Full Text HL-94-004

HEMATOLOGIC CONSEQUENCES OF HIV INFECTION OF MARROW CELLS

NIH GUIDE, Volume 22, Number 44, December 10, 1993

RFA:  HL-94-004

P.T. 34

Keywords: 
  Bone Marrow 
  AIDS 


National Heart, Lung, and Blood Institute
National Institute of Diabetes and Digestive and Kidney Diseases

Letter of Intent Receipt Date:  February 4, 1994
Application Receipt Date:  March 15, 1994

PURPOSE

The purpose of this initiative is to increase our understanding of
the cellular and molecular mechanisms that lead to the hematologic
abnormalities seen following HIV infection.  Investigators who are
well-trained in the modern techniques of cellular and molecular
biology will be encouraged to focus their expertise on issues
directly related to the understanding of cytopenia following HIV
infection, the role of hematopoietic stem and progenitor cells in the
development of AIDS, and to develop gene therapy strategies to
protect the hematopoietic stem/progenitor cell from being infected
with HIV or to promote intracellular suppression of HIV gene
expression.

HEALTHY PEOPLE 2000

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 Request
for Applications (RFA), Hematologic Consequences of HIV Infection of
Marrow Cells, is related to the priority area of HIV infection.
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 202-782-3238).

ELIGIBILITY REQUIREMENTS

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 or local
governments, and eligible agencies of the Federal government.  Awards
in response to this RFA will be made to foreign institutions only for
research of very unusual merit, need, and promise, and in accordance
with PHS policy governing such awards.  Foreign institutions are not
eligible for First Independent Research Support and Transition
(FIRST) (R29) awards.  Applications from minority individuals and
women are encouraged.

MECHANISM OF SUPPORT

This RFA will use the National Institutes of Health (NIH) individual
research grant (R01) and FIRST (R29) award and is a one-time
solicitation.  Applicants, who will plan and execute their own
research programs, are requested to furnish their own estimates of
the time required to achieve the objectives of the proposed research
project.  Up to five years of support may be requested.  At the end
of the official award period, renewal applications may be submitted
for peer review and competition for support through the regular grant
program of the NHLBI or the NIDDK.  It is anticipated that support
for the present program will begin on September 30, 1994.
Administrative adjustments in project period and/or amount of support
may be required at the time of the award.  All current policies and
requirements that govern the research grant programs of the NIH will
apply to grants awarded in connection with this RFA.

FUNDS AVAILABLE

Fiscal year 1994 financial plans for the NHLBI include $1.5 million
for this program.  The NIDDK plans to allocate an additional $1.0
million.  However, award of grants pursuant to this RFA is contingent
upon receipt of funds for this purpose.  It is anticipated that the
NHLBI will award about six new grants under this program, and the
NIDDK about five.  The specific amount to be funded will, however,
depend on the merit and scope of the applications received and on the
availability of funds.  Since a variety of approaches would represent
valid responses to this announcement, it is anticipated that there
will be a range of costs among individual grants awarded.  If
collaborative arrangements involve sub-contracts with other
institutions, the NHLBI Grants Management Staff (tel: 301-594-7436)
should be consulted regarding procedures to be followed.

RESEARCH OBJECTIVES

Background

The Centers for Disease Control estimates that approximately one
million people in the United States are infected with the HIV-1.
Therefore, AIDS and AIDS-related complex (ARC) are among the most
important public health challenges confronting not only the United
States, but the world today.

AIDS is characterized by opportunistic infections and by
opportunistic neoplasms such as Kaposi's sarcoma.  In addition, AIDS
patients frequently experience hematologic abnormalities such as bone
marrow dysplasia, anemia, thrombocytopenia, and leukopenia.  The
hematologic abnormalities contribute to worsening the clinical
condition and also limit the use of antibacterial as well as
antiviral agents.  The exact mechanisms by which HIV-1 contributes to
the characteristic cytopenias are presently unknown.

Both ineffective hematopoiesis and peripheral destruction of blood
cells may lead to cytopenia commonly seen in AIDS patients.  However,
multiple mechanisms are likely responsible for the cytopenias
observed in HIV-1 infected individuals.  Cytopenias may be due to
accelerated utilization and destruction of cells due to opportunistic
infections, reticuloendothelial cell dysfunction, or to specific
autoimmunity.  Ineffective hematopoiesis in patients with AIDS may
also be associated with the direct action of HIV-1 on (a)
hematopoietic stem/progenitor cells; (b) more mature blood cells; and
(c) bone marrow accessory cells, such as macrophages, endothelial
cells, and T-lymphocytes.  Other potential causes include the
suppressive effects of infections and the formation of circulating
inhibitors of hematopoiesis.

