Full Text HD-94-019


NIH GUIDE, Volume 23, Number 14, April 8, 1994

RFA:  HD-94-019



National Institute of Child Health and Human Development

Application Receipt Date:  July 19, 1994


The Endocrinology, Nutrition and Growth (ENG) Branch of the Center
for Research for Mothers and Children, National Institute of Child
Health and Human Development (NICHD) issues this Request for
Applications (RFA) for support of investigations of in vivo
activities of lactoferrin in human beings and animals.

Lactoferrin is a 78,000-dalton metal-binding single-chain
glycoprotein found in milk and other exocrine secretions.  It is the
major protein component of human colostral whey, with concentrations
as high as 6 mg/ml.  Since the human lactoferrin gene has been
cloned, overexpression and large scale lactoferrin production are now

A great deal of structural information has been obtained about the
lactoferrin molecule.  It has been possible to relate this structural
information to two striking in vitro properties of lactoferrin, its
ability to bind a wide variety of metal ions with extremely high
affinity, and its ability to bind to a number of different types of

Lactoferrin also has bacteriostatic properties in vitro, which have
been thought to derive from its powerful sequestration of iron; but a
small bacteriostatic peptide obtained from lactoferrin digestion is
remote from the iron binding site in the intact molecule.

Lactoferrin from mother's milk can be absorbed to some extent by the
nursing infant and excreted in the infant's urine, but no
reproducible published evidence has established any in vivo activity
of lactoferrin.  Nevertheless, there is an international trend toward
the addition of lactoferrin to infant formula.  Spiking of formula is
already being done in Japan, and there is interest in doing so in
Europe and the U.S.  European formula manufacturers have produced a
transgenic bull which carries the gene for human lactoferrin.  This
animal is being used to sire daughters and granddaughters who are
expected to produce human milk containing human lactoferrin.  The
plan is to use this milk to manufacture infant formula.

It is important to derive hypotheses about biological function from
the extensive structural knowledge about the lactoferrin molecule and
test them in vivo.  This is needed in order to plan testing of the
utility and advisability of feeding lactoferrin-containing formula to
infants.  The impending addition of lactoferrin to all infant formula
may make future definitive trials seem unethical or impractical.


The Public Health Service (PHS) is committed to achieving the health
promotion and disease prevention objectives of "Healthy People 2000,"
a PHS-led national activity for setting priority areas.  This RFA, In
Vivo Activities of Lactoferrin, is related to the priority area of
childhood nutrition.  Potential applicants may obtain a copy of
"Healthy People 2000" (Full Report:  Stock No. 017-001-00474-0) or
"Healthy People 2000" (Summary Report:  Stock No. 017-001-00473-1)
through the Superintendent of Documents, Government Printing Office,
Washington, DC 20402-9325 (telephone 202-783-3238).


Applications may be submitted by domestic and foreign for-profit and
non-profit organizations, public and private, such as universities,
colleges, hospitals, laboratories, units of State and local
governments, and eligible agencies of the Federal government.
Foreign institutions are not eligible for the First Independent
Research Support and Transition (FIRST) (R29) awards.  Applications
from minority individuals and women are encouraged.


Applications in response to this RFA will be funded through the
research project grant (R01) and FIRST Award (R29) program of the
NIH.  This RFA is for a single competition with the application
receipt deadline of July 19, 1994.  Future unsolicited competing
continuation applications will compete with all
investigator-initiated applications and be reviewed by a Division of
Research Grants (DRG) study section.  However, if the NICHD
determines that there is a sufficient continuing program need, the
NICHD may announce a request for competitive continuation
applications.  The total project period for applications submitted in
response to the present RFA may not exceed five years.  The earliest
anticipated award date is April 1, 1995.


It is anticipated that three or more grants will be awarded under
this program, contingent upon receipt of a sufficient number of
meritorious applications and the availability of funds.  To fund
these awards the NICHD has set aside $750,000 for direct costs in the
first year.



Lactoferrin has been the focus of attention in numerous
investigations of the nutrition and host defense of term and preterm
infants, but no physiological role for it has been established.
Since the human lactoferrin gene has now been cloned, overexpression
and large scale production are imminent.  Thus, there is increased
urgency for the characterization of lactoferrin function.

Three striking in vitro properties of lactoferrin may be of
importance to its biological function: its ability to bind and
release a wide variety of metal ions, especially ferric iron (Fe),
its ability to bind cations, and its binding to a number of different
types of cells.  In milk, as in other secretions, lactoferrin is
mostly iron-free, with a saturation level of about 8 to 10 percent.
In iron-free form it has pronounced bacteriostatic properties in
vitro, probably dependent on its ability to bind adventitious iron
extremely tightly, so depriving bacteria of iron essential for
growth.  The bacteriostatic properties of human milk are thought to
derive largely from the high concentrations of lactoferrin present.
In addition, sequestration of iron by lactoferrin inhibits
iron-catalyzed free radical damage to cells.  For these reasons, and
because of its widespread occurrence in an frequent association with
species such as lysozyme and immunoglobulins, lactoferrin is regarded
as a component of the body's defense mechanisms.  Lactoferrin is also
a component of neutrophil secretory granules.

