EFFECTS OF ALCOHOL ON HIV INVASION ACROSS THE BLOOD BRAIN BARRIER OR 
PLACENTAL BARRIER

Release Date:  October 30, 2001

RFA:  RFA-AA-02-008

National Institute on Alcohol Abuse and Alcoholism
 (http://www.niaaa.nih.gov)

Letter of Intent Receipt Date:  December 28, 2001
Application Receipt Date:       January 23, 2002

THIS RFA USES "MODULAR GRANT" AND "JUST-IN-TIME" CONCEPTS.  MODULAR 
INSTRUCTIONS MUST BE USED FOR RESEARCH GRANT APPLICATIONS REQUESTING LESS 
THAN $250,000 PER YEAR IN ALL YEARS. MODULAR BUDGET INSTRUCTIONS ARE PROVIDED 
IN SECTION C OF THE PHS 398 (REVISION 5/2001) AVAILABLE AT 
http://grants.nih.gov/grants/funding/phs398/phs398.html.

PURPOSE

This RFA intends to encourage innovative research leading to the development 
of new approaches, technologies, and methods to examine the effects of 
ethanol on HIV invasion across the blood brain barrier (BBB) or the placental 
barrier (PB). Alcohol is a common substance used by HIV infected individuals. 
Evaluation of the effects of ethanol exposure on HIV translocation across the 
BBB and PB is necessary before intervention strategies can be developed. 
Research in this under-explored area is needed to understand how the 
ingestion of alcohol modifies these barriers. In particular, research 
proposals may be designed to increase understanding of how ethanol 
consumption may alter physical and immunological properties of the blood-
brain or placental barriers, with special attention to how anti-retroviral 
therapies for HIV could be affected.  Mechanisms for the passage of cell-free 
virus through paracellular and transcellular routes and transmigration of 
HIV-infected leukocytes across the barriers may be investigated. The 
culturing and manipulation of these specialized cells are likely to be of 
great benefit and potential clinical significance for the treatment of HIV-
related disease among alcohol abusers. The potential for this exploratory 
work, i.e. developing relevant in vitro models of the BBB and PB, may have a 
direct impact on the amelioration of the neurodegenerative pathology 
associated with alcohol and HIV synergism or the prevention of the perinatal 
transmission of HIV.
  
HEALTHY PEOPLE 2010

The Public Health Service (PHS) is committed to achieving the health 
promotion and disease prevention objectives of "Healthy People 2010," a PHS-
led national activity for setting priority areas.  This Request for 
Applications (RFA), Title of RFA, is related to one or more of the priority 
areas.  Potential applicants may obtain a copy of "Healthy People 2010" at 
http://www.health.gov/healthypeople/.

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 and local governments, and eligible 
agencies of the Federal government.  Racial/ethnic minority individuals, 
women, and persons with disabilities are encouraged to apply as Principal 
Investigator.

MECHANISM OF SUPPORT

This RFA will primarily use the NIAAA exploratory/developmental (R21) award 
mechanism.  In addition, R01 applications will be accepted if they are within 
the scope of the announcement to develop new approaches, technologies, tools, 
or methods, and do not exceed $250,000 per year in direct costs for a period 
of up to 3 years. Exploratory/developmental grants (R21) are limited to 3 
years for up to $100,000/year for direct costs. (See Program Announcement PA- 
99-131,  NIAAA Exploratory/Developmental Grant Program,  
http://grants.nih.gov/grants/guide/pa-files/PA-99-131.html, for a complete 
description of the R21 mechanism.)  Responsibility for the planning, 
direction, and execution of the proposed project will be solely that of the 
applicant.  The anticipated award date is July 1, 2002.

This RFA is a one-time solicitation.  Future unsolicited competing 
continuation applications will compete with all investigator-initiated 
applications and be reviewed according to the customary peer review 
procedures.

FUNDS AVAILABLE

It is estimated that up to $1,000,000 will be available in FY 2002 to fund 6 
to 8 new and/or competitive continuation grants in response to this RFA.  
This level of support is dependent on the receipt of a sufficient number of 
applications of high scientific merit.  Because the nature and scope of the 
research proposed may vary, it is anticipated that the size of each award 
will also vary.  The award of grants pursuant to this RFA is contingent upon 
the availability of funds for this purpose.  At this time, it is not known if 
this RFA will be reissued.

RESEARCH OBJECTIVES

NIAAA encourages the submission of highly innovative research applications 
focusing on the development of in vitro blood brain barrier and placental 
barrier models.  It is the goal of this announcement to develop and utilize 
new technology to learn how alcohol use might alter the nature of the blood 
brain barrier to promote development of HIV-associated dementia, or how 
alcohol use might alter the placental barrier to increase susceptibility to 
HIV infection in the fetus.  Relevant in vitro blood brain barrier and 
placental barrier models may include static transwell, 
endothelial/organotypic brain slice cultures, or perfusion/hollow fiber 
models.

