for Cancer
Pharmacology, information about improvements
made to member communications, such as The
Pharmacologist and the new annual meeting website,
an introduction of ASPETs new Education Manager,
Dr. Catherine Fry, and an update on the 3 BIG IDEAS
chosen from our 2014 BIG IDEAS initiative. Members
in attendance discussed and voted on sending three
bylaws changes forward to the membership-at-
large. Reports were given from the ASPET Finance
Committee and the Board of Publications Trustees.
Finally, the 2015 ASPET award winners were
recognized and presented their awards. It was an
honor to have Mrs. Lisa Lehr and Dr. Reynold Spector,
who both gave moving speeches, with us at the
business meeting to help present the inaugural awards
for the David Lehr Research Award and the Reynold
Spector Award in Clinical Pharmacology, respectively.
Mrs. Lehr speaking at the awards ceremony
2015 ASPET Scientific Achievement Award Winners
EB 2015
The ASPET Annual Meeting at Experimental Biology 2015 took place
on March 28April 1, 2015 in Boston, MA. With over 14,300 attendees
this year, we held a highly successful meeting with very well
attended sessions, fun social events, great networking
opportunities, and a bustling booth in the exhibit hall.
IN REVIEW
Dr. Spector speaking at the awards ceremony
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June 2015 The Pharmacologist
73 73
2015 Washington
Fellows Travel Award Winners
2015 SURF Travel Award
Winners
2015 ASPET Graduate Student Travel Award Winners
2015 ASPET Young Scientist Travel Award Winners
Following the ASPET Business
Meeting, members celebrated the
start of the 2015 Annual Meeting
with an opening reception. With
over 300 people in attendance,
members enjoyed great food, an
open bar, and a lively atmosphere to
catch up with old and new friends.
Incoming President Kenneth Thummel
thanks President Annette Fleckenstein
for her services.
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The Pharmacologist June 2015
The ASPET booth in the exhibit hall had very good
traffic this year, and our booth activities were vibrant
and successful. We signed up a record number of new
members with 103 new applications. This included
10 new regular members, 2 new postdoc members,
39 new graduate student members, and 52 new
undergraduate student members. We also offered two
new products for sale in our store, a mens necktie
and a ladies scarf. We sold 124 items, with the ASPET
plush donkey still being our number one selling item. If
you didnt get a chance to purchase an ASPET logod
product at the meeting, you can make purchases
online at www.aspet.org/store. We also hosted a Meet-
a-Mentor event at our booth where students signed
up for appointments to meet with members of the
Mentoring and Career Development Committee.
ASPET Exhibit Hall Booth
Dr. Myron Toews models the new ASPET necktie.
Meet-A-Mentor
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June 2015 The Pharmacologist
The Student and Postdoctoral Best Abstract Competition gave
students and young scientists a chance to
present their work in a lively and fun atmosphere. All ASPET
divisions held their competitions simultaneously.
The competitions provided a great opportunity for students to
talk about their work and network with senior
members, colleagues, and friends. The ASPET divisions presented
their award winners with cash prizes and
award certificates. To learn who won the division competitions,
please turn to the division news section.
Also, given out at the Student and Postdoctoral Best Abstract
Competition was the 2015 Dolores C. Shockley
Best Abstract Award. Dr. Shockley was the first African American
woman to earn a PhD in pharmacology and
the first black woman appointed to chair a pharmacology
department in the US. In 2009, Dr. Shockley received
the Distinguished Alumni Award from her alma mater, Purdue
University. First place was awarded to Dominique
Jones from the University of Louisville for her
abstract entitled miR-186 Inhibition Alters
Cell Proliferation and Colony Formation in
Prostate Cancer, second place went to Alina
Monteagudo from the University of Rochester
for her abstract entitled Transglutaminase
2 as a Possible Chemotherapeutic Target in
Glioblastoma Multiforme, and third place
was shared by Kerri Pryce from SUNY-Buffalo
for his abstract entitled Regulation of the
sodium-activated potassium channel Slack by
MAGI-1 and Ariell Joiner from the University of
Michigan for her abstract entitled The Role of
Cilia in the Regulation of Olfactory Horizontal
Basal Cells.
Best Abstract Competition
Dr. Ashley Guillory presents Dominique Jones, Alina Monteagudo,
Ariell Joiner, and Kerri Pryce with the 2015 Dolores C. Shockley
Best Abstract Awards.
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The Pharmacologist June 2015
ASPET Student/Postdoc Mixer
Following the poster competition, ASPET Students and Postdocs
socialized at the Student/Postdoc Mixer.
The room was packed with young members dancing and having fun
with friends.
On Friday, March 27, 2015, ASPET members spent the day
volunteering at Cradles to Crayons, helping the children of
Boston.
To view the
full album of
EB 2015 pictures,
visit us online at: www.flickr.com/photos/
aspet_photo_gallery
76
Annual Meetings of:
American Society for Pharmacology and Experimental
Therapeutics
American Society for Investigative Pathology American Society
for Nutrition
2016
experimentalbiology.org
April 2 6San DiegoAbstract Deadline:Friday, November 6, 2015
See youNext Year!
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June 2015 The Pharmacologist
Annual Meetings of:
American Society for Pharmacology and Experimental
Therapeutics
American Society for Investigative Pathology American Society
for Nutrition
2016
experimentalbiology.org
April 2 6San DiegoAbstract Deadline:Friday, November 6, 2015
See youNext Year!
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The Pharmacologist June 2015
EB 2015 Program Highlights
We were pleased to welcome the inaugural ASPET
Presidential Symposium to our annual meeting
at EB 2015. The symposium, which focused on
Navigating the Future of Biomedical Research, was
organized by ASPET President Annette Fleckenstein
and highlighted challenging issues such as the
evolving nature of careers in science, reproducibility,
optimizing NIH-funded research, and pharmaceutical
discovery and development. The panel included
a diverse array of speakers who have worked in
academia, government, and industry during their
careers and brought to the symposium a valuable
perspective on science in the 21st century.
Michael S. Teitelbaum, senior research associate
at the Labor and Worklife Program at Harvard
Law School, discussed the patterns and trends in
science labor markets. Dr. Teitelbaum was Science
Careers Person of the Year in 2013, which honors an
individual who has made a significant and sustained
contribution to the welfare of early-career scientists.
His book Falling Behind? Boom, Bust, and the Global
Race for Scientific Talent, which examines the historic
pattern of recurrent panics over supposed shortages
of scientific talent, was released in March 2014.
Dr. Teitelbaum noted the productivity of US basic
research but emphasized the need to implement
policies to stabilize funding to avoid the recurring
alarm/boom/bust cycles noted since the 1940s. He
proposed candidate stabilizers such as aligning
graduate education more with career prospects than
research funds and emphasized the need to grow
R&D in a steady, predictable manner through longer-
term (e.g., 5 year) budget plans and counter-cyclicals
(e.g., bridge funding).
Nancy L. Desmond, office director and associate
director for Research Training and Career
Development at the National Institutes of Mental
Health (NIMH) discussed the National Institutes of
Health (NIH) and NIMH mechanisms for enhancing
the training of students and postdocs to better
prepare them for careers, including developing
professional skills that are transferrable, the use of
Individual Development Plans (IDPs) in grant progress
reports, the NIH Broadening Experiences in Scientific
Training (BEST) program, and the NIMH Biobehavioral
Research Awards for Innovative New Scientists
(BRAINS) for early stage investigators.