The lactoferrin molecules of different tissues and secretions appear
to be identical in structure and function, and the cDNA sequence of
leukocyte lactoferrin matches the amino acid sequence of milk
lactoferrin.  Lactoferrin is a member of the transferrin family of
iron binding proteins, which includes serum transferrin,
ovotransferrin, and the membrane-associated melanotransferrin.
Lactoferrin also bears a striking relationship to a sulfate-binding
protein of Salmonella typhimurium.

Lactoferrin has the capacity to bind reversibly two Fe ions
concomitantly with two carbonate (C03=) or bicarbonate (HC03-)
anions.  Three features of metal binding by lactoferrin are
particularly remarkable:  the synergistic relationship between cation
and anion binding, the extremely tight binding of iron, and the
existence of mechanisms for the release of tightly-bound iron.  Other
metals can be substituted for iron in the two specific sites; those
of similar size and charge (Ga, A1, Cr, Mn, Co) bind with affinities
close to that of Fe, but even much larger cations such as lanthanides
and some actinides (Th, Pu) can be accommodated.

Likewise, although CO3= is the anion of highest affinity, other
anions with a carboxylate group, some quite bulky, can be substituted
for it.  The two lactoferrin structural cavities in which Fe and the
anion are bound seem much larger than necessary for this function.
To some this has suggested that lactoferrin may function to bind
anionic toxins and xenobiotics.  All the Mn in human milk is bound to
lactoferrin, and lactoferrin has been suggested to have a role in Zn
binding and heavy metal absorption.  The physiological importance of
transferrin in A1 binding and its proposed therapeutic use in
detoxification may also apply to lactoferrin.

The ability of lactoferrin to bind to a variety of normal and
leukemic blood cells has led to a suggestion that the lactoferrin
released by neutrophilic leukocytes plays a role in modulating the
immune and inflammatory responses.  Lactoferrin promotes the
aggregation of neutrophils and their adhesion to epithelial cells,
and may be the agent that causes neutrophils to accumulate at
inflammatory sites.  Lactoferrin in its iron- saturated form is a
highly active inhibitor of myelopoiesis, leading to the suggestion
that lactoferrin might be useful in the treatment of leukemia.

Other observations that focus on the ability of lactoferrin to
interact with cells include its activity as an essential growth
factor for lymphocyte cell lines, its partial sequence homology with
a group of lymphoma transforming proteins, its interference in the
receptor-mediated uptake of chylomicron remnants into the liver, and
the observation that some antibacterial activities of lactoferrin
depend on actual contact with bacteria rather than simple
sequestration of iron.


The purpose of this RFA is to solicit applications for in vivo
studies of effects of lactoferrin in human beings or animals.  The
goal is to learn of the significance of human lactoferrin in human
milk to human infants or nursing women, but it is understood that
some kinds of direct experiments in humans are not advisable without
promising preclinical studies.  Animal model studies are therefore
also of interest provided they involve species-specific lactoferrin
or the use of heterologous lactoferrin that can be shown to mimic
homologous lactoferrin in adherence to receptors or effects on cells
in vitro.  A focus on effects of lactoferrin that apply to milk
rather than other external secretions is preferred.

The scope of this RFA, therefore, includes animal and human studies
of the biological effects of lactoferrin.  It does not include in
vitro bacterial or tissue culture investigations.  It is recognized
that some preliminary experiments in vitro may be required for
certain in vivo projects.  Nevertheless, the priority for funding of
each application will be determined by reviewers on the basis of the
relative merit of the in vivo studies proposed, and the likelihood
that the research will proceed to the whole animal or human level
within the terms of the recommended award.  Funded applications that
fail to demonstrate progress towards this stage may be phased out
before the recommended term of the award is completed.

The decision of the ENG Branch not to solicit applications for in
vitro studies of lactoferrin is not a result of any perception that
such studies are not important.  However, work of this kind is being
conducted widely; progress in the chemistry and cell biology of the
compound has been striking; and productive studies of lactoferrin
structure are being funded by the NICHD and others.  Studies of
direct relevance to infant nutrition are much more difficult to
execute, however, and are likely to be more speculative and uncertain
of success.  Meritorious applications of that type have been rare.
The Branch therefore seeks to encourage investigators to venture
appropriate proposals by setting aside funds for biological studies
in vivo.



It is the policy of the NIH that women and members of minority groups
and their subpopulations must be included in all NIH supported
biomedical and behavioral research projects involving human subjects,
unless a clear and compelling rationale and justification is provided
that inclusion is inappropriate with respect to the health of the
subjects or the purpose of the research.  This new policy results
from the NIH Revitalization Act of 1993 (Section 492B of Public Law
103-43) and supersedes and strengthens the previous policies
(Concerning the Inclusion of Women in Study Populations, and
Concerning the Inclusion of Minorities in Study Populations) which
have been in effect since 1990.  The new policy contains some new
provisions that are substantially different from the 1990 policies.
All investigators proposing research involving human subjects should
read the "NIH Guidelines For Inclusion of Women and Minorities as
Subjects in Clinical Research", which have been published in the
Federal Register of March 9, 1994 (FR 59 11146-11151), and reprinted
in the NIH GUIDE FOR GRANTS AND CONTRACTS of March 18, 1994, Volume
23, Number 11.

