June 2015 - The Pharmacologist

69

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

70

The Pharmacologist June 2015

Message from the President

EB 2015 in Review

EB 2015 Program Highlights

Fun Stats from EB 2015


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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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


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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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.


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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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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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

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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!

77


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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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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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.

80


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


American Society for Pharmacology and Experimental Therapeuticswww.aspet.org

Explore Pharmacology - June 2015.indd 1 6/9/2015 8:12:04 AM

81



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


American Society for Pharmacology and Experimental Therapeuticswww.aspet.org

Explore Pharmacology - June 2015.indd 1 6/9/2015 8:12:04 AM

82


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%

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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


EB 201583

21

34

5

ASPET Events

ASPET had 12 events in the top 10% of all EB events bookmarked 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:



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/.

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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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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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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

89


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.

90


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

91


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

92


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

93


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,

94


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.

95


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.

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References1. Bang F (1956) A bacterial disease of Limulus polyphemus. Bull

Johns Hopkins Hosp 98(5):325-351.

2. Howell WH (1885) Observations upon the chemical composition and coagulation of the blood of Limulus polyphemus, Callinectes hastatus, and Cucumaria sp. Johns Hopkins Univ Circulars 5(43):4-5.

3. Loeb L (1927) Amoeboid movement and agglutination of amoebocytes of limulus and the relation of these processes to tissue formation and thrombosis. Protoplasma 2(1):512-553.

4. Loeb L (1928) Amoebocyte tissue and amoeboid movement. Protoplasma 4(1):596-625.

5. ERDG (2009) The Horseshoe Crab: How does the horseshoe crab protect the public health? available from: horseshoecrab.org/med/med.html

6. Watson SW, Novitsky TJ, Quinby HL, and Valois FW (1977) Determination of bacterial number and biomass in the marine environment. Appl Environ Microbiol 33(4):940-946.

7. Williams KL (2007) Limulus amebocyte lysate discovery, mechanism, and application, in Endotoxins: Pyrogens, LAL Testing and Depyrogenation, 3rd ed (Williams KL ed) pp 191-217, Informa Healthcare, New York.

8. Shirodkar MV, Warwick A, and Bang FB (1960) The in vitro reaction of Limulus amebocytes to bacteria. Biol Bull 118(2):324-337.

9. Levin J, personal communication.

10. Thomas L (1974) The Lives of a Cell: Notes of a Biology Watcher. Viking Press, New York.

11. Levin J and Bang FB (1964) The role of endotoxin in the extracellular coagulation of Limulus blood. Bull Johns Hopkins Hosp 115 265-274.

12. Levin J and Bang FB (1964) A description of cellular coagulation in the Limulus. Bull Johns Hopkins Hosp 115:337-345.

13. Anderson RL, Watson WH, and Chabot CC (2013) Sublethal behavioral and physiological effects of the biomedical bleeding process on the American horseshoe crab, Limulus polyphemus. Biol Bull 225:137-151.

14. Morris, W (August 22, 2014) Will a shorebird knot up bacterial endotoxin assay supplies? PDA Letter; available from: www.pda.org/publications/pda-publications/pda-letter/latest-news/2014/08/22/will-a-shorebird-knot-up-bacterial-endotoxin-assay-supplies-

15. Leschen AS and Correia SJ (2010) Mortality in female horseshoe crabs (Limulus polyphemus) from biomedical bleeding and handling: implications for fisheries management. Marine and Freshwater Behavior and Physiology 43(2):135-147.

16. Marine Biological Laboratory. The horseshoe crab: Blue blood; available from: hermes.mbl.edu/marine_org/images/animals/Limulus/blood/bang.html

17. Bergner A, Oganessyan V, Muta T, Iwanaga S, Typke D, Huber R, and Bode W (1996) Crystal structure of coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor. EMBO J 15(24):6789-6797.

18. Jorgensen JH and Smith RF (1973) Preparation, sensitivity, and specificity of Limulus lysate for endotoxin assay. Appl Microbiol 26(1):43-48.

19. Levin J, Tomasulo PA, and Oser RS (1970) Detection of endotoxin in human blood and demonstration of an inhibitor. J Lab Clin Med 75:903-911.

20. US Pharmacopeial Convention (2011) Chapter , Bacterial Endotoxins Test; available from: www.usp.org/sites/default/files/usp_pdf/EN/USPNF/revisions/m98830-bacterial_endotoxins_test.pdf.

21. Watson SW and Novitsky TJ (1991) Determination of bacterial number and biomass in the marine-environment by Watson, SW, Novitsky, TJ, Quinby, HL, and Valois, VW. Current Contents/Agriculture Biology & Environmental Sciences 50:10.

22. Sullivan JD and Watson SW (1978) Limulus lysate of improved sensitivity and preparing the same. US patent 4,107,077. 1978 Aug 15.

23. Beers MH, Porter RS, Jones TV, Kaplan JL, and Berkwits M,editors (2006) The Merck Manual of Diagnosis and Therapy. Merck Research Laboratories, Whitehouse Station, New Jersey.

24. FDA (March 20, 1985) Inspection Technical Guides: Bacterial endotoxins/pyrogens, no. 40; available from: www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htm.

25. Blechov R and Pivodov D (2001) Limulus amebocyte lysate (LAL) testan alternative method for detection of bacterial endotoxins. Acta Vet Brno 70:291-296.

26. FDA (Sept. 18, 1973) Limulus amebocyte lysate: additional standards. Federal Register 38(180):26103-26132.

27. FDA (June 2012) Guidance for industry pyrogen and endotoxins testing: questions and answers; available from: www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm314718.htm.

28. Monks K (Sept 4, 2014) Why this crabs blood could save your life. CNN; available from: www.cnn.com/2014/09/04/health/this-crabs-blood-could-save-your-life/index.html.

29. Faurby S, King TL, Obst M, Hallerman EM, Pertoldi C, and Funch P (2010) Population dynamics of American horseshoe crabshistoric climatic events and recent anthropogenic pressures. Molec Ecol 19:3088-3100.

30. Levin J and Bang FB (1968) Clottable protein in Limulus: its localization and kinetics of its coagulation by endotoxin. Thromb Diath Haemorrh 19:186-197.

31. Young NS, Levin, J, and Prendergast RA (1972) An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation. J Clin Invest 51:1790-1797.

97


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

98


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.

99


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

100


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

101

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

Documents

June 2015 - The Pharmacologist