Shai Silberberg, program director for the
Extramural Research Program at the National Institute
of Neurological Disorders and Stroke (NINDS),
focused on Assuring a Bright Future for Biomedical
Research. Dr. Silberbergs lecture addressed the
issue of reproducible experimental results head-on,
describing the impact of bias, both unintentional and
ASPET Presidential SymposiumNavigating the Future of Biomedical
Research
The inaugural ASPET Presidential Symposium titled Navigating the
Future of Biomedical Research was held during the annual meeting at
EB2015.
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June 2015 The Pharmacologist
intentional, at all stages of research. He also discussed
efforts to improve the rigor of science and the
importance of professional integrity in research.
Phil Skolnick, director of the Division of
Pharmacotherapies and Medical Consequences
of Drug Abuse at the National Institutes of Drug
Abuse (NIDA), delivered a talk titled Navigating
the Future of Drug Development: Calm Seas or
Stormy Weather? Dr. Skolnick, who has extensive
experience in corporate and academic drug research,
leads a team involved in phases of therapeutic drug
development and clinical trial infrastructure. He
described the challenges faced by the pharmaceutical
industry, especially in the CNS disorders area, where
significant decreases in returns on R&D investments
have led to retreats from some therapeutic areas.
Dr. Skolnick noted contributing factors including the
increasing availability of generics and the under-
delivery of technology and genetics to contribute to
new drug discovery. To address these challenges,
he proposed an increasing decision-making role
for scientists, a focus on identifying new drugs that
improve upon current agents, and cited improved
patent protection as an important incentive for future
industry investment.
As the director of the Division of Pharmacology,
Physiology, and Biological Chemistry at the National
Institute of General Medical Sciences (NIGMS),
Michael E. Rogers oversees the administration of
more than 1500 research and training grants. Dr.
Rogers summarized NIH/NIGMS new directions in
supporting research in the context of the current
funding environment. These include programs to fund
investigators instead of projects, a new biosketch
format that emphasizes contributions to science,
and a revised grant resubmission policy at NIH. At
NIGMS, transformative efforts are directed toward
pursuing a broad and diverse research portfolio,
new guidelines for funding investigators with
substantial unrestricted research support, the MIRA
(Maximizing Investigators Research Award) program,
and mechanisms for providing training in scientific
rigor and reproducibility. Dr. Rogers noted a recent
increase in the success rate of funding at NIGMS after
a long decline.
We look forward to next years ASPET Presidential
Symposium, which will be organized by Dr. Ken
Thummel.
The Division for Translational
and Clinical Pharmacology (TCP)
sponsored a Meet the Experts
Lunch: Benchside-to-Bedside
Research at the annual meeting
in Boston. The session, which
featured four prominent clinical
pharmacologists from academia,
industry, and government, was well-
attended and gave the attendees
insight into a diverse spectrum of
viewpoints regarding where clinical
and translational pharmacology is
heading and how best to prepare
Meet-the-Experts Lunch: Benchside-to-Bedside Research
Sponsored by the Division for Translational and Clinical
Research
The inaugural Division for Translational and Clinical Research
Meet-the-Experts Lunch was held during the annual meeting at EB
2015.
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The Pharmacologist June 2015
ourselves for this transition. The lunch opened with a
general introduction of the topic and speakers by the
chair, Michael Holinstat, a translational pharmacologist
working in basic and translational research in
cardiovascular medicine at the University of Michigan.
Following general introductions, each of the speakers
described their background and view points on where
the field is heading and how best to traverse the
challenges going forward.
Andre Terzic, a physician scientist at the
Mayo Clinic who focuses on cardiovascular and
regenerative medicine, opened with insightful
comments regarding what are the important questions
of today and possibly tomorrow and where he sees
the field moving. His talk was followed by Darrell
Abernethy, the associate director for drug safety in
the Office of Clinical Pharmacology at the FDA, who
discussed his experiences both in academia as well
as government and what some of the challenges
are moving forward as well as the potential areas
for growth in the field. Next, the participants heard
from George Christ, the director of Basic Science
and Translational Research in Orthopedics at the
University of Virginia. Dr. Christ discussed the exciting
field of regenerative medicine and how this field,
which is just beginning to come of age, will blossom
over the next five to ten years and play a central role
in benchside-to-bedside medicine.
The final speaker for the session was Scott
Waldman, who is chair of the Department of
Pharmacology and Experimental Therapeutics at
Thomas Jefferson University. Dr. Waldman discussed
how he was able to take a basic science discovery in
his lab and translate this discovery into a clinical study
and eventually a clinical trial. He further discussed
the importance of following through with novel ideas
and how the lab and clinic can work symbiotically
to advance important clinical science concepts
for unmet needs. Junior investigators including
graduate students, postdoctoral fellows, and assistant
professors, especially benefited from this session
through direct interaction and question and answer
periods with each of the speakers. The Division for
Translational and Clinical Pharmacology is looking
forward to continuing this Meet the Experts lunch
series at next years meeting in San Diego.
Explore PharmacologyGraduate Studies in Pharmacology
Promote your graduate program in our Special Graduate Program
edition of Explore Pharmacology. This publication gives college
students an overview of the fundamentals and applications of
pharmacology.
In addition, it describes the many employment opportunities that
await graduate pharmacologists and outlines the academic path that
they are advised to follow. There is no better place to advertise
your graduate program!
Bene ts of Advertising with Explore Pharmacology:Distributed to
1,100+ undergraduate students and ASPET Undergraduate Student
Members who have a direct interest in pharmacology and related
graduate programs
Distributed at the Annual Biomedical Research Conference for
Minority Students (ABRCMS), the Society for Advancement of Chicanos
and Native Americans in Science (SACNAS) meeting, and the Society
for Neuroscience Annual Meeting where over 30,000 attendees are
expected
Copies will be sent to each of the 21 universities that
participate in ASPETs Summer Undergraduate Research Fellowship
(SURF) program
Advertising OpportunitiesAdvertise with a page, page, or full
page, 4-color display ad
Enhance your visibility by advertising on one of the covers
(inside front, inside back, or back cover) with a full page,
4-color ad
Your ad will be highlighted on the ASPET Departments and
Training Programs in Pharmacology webpage with a link to your
website from September 1 - December 31, 2015
Act quickly, the Space and Materials deadline is Wednesday, July
15.
If you have questions or would like to see sample ads, contact
ASPETs advertising department:
Jason WellsAdvertising [email protected]
301.634.7117www.faseb.org/adnet/aspet
Explore PharmacologyGraduate Studies in Pharmacology
American Society for Pharmacology and Experimental
Therapeuticswww.aspet.org
Explore Pharmacology - June 2015.indd 1 6/9/2015 8:12:04 AM
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June 2015 The Pharmacologist
Explore PharmacologyGraduate Studies in Pharmacology
Promote your graduate program in our Special Graduate Program
edition of Explore Pharmacology. This publication gives college
students an overview of the fundamentals and applications of
pharmacology.
In addition, it describes the many employment opportunities that
await graduate pharmacologists and outlines the academic path that
they are advised to follow. There is no better place to advertise
your graduate program!
Bene ts of Advertising with Explore Pharmacology:Distributed to
1,100+ undergraduate students and ASPET Undergraduate Student
Members who have a direct interest in pharmacology and related
graduate programs
Distributed at the Annual Biomedical Research Conference for
Minority Students (ABRCMS), the Society for Advancement of Chicanos
and Native Americans in Science (SACNAS) meeting, and the Society
for Neuroscience Annual Meeting where over 30,000 attendees are
expected
Copies will be sent to each of the 21 universities that
participate in ASPETs Summer Undergraduate Research Fellowship
(SURF) program
Advertising OpportunitiesAdvertise with a page, page, or full
page, 4-color display ad
Enhance your visibility by advertising on one of the covers
(inside front, inside back, or back cover) with a full page,
4-color ad
Your ad will be highlighted on the ASPET Departments and
Training Programs in Pharmacology webpage with a link to your
website from September 1 - December 31, 2015
Act quickly, the Space and Materials deadline is Wednesday, July
15.