Investigators may obtain copies from these sources or from the
program staff or contact person listed below.  Program staff may also
provide additional relevant information concerning the policy.


The research grant application form PHS 398 (rev. 9/91) is to be used
in applying for these grants.  These forms are available at most
institutional offices of sponsored research and from the Office of
Grants Information, Division of Research Grants, National Institutes
of Health, 5333 Westbard Avenue, Room 449, Bethesda, MD 20892,
telephone 301-435-0714.

The RFA label available in the PHS 398 application form must be
affixed to the bottom of the face page.  Failure to use this label
could result in delayed processing of an application such that it may
not reach the review committee in time for review. In addition, the
RFA title and number must be typed on line 2a of the face page of the
application form and the YES box checked.

Applications for the FIRST Award (R29) must include at least three
sealed letters of reference attached to the face page of the original
application.  FIRST Award (R29) applications submitted without the
required number of reference letters will be considered incomplete
and will be returned without review.

Submit a signed, typewritten original of the application, including
the Checklist, and three signed, exact photocopies, in one package

Division of Research Grants
National Institutes of Health
Westwood Building, Room 240
Bethesda, MD  20892**

At the time of submission, two additional copies of the application
must also be sent to:

Susan Streufert, Ph.D.
Division of Scientific Review
National Institute of Child Health and Human Development
6100 Building, Room 5E01
Bethesda, MD  20892

Applications must be received by July 19, 1994.  If an application is
received after that date, it will be returned to the applicant.  The
Division of Research Grants (DRG) will not accept any application in
response to this RFA that is essentially the same as one currently
pending initial review, unless the applicant withdraws the pending
application.  If the application submitted in response to this RFA is
substantially similar to a research grant application already
submitted to the NIH for review, that has not yet been reviewed, the
applicant will be asked to withdraw either the pending application or
the new one.  Simultaneous submission of identical applications will
not be allowed, nor will essentially identical applications be
reviewed by different review committees.  Therefore, an application
cannot be submitted in response to this RFA that is essentially
identical to one that has already been reviewed.  This does not
preclude the submission of substantial revisions of applications
already reviewed, but such applications must include an introduction
addressing the previous critique.


Applications will be reviewed by NICHD staff for responsiveness to
the RFA.  Applications judged to be nonresponsive will be returned.
The applicant may resubmit the application and have it assigned for
review in the same manner as unsolicited grant applications.

Responsive applications may be subjected to a triage by a peer-review
group to determine their scientific merit relative to the other
applications received in response to this RFA.  The NIH will withdraw
from competition those applications judged to be noncompetitive and
notify the applicant and institutional business official.  Those
applications judged to be competitive will be further evaluated for
scientific/technical merit by a review group convened solely for this
purpose by the Division of Scientific Review, NICHD.  Criteria for
the initial review will include the significance and originality of
research goals and approaches; the feasibility of research and
adequacy of the experimental design; the research experience and
competence of the investigator(s) to conduct the proposed work; the
adequacy of investigator effort devoted to the project; and the
appropriateness of the project duration and cost relative to the work
proposed.  Following review by the Initial Review Group, applications
will be evaluated by the National Advisory Child Health and Human
Development Council for program relevance and policy issues before
awards for meritorious proposals are made.


The anticipated award date is April 1, 1995.  Scientific merit and
technical proficiency, based on the demonstrated and projected
capabilities described in the application will be the predominant
criteria for determining funding priorities.


Written and telephone inquiries concerning this RFA are encouraged.
The opportunity to clarify any issues or questions for potential
applicants is welcomed.

Direct inquiries regarding programmatic issues to:

Ephraim Y. Levin, M.D.
Center for Research for Mothers and Children
National Institute of Child Health and Human Development
6100 Building, Room 4B11
Bethesda, MD  20892
Telephone:  (301) 496-5593

Direct inquiries regarding fiscal matters to:

Mr. E. Douglas Shawver
Office of Grants and Contracts
National Institute of Child Health and Human Development
6100 Building, Room 8A17
Bethesda, MD  20892
Telephone:  (301) 496-1303


This program is described in the Catalog of Federal Domestic
Assistance No. 93.865, Research for Mothers and Children.  Awards are
made under the authority of the Public Health Service Act, Title IV,
Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC
241 and 285) and administered under PHS grants policies and Federal
Regulations 42 CFR Part 52 and 45 CFR Part 74.  This program is not
subject to the intergovernmental review requirements of Executive
Order 12372 or Health Systems Agency review.

The Public Health Service (PHS) strongly encourages all grant
recipients to provide a smoke-free workplace and promote the non-use
of all tobacco products.  This is consistent with the PHS mission to
protect and advance the physical and mental health of the American


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