Differences remain in the field regarding direct infection of CD34+
bone marrow cells by HIV-1 with some groups reporting infectibility
(1,2) and other groups reporting an absence of infectibility (3,4).
The low levels of infectibility reported is insufficient to explain
the defective biological activity of stem/progenitor cells (5,6).
Exposure of normal CD34+ bone marrow cells to certain HIV-1 isolates
induces defects in BFU-E, CFU-GM, CFU-Meg, and CFU-TL derived colony
formation (7,8).  Therefore, more pertinent to mechanisms of
cytopenias in HIV-infected individuals may be the ability of HIV-1 to
directly induce defects in stem/progenitor cells.

Symptomatic anemia is the most common cytopenia and occurs in the
presence and absence of myelosuppressive drug therapy such as AZT.
The lack of reticulocyte response seen in these patients may be due
to a hypoproliferative anemia or to ineffective hematopoiesis.  Low
erythropoietin levels and associated abnormalities seen in some AIDS
patients may be a contributing factor to anemia. Thrombocytopenia is
also commonly seen in HIV infected patients, and immune
thrombocytopenic purpura has been recognized as a diagnostic category
of ARC.  Separate studies have shown that megakaryocytes may be a
target for HIV infection (9,10) suggesting a role for megakaryocytes
as viral reservoirs in the bone marrow and a potential mechanism for
defective thrombocytopoiesis.  Neutropenia is also common in HIV
infected individuals.

Attempts to reduce the impact of AIDS on the bone marrow failure
discussed above have focused on dose reduction of ganciclovir, AZT,
and chemotherapy, and the use of recombinant hematopoietic growth
factors and cytokines such as erythropoietin, G-CSF (granulocyte
colony-stimulating factor), GM-CSF (granulocyte macrophage
colony-stimulating factor), M-CSF (macrophage colony-stimulating
factor), IL-3 (interleukin-3), interferons, and stem cell factor.
These efforts have been successful in correcting some of the
cytopenias associated with HIV infections.  However, compounds that
interfere with viral replication, also appear to have damaging
effects on the bone marrow (11,12).  Thus, a number of AIDS patients,
although under treatment with current anti-viral compounds, will
become transfusion dependent.  Combined therapy, using cytokines and
anti-viral drugs, may allow the use of anti-viral compounds in
patients intolerant of the hematologic effects that they cause.

With the widespread use of recombinant growth factors and cytokines
in AIDS treatment, there is a need to elucidate their role in
modulating hematopoiesis and HIV infection.  The growth and
differentiation of hematopoietic stem/progenitor cells is primarily
regulated by cytokines.  Direct infection of other cell types in the
marrow could lead to decreased production of stimulatory cytokines or
increased production of inhibitory cytokines that act on
stem/progenitor cells.  Thus, it is likely that altered cytokine
production in HIV-1 seropositive individuals could play an important
role in the pathogenesis of the hematologic abnormalities
characteristic of HIV-1 infected individuals.  T-lymphocytes (CD4+)
appear to be the primary reservoir for HIV in vivo although marrow
cells belonging to the macrophage lineage also were infected at a
lower level with HIV-1 (13,14,15).  The ability of the HIV-1 tat
protein to induce marrow macrophages to increase their expression of
TGF-beta-1, a potent inhibitor of stem/progenitor cells has been
reported (16).  Therefore, determining the role of HIV viral proteins
in the negative regulation of hematopoiesis is important to pursue.
The use of the long term bone marrow culture system (17) will also
continue to be extremely valuable in elucidating the hematologic
abnormalities seen following HIV infection in the context of the
stromal environment.

Objectives

The objective of this initiative is to solicit research grant
applications on the cellular and molecular mechanisms that lead to
the hematologic abnormalities seen following HIV infection.

The following are only examples of the types of research approaches
that would be responsive to this program:  (a) determine the role of
hematopoietic stem/progenitor cells in the development of AIDS; (b)
elucidate the pathways of signal transduction in hematopoietic cells
activated by cytokines that may regulate HIV processes in the
infected cell; (c) develop gene therapy strategies using the
hematopoietic stem cell as the target for transfection as a means for
protecting the hematopoietic stem cell, and thus all blood cells,
from becoming infected with HIV; (d) determine the role of HIV viral
proteins in the negative regulation of hematopoiesis; and (e)
development and testing of therapeutic modalities for bone marrow and
hematologic abnormalities associated with HIV infection and AIDS.