If you have questions or would like to see sample ads, contact
ASPETs advertising department:
Jason WellsAdvertising [email protected]
301.634.7117www.faseb.org/adnet/aspet
Explore PharmacologyGraduate Studies in Pharmacology
American Society for Pharmacology and Experimental
Therapeuticswww.aspet.org
Explore Pharmacology - June 2015.indd 1 6/9/2015 8:12:04 AM
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The Pharmacologist June 2015
Find Some Fun Stats Below to See How Well We Did at EB 2015
Fun Stats at EB 2015
ASPET Booth
Thank you for visiting us at the ASPET booth!
We received double the number of new member
applications from students at the ASPET booth in
Boston than we had at EB 2014. The ASPET store did
well too!
of shoppers
purchased an
ASPET donkey
of shoppers purchased
the Einstein T-shirt
of shoppers purchased a
child-size T-shirt
28%
19%
13%
82
1 Julius Axelrod Award in Pharmacology Lecture2 Graduate
Student-Postdoctoral Colloquium:
How to Get Started3 Julius Axelrod Symposium: The Ins and Outs
of
G Protein-Coupled Receptor Signaling4 Biased GPCR Signaling in
Drug Development:
From Theory to Physiology5 Cardiac Fibroblasts: Fair-Weather
Friends in
Myocardial Fibrosis and Repair
ASPET SessionsMost Popular ASPET Events
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June 2015 The Pharmacologist
EB 201583
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5
ASPET Events
ASPET had 12 events in the top 10% of all EB events book- marked
on the Mobile App
ASPET had 1 event in the top 2% of all EB events bookmarked on
the Mobile App (Colloquium)
ASPET abstract submissions increased by 8% over last year.
ASPET registrants increased by 13% over last year.
The number of submissions to ASPET Travel Awards increased by
29%.
The number of submissions to ASPETs Best Abstract Awards
increased by 15%.
Interest in Pharmacology is increasing at EB!
Graduate Student-Postdoctoral Colloquium: How to Get Started
Julius Axelrod Award in Pharmacology Lecture
The Human Microbiome: Systems Pharmacology Insights and the
Potential for New Drug Discovery
ASPET Opening and Awards Reception
Speed Networking for Careers beyond the Academic Bench
ASPET Members Go Mobile!
Over 76% of EB participants downloaded
the EB 2015 Mobile App!
Top Bookmarked ASPET Events on the
EB Mobile App:
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The Pharmacologist June 2015
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June 2015 The Pharmacologist
Call for Award Nominations
ASPET Scientific Achievement Awards
ASPET presents several major awards to recognize accomplishments
either in specific areas of
pharmacology or contributions to and accomplishments in the
discipline in general. We will be launching a
new online award nomination system by this summer. The deadline
for nominations is September 15, 2015.
The John J. Abel Award in
Pharmacology is presented for
original, outstanding research in
the field of pharmacology and/
or experimental therapeutics by
a candidate who is younger than
45. This award, named after the
founder of ASPET, was established
in 1946 to stimulate fundamental
research in pharmacology and
experimental therapeutics by
young investigators.
The Julius Axelrod Award in
Pharmacology is presented
for significant contributions to
understanding the biochemical
mechanisms underlying the
pharmacological actions of
drugs and for contributions to
mentoring other pharmacologists.
This award was established in
1991 to honor the memory of the
eminent American pharmacologist
who shaped the fields of
neuroscience, drug metabolism,
and biochemistry and who served
as a mentor for numerous eminent
pharmacologists around the world.
The Pharmacia-ASPET Award
in Experimental Therapeutics
recognizes and stimulates
outstanding research in
pharmacology and experimental
therapeutics, basic laboratory,
or clinical research that has had,
or potentially will have, a major
impact on the pharmacological
treatment of disease.
For more information about these awards and to access the new
online nomination system, please visit:
www.aspet.org/awards/.
85
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The Pharmacologist June 2015
The Robert R. Ruffolo Career
Achievement Award in
Pharmacology recognizes
the scientific achievements of
scientists who are at the height
of their careers (typically mid-to
late-career) and who have made
significant contributions to any
area of pharmacology. This
award was established in 2011 in
recognition of the contributions
made to drug discovery and
development by Dr. Ruffolo.
The Louis S. Goodman and
Alfred Gilman Award in Receptor
Pharmacology was established
in 1980 to recognize and
stimulate outstanding research
in pharmacology of biological
receptors. Such research might
provide a better understanding
of the mechanisms of biological
processes and potentially provide
the basis for the discovery of
drugs useful in the treatment of
diseases.
Sponsored by the ASPET Division
for Drug Metabolism
The B. B. Brodie Award in
Drug Metabolism recognizes
outstanding original research
contributions in drug metabolism
and disposition, particularly
those having a major impact on
future research in the field. This
award was established to honor
the fundamental contributions of
Bernard B. Brodie in the field of
drug metabolism and disposition.
Sponsored by the ASPET Division
for Behavioral Pharmacology
The P. B. Dews Award for
Research in Behavioral
Pharmacology recognizes
outstanding lifetime achievements
in research, teaching, and
professional service in the field
of behavioral pharmacology.
The award honors Peter Dews
for his seminal contributions to
the development of behavioral
pharmacology as a discipline.
ASPET Division Sponsored Awards
Sponsored by the ASPET Division for
Cardiovascular Pharmacology
The Paul M. Vanhoutte Distinguished Lectureship in
Vascular Pharmacology was established to honor Dr.
Vanhouttes lifelong scientific contributions to our better
understanding and appreciation of the importance of
endothelial cells and vascular smooth muscle function
in health and disease and for his mentoring of countless
prominent endothelial and vascular biologists and
pharmacologists. This award includes a state-of-the-
art lecture on recent advances in vascular biology and
pharmacology at the ASPET Annual Meeting.
Proposed amendments to ASPET bylaws have been approved by the
membership-at-large. Thank you for voting! View the amendments
here: www.aspet.org/2015_Proposed_Changes_to_Bylaws/
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June 2015 The Pharmacologist
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Nominate Candidates for ASPET Awards
Did your mentor have a profound impact on you and the
pharmacology community? Nominate them for the Axelrod Award.
Have you mentored a young investigator whose original research
is outstanding? Nominate them for the Abel Award.
Do you have a colleague who has made a major impact on the
pharmacological treatment of disease?Nominate them for the
Pharmacia-ASPET Award.
Is the head of your department or lab at the height of their
career having made significant contributions to an area of
pharmacology?Nominate them for the Ruffolo Award.
Do you know someone who is performing outstanding research in
the pharmacology of biological receptors?Nominate them for the
Goodman and Gilman Award.
Exercise your membership benefits! Nominate someone who has made
an impression on you.
Face itmost of the time, research is a thankless job. What
better way to give long deserved
kudos to our everyday unsung heroes! Only ASPET members may
nominate candidates for
awards, so please make sure your membership is up-to-date.
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The Pharmacologist June 2015
It all started with one sick crab.
Frederik Bang was spending the
summer at the Marine Biological
Laboratory in Woods Hole,
Massachusetts, and contemplating
his choices for research. He had
his pick among a treasure trove
of marine species in MBLs Supply
Department, stocked daily from the
local fish catch. Horseshoe crabs
were plentiful, and the sick one, in
particular, piqued his interest.
Bang was a pathologist and
routinely used marine organisms
to gain insights into biological
mechanisms of clinical significance. In
the US Army Medical Corps, he had
directed research studies on malaria and other tropical
diseases
in the South Pacific. After World War II, he joined the
Medical
School faculty at Johns Hopkins University and spent his
summers
rotating between field laboratories in France (Station
biologique de
Roscoff); Calcutta, India; and Woods Hole. At Roscoff, he
studied a
marine worm that produced a thick mucus, hoping to elucidate
the
mechanisms responsible for cystic fibrosis. In Calcutta, he
studied
parasites and diarrhea. In Woods Hole, he played around with
oysters and marine worms, among other things.
In 1953, Bang was appointed chairman of the Department of
Pathobiology at the Johns Hopkins School of Public Health.
For
his summertime research, he headed to Woods Hole, where he
spotted that sick horseshoe crab. When it died, Bang
conducted
a necropsy to determine why. He discovered that the crabs
entire
blood volume had clotted into a semi-solid gel (1). Thus
began
a cascade of landmark investigations and the discovery of a
substance that has protected the lives of millions of
patients.
Photo by unidentified photographer.
Alan Mason Chesney Medical
Archives of The Johns Hopkins
Medical Institutions.
Blue Bloods: How the Humble Horseshoe Crab Helps Save Human
Lives Rebecca J. Anderson
Frederik Bang
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June 2015 The Pharmacologist
Characterizing the ClotsIn 1885, W. H. Howell first reported
that blood
withdrawn from the horseshoe crab almost
immediately forms a solid clot (2). But most of what
Bang learned about the jelly-like clots came from Leo
Loeb. During his summer visits to Woods Hole in the
1920s, Loeb extensively studied horseshoe crabs and
characterized the clotting phenomenon.
Blood circulating in the horseshoe crab (Limulus
polyphemus) contains only one type of cell, the
amebocyte: a clear, nucleated, oblong cell that is
packed with granules. When Loeb placed blood
on a glass slide, he observed that the amebocytes
changed their shape and released their granules,
which then formed a gel (3, 4). Loeb also observed
that the amebocytes collected around certain foreign
bodies, but he did not pursue studies to determine
whether natural pathogens could trigger granule
release and gel formation. Bang suspected the trigger
was marine bacteria (1).
Horseshoe crabs live on the soft sandy and muddy
bottoms of near-shore seawater, which is teeming
with bacteriaone billion bacteria per ml (5, 6). Some
sort of bacterial infection had likely made the crab
sick and caused the intravascular coagulation that
Bang observed. Pursuing this possibility, he followed
the guidelines established by Robert Koch, who had
proposed criteria for determining that bacteria cause a
given disease (7).
During the summers of 1953 and 1954, Bang
extracted bacteria at random from fresh seawater and
injected samples into a series of horseshoe crabs.
The injected bacteria caused an active progressive
disease marked by extensive intravascular clotting
and death (1).
In subsequent experiments, he isolated bacteria
from naturally diseased animals and identified Vibrio,
a rod-shaped Gram-negative bacterium (8). Under
the microscope, Bang saw that bacteria caused
amebocytes to change their shape and release their
granules (1). When he mixed pure cultures of Vibrio
with amebocytes, a much heavier gel-like material
formed (8). The gel trapped and immobilized live
bacteria within minutes.
All of these findings satisfied Kochs postulates,
but some of Bangs observations seemed to refute
the idea that his horseshoe crabs were suffering from
a bacterial infection. He boiled bacterial suspensions
to kill the bacteria and found to our surprise that
the sterile liquid still caused extensive intravascular
clotting in a few minutes after injection and killed
the crabs within a few hours (1, 7). Also, when he
added his sterile liquid to blood removed from healthy
horseshoe crabs, it induced a stable gel (1). Bang
concluded that the pathogen responsible for these
effects was a heat-stable bacterial toxin (1).
By the early 1960s, Bang had learned as much as
he could about the pathology of bacterial infections
in the horseshoe crab. To study the clotting reaction
further and to characterize the putative toxin, he
needed the expertise of a hematologist.
While planning his next trip to Woods Hole, Bang
called C. Lockard Conley, the founder and chairman of
the Division of Hematology at Johns Hopkins. Conley
headed an active research group studying blood
coagulation, platelets, and hemorrhagic diseases, and
Bang asked if Conley could recommend someone to
assist with the blood clotting experiments.
To everyones surprise, the rather straight-laced
and old-school Conley proposed Jack Levin, a
young hematologist who had only recently joined his
research group. Starting research fellows in Conleys
lab just didnt get selected to spend a summer at
such a prestigious facility (9). The Marine Biological
Laboratory attracted biologists from all over the
world, and its productivity was equivalent to that of
many of the countrys universities combined (10).
Without exaggeration, Lewis Thomas said, If you
can think of good questions to ask about the life of
the earth, it should be as good a place as any to go
for answers (10). So, when Conley asked him, Levin
jumped at the opportunity. Joining Dr. Bang, Levin
A colony of horseshoe crabs off the Atlantic coast.
Photo by Breese Greg, U.S. Fish and Wildlife Service. In the
Public Domain.
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The Pharmacologist June 2015
said, was one of the smartest professional decisions
I ever made (9).
The Big BangIn the 1960s and 1970s, the Marine Biological
Laboratory was a place put togetherby what can
only be described as a bunch of people...It seems to
have a mind of its own, which it makes up in its own
way (10). When Levin arrived, Bang handed him a
horseshoe crab and said, Go to it (9). The large,
intimidating creature had nine eyes, ten legs, and a
long spikey tail protruding from its massive shell. But
Levin soon discovered his experimental subject was
docile, cooperative, and well suited to his studies.
Hemocyanin accounts for 90% of the cell-free
protein floating in the plasma of horseshoe crab
blood. This copper-containing protein turns blue
when oxygenated, making the crabs blood a milky
shade of blue. When Levin separated the blood cells
from plasma, he saw that (unlike the platelets in mammalian
plasma) the horseshoe crabs blue plasma
did not clot. The clotting factor, whatever it was,
resided in the amebocytes.
Levin also discovered, to his dismay, that the
clotting process was remarkably robust. In all of his
early experiments, blood extracted from the crabs
clotted spontaneously. Levin tried every anti-coagulant
treatment, but nothing prevented the blood from
clotting (9).
Finally, as a last resort, he made use of his studies
in Conleys laboratory, where he had seen rabbit blood
coagulate after exposure to bacterial endotoxins.
Bacterial endotoxins survive the standard wet
sterilization procedure that kills bacteria. Levin thought
perhaps endotoxin contamination of his laboratory
equipment was causing the persistent blood clotting.
To test his theory, he baked all of his glassware
with sustained dry heat (the only way to ensure
inactivation of endotoxins) and found that blood
extracted and placed in his heat-treated glassware
did not clot. Subsequently, he conducted all of his
experiments with glassware that had been rigorously
decontaminated in a drying oven at 180-190C for over
2 h. In addition, he used only distilled water and other
solutions that had been certified as endotoxin-free for
human use (9).
Other than those precautions, though, Levin
worked under Spartan conditions. Despite its stellar
reputation, Woods Hole operated like a summer
camp. One cold water spigot served the whole
lab. For hot water, they boiled it over a gas flame. Jack Levin
collecting blood from a horseshoe crab at the Marine Biological
Laboratory.
Jack
Le
vin
Flasks of blue blood collected from horseshoe crabs. The white
layer in the flask on the right shows amebocytes settling from the
blood plasma.