These examples are not meant to be all inclusive or restrictive.
Investigators are encouraged to develop their own innovative
approaches.

Epidemiological studies, large-scale clinical trials, and large
multi-project grant applications (program project grants) are
specifically excluded from this RFA.  Also, research focused on
testing the effectiveness of vaccines for HIV infection is excluded.

SPECIAL REQUIREMENTS

Upon initiation of the program, the NHLBI and the NIDDK will sponsor
annual meetings to encourage the exchange of information among
investigators who participate in this program.  In the preparation of
the budget for the grant application, applicants may request
additional travel funds for one meeting each year to be held in
Bethesda, Maryland.  Applicants may also include a statement in the
applications indicating their willingness to participate in such
meetings.

STUDY POPULATIONS

SPECIAL INSTRUCTIONS TO APPLICANTS REGARDING IMPLEMENTATION OF NIH
POLICIES CONCERNING INCLUSION OF WOMEN AND MINORITIES IN CLINICAL
RESEARCH STUDY POPULATIONS

NIH policy is that applicants for NIH clinical research grants and
cooperative agreements will be required to include minorities and
women in study populations so that research findings can be of
benefit to all persons at risk of the disease, disorder or condition
under study; special emphasis should be placed on the need for
inclusion of minorities and women in studies of diseases, disorders
and conditions which disproportionately affect them.  This policy is
intended to apply to males and females of all ages.  If women or
minorities are excluded or inadequately represented in clinical
research, particularly in proposed population-based studies, a clear
compelling rationale should be provided.

The composition of the proposed study population must be described in
terms of gender and racial/ethnic group.  In addition, gender and
racial/ethnic issues must be addressed in developing a research
design and sample size appropriate for the scientific objectives of
the study.  This information must be included in the form PHS 398
(rev. 9/91) in Sections 1-4 of the Research Plan AND summarized in
Section 5, Human Subjects.  Applicants are urged to assess carefully
the feasibility of including the broadest possible representation of
minority groups.  However, NIH recognizes that it may not be feasible
or appropriate in all research projects to include representation of
the full array of United States racial/ethnic minority populations
(i.e., Native Americans [including American Indians or Alaskan
Natives], Asian/Pacific Islanders, Blacks, and Hispanics).

The rationale for studies on single minority population groups should
be provided.

For the purpose of this policy, clinical research includes human
biomedical and behavioral studies of etiology, epidemiology, (and
preventive strategies), diagnosis, or treatment of diseases,
disorders or conditions, including but not limited to clinical
trials.

The usual NIH policies concerning research on human subjects also
apply.  Basic research or clinical studies in which human tissues
cannot be identified or linked to individuals are excluded.  However,
every effort should be made to include human tissues from women and
racial/ethnic minorities when it is important to apply the results of
the study broadly, and this should be addressed by applicants.

For foreign awards, the policy on inclusion of women applies fully;
since the definition of minority differs in other countries, the
applicant must discuss the relevance of research involving foreign
population groups to the United States' populations, including
minorities.

If the required information is not contained within the application,
the application will be returned.

Peer reviewers will address specifically whether the research plan in
the application conforms to these policies.  If the representation of
women or minorities in a study design is inadequate to answer the
scientific question(s) addressed AND the justification for the
selected study population is inadequate, it will be considered a
scientific weakness or deficiency in the study design and will be
reflected in assigning the priority score to the application.

All applications for clinical research submitted to NIH are required
to address these policies.  NIH funding components will not award
grants or cooperative agreements that do not comply with these
policies.

LETTER OF INTENT

Prospective applicants are asked to submit, by February 4, 1994, a
letter of intent that includes a descriptive title of the proposed
research, the name, address, and telephone number of the Principal
Investigator, the identities of any other key personnel and
participating institutions and the number and title of the RFA in
response to which the application may be submitted.   Such letters
are requested only for the purpose of providing an indication of the
number and scope of applications to be received; therefore their
receipt is usually not acknowledged.  A letter of intent is not
binding, and it will not enter into the review of any application
subsequently submitted, nor is it a necessary requirement for the
application.

This letter of intent is to be sent to:

Acting Chief, Centers and Special Projects Review Section
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 553
Bethesda, MD  20892
Telephone:  (301) 594-7448
FAX:  (301) 594-7407

APPLICATION PROCEDURES

Applications are to be submitted on the  research grant application
form PHS 398 (rev. 9/91).  This form is available in an applicant
institution's office of sponsored research and from the Office of
Grant Information, Division of Research Grants, National Institutes
of Health, 5333 Westbard Avenue, Room 449, Bethesda, MD 20892,
telephone (301) 435-0714.  Use the conventional format for research
grant applications and ensure that the points identified in the
section on REVIEW CONSIDERATIONS are fulfilled.  FIRST (R29)
applications must include at least three sealed letters of reference
attached to the face page of the original application.  FIRST
applications submitted without the required number of reference
letters will be considered incomplete and will be returned without
review.