Jack
Le
vin
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June 2015 The Pharmacologist
Open windows provided the only air conditioning
and ventilation, and Levin conducted all of his
experiments bare-handed. Other workers routinely
carted fresh marine specimens in and out for their
experiments. There was no fume hood, and Levins
use of N-ethyl-maleimide (a potent eye irritant he
needed to prepare his amebocytes and prevent
them from clumping) annoyed his lab mates and
discouraged visitors.
Despite the makeshift environment, Levin
succeeded in controlling all of his subsequent
experiments, thanks to the baked glassware and
careful laboratory technique. My most valuable
laboratory instrument was that drying oven (9).
Levin and Bang reported their initial findings in
1964. They demonstrated that the amebocyte is
necessary for clotting horseshoe crab blood. The
clotting factors are located in the granules of the
amebocytes and not in the blood plasma. And the gel-
clot reaction occurs when those clotting factors are
exposed to bacterial endotoxins (11, 12).
Endotoxins are lipopolysaccharides, which are
found in the outermost cell wall layer of Gram-negative
bacteria. Little bits of lipopolysaccharides break off
as the bacteria move in their environment. When the
bacteria are killed or crushed, they release larger
amounts. Levins results were consistent with Bangs
earlier observation that horseshoe crab blood clotted
when exposed to dead Gram-negative bacteria. Bang
had also observed that Gram-positive bacteria do not
induce a clotting reaction (1). (The cell walls of Gram-
positive bacteria do not contain lipopolysaccharides.)
Surviving in the Bacterial SoupHorseshoe crabs are a resilient
species. They
can trace their ancestry back more than 400 million
years to the Paleozoic erabefore humans, before
the dinosaurs, and even before flowering plants.
Along the Atlantic and Gulf coasts, they contribute
significantly to the marine ecosystem. Horseshoe
crabs are a predator of mollusks and marine
worms, and their eggs provide a food source for
shorebirds, fish, and crustaceans (13, 14). They also
churn and aerate ocean sediments, which facilitates
oxygenation of the estuaries and marine food
production (15).
Somehow, horseshoe crabs have adjusted to
life in a bacterial soup in which 95% of the single-
cell organisms are Gram-negative bacteria (6).
Crabs lack an immune system and cannot produce
antibodies, but they have developed simple, efficient
mechanisms for fending off environmental threats
(5). The amebocytes in their blood serve many of
the same functions as white blood cells in mammals.
Amebocytes engulf foreign or dead cells, transport
and store digested materials, and repair wound sites,
among other things (16).
The horseshoe crabs rudimentary circulatory
system is highly functional. Rather than an extensive
network of arteries, veins, and capillaries, it is
characterized by large sinuses that allow direct
Horseshoe crabs are a resilient
species. They can trace their ancestry
back more than 400 million years to
the Paleozoic erabefore humans,
before the dinosaurs, and even before
flowering plants.
A view of the horseshoe crabs anatomy
In t
he
Pu
blic
Do
ma
in
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The Pharmacologist June 2015
contact of blood with the crabs internal tissues.
However, these sinuses also give bacteria easy
and extensive access to internal organs if the crab
is wounded or its helmet-shaped shell is cracked.
Fortunately, over the eons, the horseshoe crab has
developed an exquisitely sensitive mechanism for
detecting endotoxin and combatting invasion by even
minute amounts of bacteria.
In response to a bacterial threat, amebocytes
release their granules and liberate clotting proteins. A
gel forms to trap and prevent further entry of bacteria at
the trauma point, as well as block the leakage of blood.
Other antimicrobial substances (such as tachyplesins
and big defensins) are also released by the amebocytes
and liquidate the trapped microbes (17).
Reducing to PracticeLevin and Bang developed methods for
extracting
and isolating the clotting factors from the amebocyte
granules (12, 18). A needle is inserted in the crabs
cardiac chamber from the dorsal side, and the
blue blood is collected. After centrifuging, the blue
supernatant fluid is discarded and the packed
amebocytes are washed with saline. The cells are then
osmotically lysed by adding distilled water, releasing
the substances responsible for gel formation. The
cellular debris is removed by centrifugation and the
supernatant lysate is stored.
The resulting protein mixture was named Limulus
amebocyte lysate, or LAL, a very descriptive moniker
comprised of the generic name of the horseshoe
crab (Limulus), the blood cell that contains the clotting
substances (amebocyte), and the process Levin and
Bang used to harvest them (lysis).
The horseshoe crab is not the only species whose
blood will clot in the presence of Gram-negative
bacteria or their endotoxins. Investigators observed
the same clotting mechanism in lobsters, oysters, and
even some insects, but blood extraction from those
animals was challenging (7). Levin found horseshoe
crabs ideal: they are large, have a large blood volume,
and have only one type of blood cell, from which the
clotting substances can be easily extracted (9).
Levin was the first to use LAL in an assay for
detecting bacterial endotoxins (19). A small amount
of LAL was mixed with a sample solution in a test
tube (18). If endotoxin was present in the sample, the
solution gelled and stuck to the bottom of the tube
when inverted. Although the rate of gel formation can
be used to determine endotoxin concentration, more
recent quantitative methods using photometric and
turbidimetric endpoints have also been developed
and certified by the US Pharmacopeia (20).
LAL Lift-OffAcross Eel Pond from the Marine Biological
Laboratory where Levin and Bang were conducting
their experiments, Stanley Watson, a microbiologist
at the Woods Hole Oceanographic Institution, was
studying the role of bacteria in the marine nitrogen
cycle. He had isolated membrane fractions from some
Gram-negative marine bacteria and was looking for a
way to assess the purity of his samples (21).
Photomicrograph of amebocytes from a horseshoe crab. Each ovoid
cell is packed with granules.
Jack
Le
vin
LAL test. Test tubes on the left are control samples of LAL; on
the right, endotoxin has been added. The upper panel shows gel
formation. The lower panel shows flocculent that forms early in the
clotting reaction or in samples containing low concentrations of
endotoxin.
Jack
Le
vin
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June 2015 The Pharmacologist
A colleague told him about LAL, which Levin and
Bang had recently isolated at MBL. Commercial
sources of LAL were not available, so Watson
obtained horseshoe crabs from the MBL Supply
Department and set up production in his garage (9,
21). Unfortunately, his first batches were not sensitive
enough for his purposes (21).
Watson decided to spend a few weeks trying
to improve the sensitivity and reproducibility of his
LAL batches (21). The weeks turned into months and
a major research effort. Ultimately, he succeeded.
Watsons LAL could reliably detect as little as 10-12 g of
endotoxin (22). He not only produced LAL for his own
research but also shared excess samples with other
scientists who were studying bacterial endotoxins
(5). When demand outpaced supply, he patented his
procedure, set up a small company (Associates of
Cape Cod, Inc.), and produced LAL as a lyophilized
product (22).
Protecting PatientsAll Gram-negative bacteria, including
Pseudomonas, Salmonella, and Escherichia, release
endotoxin fragments from their cell walls. The
immune systems of healthy people routinely handle
these microorganisms when ingested, but in the
bloodstream, as little as 10-6 g of endotoxin can cause
endotoxemia: a high fever, organ failure, and possibly
septic shock (22, 23). Endotoxin contamination is the
most common cause of fever induced by intravenous
drugs and fluids, blood products, and disposable
pharmaceutical devices (24, 25).
Since the 1940s, pharmaceutical manufacturers
had relied on the rabbit Pyrogen Test for detecting
endotoxins in injectable drugs because, like humans,
rabbits exhibit a pyrogenic response to endotoxin
exposure. In the Pyrogen Test, rabbits are injected
with a small amount of solution from a sterile drug
batch. If the animals develop a fever, the batch is
considered pyrogenic and is rejected. The Pyrogen
Test and a test for sterility became the two most
important tools in parenteral drug manufacturing (5).
Unfortunately, the Pyrogen Test has inherent
disadvantages. It is time-consuming, expensive, and
nonspecific. Also, the method produces results that
are only qualitative, and the induced fever varies
between animals due to differences in animal handling
and interlaboratory factors. Some critics raised
concerns about excessive use of animals.
Regulatory officials still accept the Pyrogen Test as
a method for detecting bacterial endotoxin, but they
were willing to consider alternatives. Legionnaires
Disease provided a compelling argument in favor of
LAL use. The endotoxin of the Legionnaires bacillus
has a different spectrum of toxicity than other, more
common, Gram-negative bacteria. It induces only a
weak pyrogenic response in rabbits, but it is readily
detected by LAL1000-fold greater sensitivity (24).
Regulatory officials saw the LAL test as a simple,
reproducible, inexpensive, and highly sensitive
alternative to the Pyrogen Test. Also, LAL could be
used to assay for endotoxin in products (such as
radiopharmaceuticals, cancer chemotherapy agents,
vaccines, and intrathecal drugs) that are not amenable
to testing in rabbits (24, 25).
The Food and Drug Administrations Office of
Biologics established a reference standard for use in
determining the sensitivity of each batch of LAL, and
quantitation of endotoxin was defined in Endotoxin
Units (7, 18, 20). In 1973, the FDA published the first
guidelines for the manufacture of LAL (26). In 1977,
Associates of Cape Cod received the first commercial
license from the FDA to manufacture LAL for use in
pharmaceutical assays (5, 27).
Also in 1977, the FDA issued the first in a series of
guidance documents regarding validation and use
of LAL to detect endotoxins in medical products, and
regulatory officials began accepting data from the LAL
test as an alternative to the Pyrogen Test (5, 24, 27).
In parallel, the US Pharmacopeia issued a series of
monographs that established specific limits for bacterial
endotoxin contamination in various parenteral products
(e.g., intravenous drugs, intrathecal drugs, sterile water
for injection, and radiopharmaceuticals) (20, 24).
Some manufacturers were reluctant to employ
LAL because it was too sensitive, but most of them
readily adopted LAL as their preferred quality control
method for parenteral drugs (24). Unlike the Pyrogen
Test, which (for practical reasons) was only used to
assess the end product, LAL tests could be applied
across the entire manufacturing process of both
the drug substance and formulated product (7). This
series of quality control tests was especially beneficial
for biological drugs, which are expensive to produce.
Rejecting an entire lot of finished biological product
was much more costly than detecting and addressing
contamination at earlier stages of production.
Virtually all intravenous drugs, as well as in-process
materials (i.e., containers and closures, sterile water,
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The Pharmacologist June 2015
bulk drug materials, and excipients), must
now pass these multiple LAL checkpoints
before marketing (7). In addition, the
needles and tubing used to deliver those
drugs, as well as implantable devices (e.g.,
pacemakers) and artificial kidneys used
for renal dialysis, are also checked for
endotoxins using the LAL test (5).
LAL has some limitations. It cannot
distinguish between live and dead
bacteria, nor differentiate between
species of bacteria-generated endotoxins.
Fungi, as well as endotoxins, will elicit
the clotting reaction. Still, the LAL test
has been widely used not only for quality
control of injectable drugs but also
in many other situations. It is a handy
method for rapid diagnosis of urinary tract
infections and spinal meningitis. Other
analysts have used LAL to assess food
spoilage (fish, milk, and ground beef), as
well as air and water quality (5).
In recognition of Frederik Bangs
insightful research and its healthcare
impact, the International Endotoxin and
Innate Immunity Society and the Stanley
Watson Foundation established the
Frederik Bang Award in 1985. The biennial
award recognizes scientists for lifetime
achievements in endotoxin research.
LAL on an Industrial ScaleFrom one sick crab, a new industry
emerged based on Bang and Levins
discoveries. Specialist facilities in the
United States, Japan, and China (including
Associates of Cape Cod, Charles River,
Lonza, Wako Chemicals, and Hyglos) now
produce LAL commercially. They collect
600,000 horseshoe crabs each year and
harvest the blood, which is worth $60,000
per gallon (5, 28).
Along the eastern coast of the United
States, horseshoe crabs are caught in the
spring when they swim into very shallow
water to spawn. Although industrial-scale
production of LAL has been streamlined,
the method remains essentially the same
as that first described by Levin and Bang.
Technicians extract no more than 30% of
each crabs blood, and the animals are
then released back into the sea.
Studies have shown that the horseshoe
crabs blood volume rebounds in about
a week. Hemocyanin takes more than 6
weeks to recover, and the blood cell count
returns to normal in about 2-3 months (5,
13). Theoretically, horseshoe crabs could
be bled several times a year, but the New
England LAL manufacturers collect them
only once a year. This restricted bleeding
schedule allows the animals to recover,
and they may be recaptured and bled
again in subsequent years.
When released, the horseshoe crabs
return to their natural spawning areas,
but the impact of biomedical bleeding on
spawning productivity is unknown. Recent
studies have shown that horseshoe crabs
are more lethargic, slower, and less likely
to follow the tides for several weeks
after being bled (13). The bleeding and
catch-and-release procedures result in an
estimated 8-15% mortality in males and 10-
29% mortality in females (13).
Commercial harvesting of horseshoe
crabs by fisheries (for bait) and by the
biomedical industry (for LAL) is closely
monitored and regulated in the United
States (15). Of particular concern are
decreases in the proportion of spawning
females (from 30% to 10%) and in the
number of eggs deposited in spawning
beaches (13, 14). Research conducted
by the US Geological Survey along the
Atlantic and Gulf coasts suggested that
multiple factors are likely responsible for
these declines, including overharvesting
of the crabs for fishing bait (a preferred
bait for eels and predatory mollusks) and
perhaps climate change (29).
Off the shores of Cape Cod where
biomedical harvesting is concentrated,
bait fishing has not been allowed since
Biosketch:
Rebecca J. Anderson
holds a bachelors
in chemistry from
Coe College and
earned her doctorate
in pharmacology
from Georgetown
University. She has 25
years of experience
in pharmaceutical
research and
development and now
works as a technical
writer. Her most recent
book is Nevirapine
and the Quest to End
Pediatric AIDS. Email
rebeccanderson@msn.
com.
In the next issue of The Pharmacologist
Dr. Anderson will be
exploring a story about
the sleepy sickness,
Oliver Sacks, and the
early days of L-DOPA.
Dont miss the exciting
September 2015 issue.
From one sick crab, a new
industry emerged based on
Bang and Levins discoveries.
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June 2015 The Pharmacologist
2000 (5). Yet, despite the catch-and-release practices
of LAL manufacturers, the loss of horseshoe crabs
in this area, especially females, has been a growing
concern (15). The Massachusetts Division of Marine
Fisheries examined the factors contributing to
horseshoe crab mortality resulting from biomedical
bleeding. Based on the results of these studies,
LAL manufacturers implemented gentler handling
procedures in 2009, aimed at restoring the horseshoe
crab population (15).
Synthetic AlternativesThe declining horseshoe crab population
poses
a serious threat to both the marine ecosystem and
pharmaceutical manufacturers who rely on the LAL
test for quality control. Consequently, researchers
have been exploring endotoxin detection alternatives
that are not dependent on extraction of LAL from
horseshoe crab blood.