To identify the application as a response to this RFA, check "YES" on
Item 2a of page 1 of the application and enter the title and RFA
Number:  HEMATOLOGIC CONSEQUENCES OF HIV INFECTION OF MARROW CELLS
RFA HL-94-004-B

The RFA label available in the PHS 398 application kit must be
affixed to the bottom of the face page of the original copy of the
application.  Failure to use this label could result in delayed
processing of the application such that it may not reach the review
committee in time for review.

Send or deliver the completed application and three signed, exact
photocopies of it to the following, making sure that the original
application with the RFA label attached is on top:

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

Send an additional two copies of the application to the Chief,
Centers and Special Projects Review Section at the address listed
under LETTER OF INTENT.  It is important to send these two copies at
the same time as the original and three copies are sent to the
Division of Research Grants.  Otherwise the NHLBI cannot guarantee
that the application will be reviewed in competition for this RFA.

Applications must be received by March 15, 1994.  An application not
received by this date will be returned to the applicant without
review.  The Division of Research Grants (DRG) will not accept any
application in response to this announcement that is essentially the
same as one currently pending initial review, unless the applicant
withdraws the pending application.  The DRG will not accept any
application that is the same as one already 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.

REVIEW CONSIDERATIONS

Upon receipt, applications will be reviewed for their responsiveness
to the objectives of this RFA by the NHLBI and the NIDDK.  Grant
applications will be assigned according to standard referral
guidelines.  If an application is judged unresponsive to the RFA, the
staff will contact the applicant to determine whether to return the
application to the applicant or submit it for review in competition
with unsolicited applications at the next review cycle.

Applications may be triaged by an NHLBI peer review group on the
basis of relative competitiveness.  The NIH will withdraw from
further competition those applications judged to be non-competitive
for award and notify the Principal Investigator and institutional
official.  Those applications judged to be competitive will undergo
further scientific merit review.  Those applications that are
complete and responsive will be evaluated in accordance with the
criteria stated below for scientific/technical merit by an
appropriate peer review group convened by the Division of Extramural
Affairs, NHLBI, solely to review these applications.  The second
level of review will be provided by the National Heart, Lung, and
Blood Advisory Council, and the National Diabetes and Digestive and
Kidney Diseases Advisory Council.

The criteria used in the evaluation of scientific merit of each
application will be similar to those used in the review of
traditional research-project grant applications, including the
novelty, originality, and feasibility of the approach; the training,
experience and research competence of the investigator(s); the
adequacy of the experimental design; the suitability of the
facilities; and the appropriateness of the requested budget to the
work proposed.

AWARD CRITERIA

Funding decisions will be made on the basis of scientific and
technical merit as determined by peer review, program needs and
balance, and the availability of funds.

Awards in response to this RFA will be made to foreign institutions
only for research of very unusual merit, need, and promise, and in
accordance with PHS policy governing such awards.

INQUIRIES

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

Inquiries regarding this programmatic issues may be directed to:

Helena O. Mishoe, Ph.D.
Division of Blood Diseases and Resources
National Heart, Lung, and Blood Institute
Federal Building, Room 5A12
Bethesda, MD  20892
Telephone:  (301) 496-5911
FAX:  (301) 496-9940

Ralph L. Bain, Ph.D.
Division of Kidney, Urologic, and Hematologic Diseases
National Institute of Diabetes and Digestive and Kidney Diseases
Westwood Building, Room 3A05
Bethesda, MD  20892
Telephone:  (301) 594-7556
FAX:  (301) 594-7501

Inquiries regarding fiscal and administrative matters may be directed
to:

Ms. Jane R. Davis
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 4A11
Bethesda, MD  20892
Telephone:  (301) 594-7436
FAX:  (301) 594-7492

AUTHORITY AND REGULATIONS

The programs of the Division of Blood Diseases and Resources, NHLBI,
are described in the Catalog of Federal Domestic Assistance number
93.839.  Awards are made under the authority of the Public Health
Service Act, Section 301 (42 USC 241) and administered under PHS
grants policies and Federal regulations, most specifically 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.