Levin and his colleagues proved that the reaction
between endotoxin and LAL was enzymatic and
described essentially all of the endpoints that are
currently in use (30, 31). They also isolated, partially
purified, and described coagulogen, the gel-producing
protein in LAL (31). Subsequent researchers identified
five LAL proteins that are involved in clot formation.
The first four proteins in the clotting cascade (Factors
C, B, G, and proclotting enzyme) are serine proteinase
pro-enzymes. The final substance is coagulogen, a
soluble protein that is cleaved to produce coagulin, an
insoluble gel (17).
Factor C is highly sensitive for detecting the
lipopolysaccharides found in the cell walls of Gram-
negative bacteria, whereas Factor G is highly sensitive
to the (1,3)--glucan present in the cell walls of fungi.
Invading pathogens trigger activation of these factors,
resulting in the sequential activation of Factor B
and the proclotting enzyme. In the final step of this
cascade, the activated clotting enzyme converts
coagulogen to coagulin (17).
Several research groups have devised assays
using recombinant Factor C. The rFC reagent has
been designed to activate a fluorogenic substrate in
the presence of endotoxin and produce a fluorescent
product. Commercial kits utilizing recombinant
Factor C are available from Lonza (PyroGeneTM)
and Hyglos (EndoZyme rFC). Unlike LAL, which is
activated by both the lipopolysaccharides of Gram-
negative bacteria and the glucans from fungi, the
rFC fluorescence assays are selective for bacterial
endotoxins.
While the rFC assays provide researchers with a
valuable tool for endotoxin detection in laboratory
research, pharmaceutical manufacturers still rely
almost exclusively on the LAL test. The FDA permits
the use of alternative endotoxin assays if the methods
have been validated according to US Pharmacopeia
compendial procedures (20, 27). But compendial
validation is a long and challenging process,
compared to the already-accepted LAL standard (5, 7).
Consequently, the lowly horseshoe crab, with
its helmet-shaped shell, prehistoric ancestry, and
blue blood, remains the sole sentry protecting
millions of patients from otherwise deadly endotoxin-
contaminated drugs.
Kochs Postulates The bacteria must be present in every case
of
the disease.
The bacteria must be isolated from the host with
the disease and grown in pure culture.
A pure culture of the bacteria causes the specific
disease when it is inoculated into a healthy
susceptible host.
The bacteria must be recoverable from the
experimentally infected host.
The clotting cascade in LAL. Bacterial endotoxins activate
Factor C, which in turn activates Factor B. Similarly, fungal
glucans activate Factor G. When activated by either Factor B or
Factor G, the clotting enzyme is activated, resulting in cleavage
of coagulogen to produce coagulin.
Re
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25. Blechov R and Pivodov D (2001) Limulus amebocyte lysate
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29. Faurby S, King TL, Obst M, Hallerman EM, Pertoldi C, and
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Science Policy
NIH FY 2016 Funding Now in Appropriators Hands
Final Spending Numbers De-pend on Congress Lifting of Budget
Caps
As the weather gets hotter in
Washington so too will the budget
battles facing Congress as it
continues to move forward to pass
spending bills funding the NIH and
other federal agencies and programs.
Last May found Congress in an
unusual position, having passed
the first joint budget resolution in
over five years. After a lot of self-
congratulations by members of
congress over passing what should
be an annual event, Congress
must now turn to a far more
importantand problematic issue
such as drafting and approving
real appropriations bills funding
government agencies for FY 2016.
Despite Congress genuflecting
over passing the budget resolution,
the bill is merely a guide to spending
and has no force of law. The budget
resolution offers suggested spending
levels, but the Appropriations
Committees will have their say at
the end of the dayeven if that day
might not come until or after FY 2016
begins on October 1. But the budget
resolution does reveal the political
fault lines in Congress and the
difficulty of finding a compromise on
spending that allows for growing the
NIH and other federal agencies.
For instance, the budget
resolution keeps sequestration
in place, continuing the squeeze
on non-defense discretionary
programs while increasing defense
spending by more than $40 billion
in an account not subject to the
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The Pharmacologist June 2015
2011 Budget Control Act spending caps. Senator
Patty Murray (D-WA), whose leadership helped
provide for some sequestration relief during the
past two years criticized the budget agreement
stating, Instead of working with us to build on the
bipartisan budget deal we struck last Congress,
Republicans have introduced a budget that would
lock in sequestration, hollow out defense and
non-defense investments, and use gimmicks and
games to paper over the problems. Murrays
comments also reveal the very real policy
fractures between Republicans and Democrats
that will play out through the remainder of this
summer and into fall. President Obama has
stated that he would veto any funding bill that
retains sequestration levels of funding. Office of
Management and Budget (OMB) Director Shaun
Donovan stated that the president has been
clear that he is not willing to lock in sequestration
going forward, nor will he accept fixes to defense
without also fixing non-defense.
Political Compromise NecessaryEven some Republicans admitted
that
passing spending bills that fall within the budget
resolution guidelines will almost certainly never
happen. Their strategy, according to Senate
Majority Leader Mitch McConnell (R-KY) is To try
to delay, reign in, and restrict various bureaucratic
overreach through the appropriation bills that
spend the money. The House has a large
enough Republican majority to pass spending
bills that more closely align with the budget
resolution. However, even in the House that is
not a given. House Appropriation Committee
Chair Hal Rogers (R-KY) has stated that there
likely will not be enough votes in his committee to
approve appropriations spending levels that are
already too low. However, the Senate will need
Democrats to pass spending bills, and that will
not happen unless the spending caps are lifted
for both non-defense discretionary spending and
defense spending.
Several weeks before the budget resolution
was approved, the House Appropriations
Committee approved its subcommittee allocations
for FY 2016. Not surprisingly, the total allocation
follows the discretionary (non-defense and
defense spending) $1.017 trillion spending cap
mandated by the 2011 Budget Control Act, as well
as adopted in the FY 2016 budget resolution. The
House allocation for the Labor-HHS-Education
subcommittee (the subcommittee that funds
NIH) is $153.05 billion. This is a decrease of $3.7
billion (2.4 %) from the current years level. The
research community recommended funding
levels of at least $163.6 billionthe level it was
in FY 2010. The FY 2016 spending allocations
essentially prevent any chance of the NIH, or any
other program, of receiving a modest increase.
Even if the NIH were to receive inflation increases,
it would mean more sizeable cuts to other
worthy programs. How or whether the Labor/
HHS subcommittee makes these programmatic
spending decisions remains to be seen, as
Chairman Rogers has indicated. The Committees
Ranking Democrat, Nita Lowey (D-NY) offered
an amendment to increase the allocations to
the Appropriations Committee by $75 billion
that would have brought the total allocation to
the spending levels in the Presidents FY 2016
budget. But that effort was rejected along a party
line vote. And Rep. Duncan Hunter (R-CA) told
the San Diego Union-Times that, As Congress
prioritizes spending under federal budget caps,
one area that deserves a bigger slice of pie is
NIH, which leads in medical advancement and
innovation, there are plenty of things that should
see less funding, but NIH isnt one of them.
At press time, the Senate had not approved its
subcommittee allocations.
Given the comments mentioned above by
Appropriations Chairman Rogers, Rep. Hunter,
and the efforts of Rep. Lowey, there appears to
The House allocation for the Labor-
HHS-Education subcommittee (the
subcommittee that funds NIH) is
$153.05 billion. This is a decrease of
$3.7 billion (2.4 %) from the current
years level.
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June 2015 The Pharmacologist
be some momentum to fix sequestration, lifting
the budget caps to allow agencies like the NIH to
begin the process of restoring funding that was
lost in the past decade. How to get there is both
the question and the problem. Congressional
leadership has said they hope to fast track
appropriations bills and finish well before the
current fiscal years ends on September 30. That
might be a pipe dream and at a minimum is
probably too much to hope for. More likely, as
we get closer to the end of this fiscal year, we
will hear, once again, talk of shutdowns and the
increased likelihood of continuing resolutions to
fund the government beyond the start of the new
fiscal year on October 1. What remains clear and
certain is the need for ASPET members to continue
to reach out to their Congressional delegation
during this process, reinforcing the need for
steady and sustained increases for the NIH to help
meet the many scientific opportunities that will
never be fulfilled under current funding levels.
What remains clear and certain is the
need for ASPET members to continue
to reach out to their Congressional
delegation during this process,
reinforcing the need for steady and
sustained increases for the NIH to help
meet the many scientific opportunities
that will never be fulfilled under current
funding levels.
Fiscal Year 2016 Appropriations for the National Institutes of
Health
The American Society for Pharmacology and
Experimental Therapeutics (ASPET) is pleased to
submit written testimony in support of the National
Institutes of Health (NIH) FY 2016 budget. ASPET
recommends a FY 2016 NIH budget of at least
$32 billion.
Steady and sustained investment in the NIH is
critical to improving human health, stimulating state
and local economies, and improving the nations
global competitiveness. We call upon Congress
to ensure that the NIH remains a national priority.
ASPET appreciates Congressional action in providing
NIH-needed increases in the FY 2014 and FY
2015 omnibus appropriations bills. However, these
increases did not restore the purchasing power
lost to sequestration in FY 2013. From 2003-2013,
the NIH budget failed to keep pace with inflation in
research costs leading to nearly a 25% reduction in
the agencys purchasing power and a 34% reduction
in the primary grant mechanism for supporting
investigator-initiated research. A FY 2016 budget
of $32 billion would enable the NIH to fund 465
more research grants and help restore the agencys
lost purchasing power that has occurred over the
past decade.
Additionally, if funding for the next ten years is
similar to that of the past decade, the nation will
lose a generation of young scientists. Increasingly,
these individuals, seeing no prospects for careers
in biomedical research, will leave the research
enterprise or look for employment in foreign
countries. Not only are jobs increasingly limited in
the academic sector, but the health industry too is
under significant stress. The brain drain of young
scientific talent jeopardizes the nations leadership in
biomedical research. A 2013 survey of ASPETs own
Written Testimony of the American Society for Pharmacology &
Experimental Therapeutics
Submitted to the House and Senate Appropriations Subcommittee on
Labor, Health and Human Services, Education & Related
Agencies
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The Pharmacologist June 2015
graduate students and post-doctoral researchers
revealed that 45% of post-doctoral trainees and
25% of graduate students say they are no longer
considering a career in biomedical research due to
the restrictive funding environment; 50% of graduate
students and 29% of post-doctoral trainees say they
are willing to consider leaving the United States to
pursue a career in biomedical research.
A $32 billion budget for the NIH in FY 2016 is
an important start to help restore NIHs biomedical
research capacity. Currently, the NIH only can fund
one in six grant applications, the lowest rate in the
agencys history. Many highly innovative proposals
that have important implications for human health go
unfunded as a consequence of limited NIH funding.
A budget of at least $32 billion in FY 2016 will
help the agency manage its research portfolio more
effectively without having to withhold funding for
existing grants to researchers throughout the country.
Only through steady, sustained, and predictable
funding increases can the NIH continue to fund the
highest quality biomedical research to help improve
the health of all Americans and continue to make
significant economic impact in many communities
across the country.
There is no substitute for a steady, sustained
federal investment in biomedical research. Industry,
venture capital, and private philanthropy can
supplement some elements of health research,
but they cannot replace the investment in basic,
fundamental biomedical research provided by the
NIH. Neither the private sector nor industry will be
able to fill a void for NIH-funded basic biomedical
research. Much of the research undertaken by
industry builds upon the discoveries generated from
NIH-funded projects. The majority of the investment
in basic biomedical research that NIH provides
is broad and long-term, providing a continuous
development platform for industry, which would not
typically invest in research that may be of higher risk
and require several years to fully mature. In addition
to this long-term view, the NIH also has mechanisms
in place to rapidly build upon key technologies and
discoveries that have the ability to have significant
impact on the health and well-being of our citizens.
Many of the basic science initiatives supported by
the NIH have led to totally unexpected discoveries
and insight that have transformed our mechanistic
understanding of and our ability to treat a wide range
of diseases.
Diminished Support for NIH Will Negatively Impact Human
Health
Additional cuts or limited growth in the NIH budget
will further reduce the NIHs purchasing power
and accelerate the loss of scientific opportunities
to discover new therapeutic targets. Without a
steady, sustained federal investment in fundamental
biomedical research, scientific progress will be
slower, and potentially helpful diagnostic methods,
therapies or cures will not be developed. For
example, more research is needed on Parkinsons
disease to help identify the causes of the disease and
help develop better therapies. As another example,
discovery of gene variations in age-related macular
degeneration could result in new screening tests
and preventive therapies. More basic research is
needed to focus on new molecular targets to improve
treatment for Alzheimers disease. As yet another
example, diminished support for NIH will prevent new
and ongoing investigations into rare diseases that the
Food and Drug Administration estimates almost 90%
are serious or life-threatening.
Historically, our past investment in basic
biological research has led to many innovative
medicines. The National Research Council reported
that of the 21 drugs with the highest therapeutic
impact, only five were developed without input from
the public sector. The significant past investment in
the NIH has provided major gains in our knowledge
of the human genome, resulting in the promise of
pharmacogenomics and a reduction in adverse
drug reactions that currently represent a major
worldwide health concern. The NIH is the world
leader in efforts to prevent and treat HIV-AIDS.
Several completed human genome sequence
analyses have pinpointed disease-causing variants
that have led to improved therapy and cures, but
further advances and improvements in technology
will be delayed with diminished NIH funding. The
evolution of patient care into what has been termed
personalized medicine or precision medicine
and its application to a wide range of clinical
disorders requires research to identify and test
optimal diagnostic and therapeutic approaches for
each individual. Our past support for the NIH has
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revealed new frontiers of immunopharmacology
and regenerative medicine which are producing
cost savings by reducing in-patient hospital care for
debilitative autoimmune diseases like rheumatoid
arthritis and restoring movement and function
through regenerative interventions. Furthermore, the
NIH must continue its support of research to prevent
and treat infectious diseases.
Investing in the NIH Helps America Compete Economically
A $32 billion budget in FY 2016 will also help
the NIH train the next generation of scientists
and provide a platform for broader workforce
development that is so critical to our nations
growth. While most NIH trainees follow a career
path in research, many individuals trained in the
sciences through NIH support become educators in
high schools and colleges. These individuals also
enter into other areas of technology development
and evaluation in the public and private sectors,
further enriching the community and accelerating
economic development.
NIH research funding catalyzes private sector
growth. More than 83% of NIH funding is awarded to
over 3,000 universities, medical schools, teaching
hospitals, and other research institutions in every
state. One national study by an economic consulting
firm found that federal (and state) funded research at
the nations medical schools and hospitals supported
almost 300,000 jobs and added nearly $45 billion
to the U.S. economy. NIH funding also provides
the most significant scientific innovations of the
pharmaceutical and biotechnology industries.
Thus, this investment will help to create jobs and
promote economic growth. A stagnating NIH budget
will mean forfeiting future discoveries and jobs to
other countries.
It is a sobering fact that the US share of global
research and development investment from
19992009 was only 31%, representing a decline
of 18%. In con