References

1.  Folks TM, Kessler SW, Orenstein JM, Justement JS, Jaffe ES, Fauci
AS:  Infection and replication of HIV-1 in purified progenitor cells
of normal human bone marrow. Science 242:919-922, 1988.

2.  Kitano K, Abboud CN, Ryan DH, Quan SG, Baldwin GC, Golde DW:
Macrophage-active colony-stimulating factors enhance human
immunodeficiency virus type 1 infection in bone marrow stem cells.
Blood 77:1699-1705, 1991.

3.  Molina JM, Scadden DT, Sakaguchi M, Fuller B, Woon A, Groopman
JE: Lack of evidence for infection of or effect on growth of
hematopoietic progenitor cells after in vivo or in vitro exposure to
human immunodeficiency virus. Blood 76:2476, 1990.

4.  Zauli G, Re MC, Visani G, Furlini G, Mazza P, Vignoli M, La Placa
M: Evidence for an HIV-1 mediated suppression of uninfected
hematopoietic (CD34 (+)) progenitor cells in vivo. J. Infect.
Diseases 1666:710-716, 1992.

5.  Zauli G, Re MC, Davis B, Sen L, Visani G, Guliotta L, Furlini G,
La Placa M: Impaired in vitro growth of purified (CD34 (+))
hematopoietic progenitors in human immunodeficiency virus-1
seropositive thrombocytopenic individuals. Blood 79:2680, 1992.

6.  Louache F, Henri A, Bettaieb A, Oksenhendler E, Raguin G, Tulliez
M, Vainchenker W: Role of human immunodeficiency virus replication in
defective in vitro growth of hematopoietic progenitors. Blood
80:2991-2999.

7.  Ho DD, Moudgil T, Alam M: Quantitation of human immunodeficiency
virus type 1 in the blood of infected persons. N. Eng. Jour. Med.
321:1621, 1989.

8.  Steinberg HN, Crumpacker CS, Chatis PA: In vitro suppression of
normal human bone marrow progenitor cells by human immunodeficiency
virus. J. Virol 65:1765, 1991.

9.  Zucker-Franklin D, Seremetis S, Zheng ZY: Internalization of
human immunodeficiency virus type I and other retroviruses by
megakaryocytes and platelets. Blood 75:1920-1923, 1990.

10.  Kouri YH, Borkowsky W, Nardi M, Karpatkin S, Basch RS: Human
megakaryocytes have a CD4 molecule capable of binding human
immunodeficiency virus-1. Blood 81:2664-70, 1993.

11.  Mitsuya H, Broder S: Inhibition of the in vivo infectivity and
cytopathic effect of human T-lymphotropic virus type
III/lymphadenopathy-associated virus (HTLV- III/LAV) by
2'3'-dideoxynucleosides. Proc. Natl. Acad. Sci. USA 83:1911-1915,
1986.

12.  Richman DD, Fischl, MA, Grieco MH, Gottlieb MS, Volberding PA,
Laskin OL, Leedom, JM, Groopman JE, Mildvan D, Hirsch MS, Jackson GG,
Durack DT, Nusinoff-Lehrman S, and AZT Collaborative Working Group.
The toxicity of azidothymidine (AZT) in the treatment of patients
with AIDS and AIDS-related complex. A double-blind, placebo-
controlled trial. N. Engl. J Med. 317:192-197, 1987.

13.  Davis BR, Schwartz DH, Marx JC, Johnson CE, Berry JM, Lyding J,
Merigan TC, Zander A: Absent or rare human immunodeficiency virus
infection of bone marrow stem/progenitor cells in vivo. J Virol,
65:1985, 1991.

14.  von Laer D, Hufert FT, Fenner TE, Schwander S, Dietrich M,
Schmitz H, Kern P: CD34 (+) hematopoietic progenitor cells are not a
major reservoir of the human immunodeficiency virus. Blood 76:1281,
1990.

15.  McElrath MJ, Pruett JE, Cohn ZA: Mononuclear phagocytes of blood
and bone marrow: comparative roles as viral reservoirs in human
immunodeficiency virus type I infections. Proc. Natl. Acad. Sci, USA
86:675, 1989.

16.  Zauli G, Davis BR, Re MC, Visani G, Furlini G, La Placa M: Tat
protein stimulates production of transforming growth factor-beta-1 by
marrow macrophages: A potential mechanism for HIV-1 induced
hematopoietic suppression. Blood 80:3036-3043, 1992.

17.  Geissler RG, Ottman OG, Kleiner K, Mentzel U: Decreased
haematopoietic colony growth in long-term bone marrow cultures of
HIV-positive patients. Res Virol 144:69-73, 1993.

.

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