Background Information on the Blood Brain Barrier: The human immunodeficiency 
virus (HIV) associated dementia (HAD) is thought to originate as virus-
infected monocytes infiltrate into the brain and cause neurologic disease.  
The mechanism(s) for monocyte penetration across the blood-brain barrier 
(BBB) remains ill defined but is likely to involve stimulation of 
transmigration by chemokines secreted by astrocytes, endothelial cells and 
the monocytes themselves.  Many questions about this process, and its 
relation to immune deterioration and pathogenesis, remain to be investigated. 
NIAAA is particularly interested in contributing to efforts to expand 
awareness of how alcohol abuse and alcoholism affects human health and can 
complicate the illness in those who suffer from HIV and their children.  
Alcohol has been identified as a cofactor in HIV disease.  Since alcohol is a 
commonly used drug in HIV-infected persons, evaluation of the exacerbating 
effects of ethanol on HIV neuroinvasion is important and necessary before an 
appropriate therapeutic intervention strategy can be designed. Moreover 
techniques for isolation and culture of brain endothelial cells could be of 
broad benefit for both HIV-related studies and neuropathology in general.  
Here we attempt to stimulate research which focuses more specifically on 
learning about how alcohol use might alter the nature of the BBB and could 
promote development of HAD.  Alcohol could cause increase in viral load, 
change in membrane permeability or transmigration and/or reduced 
immunological function.  Any increase in the titer of viremia caused by 
alcohol or any change in the titer caused by anti-retroviral therapy may have 
profound effects on the future course of HIV disease. 
   
Some of the questions that the investigator may seek to answer include:
- How does the combination of alcohol and HIV restructure microvascular 
endothelial cells?
- How may permeability to serum proteins be effected? 
- Will rates of migration of mononuclear cells increase across the BBB?
- What is the impact of alcohol"s immunosuppressive effects?
- What is the effect of alcohol on T-cell maturation?

Background Information on the Placental Barrier: The placenta provides a 
barrier between the maternal and fetal circulations but its role in HIV 
transmission remains unclear.  The placenta consists of a vast array of villi 
which are bathed directly in circulating maternal blood which is thereby in 
extensive and intimate contact with fetal tissues.  Underlying the villi is a 
continuous epithelium generated from and maintained by a layer of 
trophoblastic cells. Several trophoblast cell lines have been derived from 
full-term placentae and these can be used to construct in vitro models of the 
placental barrier structure. Studies are currently underway to determine if 
cells in the full-term placentae of HIV-seropositive mothers are infected and 
which cell types are involved.  At the most fundamental level, the mechanism 
by which HIV enters and/or traverses the trophoblast layer is not known.  
Chemokine receptor expression by trophoblasts and other placental cells is 
currently an area of intense investigation. Placentae of HIV-infected mothers 
have been found to express a different repertoire of cytokines and chemokines 
than seen in non-infected controls.  Since chemokine receptors are now 
thought to be co-receptors for HIV entry this work has important implications 
for our understanding of maternal-fetal transmission. Ruptures in the 
placental barrier do occur, particularly during delivery, and it is important 
to understand more about what promotes or disturbs the stability of this 
barrier.  Documented effects of ethanol in weakening cell adhesion could play 
a role in this context. Discontinuities in the trophoblastic layer in 
placenta occurring due to apoptosis have recently been described and this is 
another modification that has been linked to ethanol in other systems.  

Some questions that the investigator may seek to answer include:
- Does alcohol affect the invasion of HIV across the placental barrier?  
- Can cellular models of this barrier be developed in vitro to test the 
impact of alcohol on membrane permeability and/or immunological functioning?
- Can alcohol affect the immature immune systems of children of HIV-infected 
mothers? 
- Can an increase in viral strain diversity be seen in such children?
- Does alcohol increase the risk of "trauma" to the membrane as a whole?
- While alcohol is well known as a prenatal teratogen, can mechanisms of 
neuropathogenesis, as seen in fetal alcohol syndrome as well as HIV-dementia, 
be studied using in vitro models for barrier permeability?  

Technical considerations: Since primary or permanent cultures of endothelial 
cells that exhibit true BBB and PB characteristics are difficult to generate 
and maintain, applicants may consider the potential problems and solutions 
when developing comprehensive physical models for these barriers.

Researchers may also wish to address some of the following topics in the 
course of their investigation:

Maintaining brain microvascular endothelial cells 
- New sources and methods for isolating and maintaining BMVEC in a 
differentiated state
- Use of BMVEC from gene modified or knockout animals
- Characterization and quality control of BMVEC lines

Culturing astrocytes and other co-cultivated cells lines
- Sources and culturing methods for astrocyte and other BBB 
- Use of support cell lines from gene modified or knock out animals

Performance of perfusion Devices
- Commercially available hollow fiber bioreactors
- Custom perfusion devices or chambers
- Multiple or single fiber configurations
- Pulsatile or peristaltic medium flow with variable shear stress parameters
- Compatibility with confocal microscopy

Performance of transwell Devices
- Commercially available transwell

Development of scaffolding materials and biocompatible substrates
- Synthetic or biomaterial derived
- Biodegradable
- Extracellular matrix proteins, adhesion molecules, etc.

Improvement of standardization and quality control
- Time course for development and maintenance of EC barrier
- Criteria for EC tight junction testing
- High electrical resistance across EC barrier
- Appropriate and selective transport of ions from the lumenal to the 
extracapillary compartments

Comparison with in vivo models
- Correlation of in vitro BBB and PB models with in vivo animal models
- Internal carotid artery perfusion method
- Injection and recovery method
- Use of standard test compounds for transcellular and paracellular route of 
entry	

Application of analytical Techniques
- Scanning electron microscopy
- Transmission electron microscopy
- Confocal microscopy
- Immunocytochemistry and in situ hybridization
- Permeability studies
- Pharmacokinetic studies

Special Concerns about Biohazards:  The use of HIV and EC, monocytes and 
other cell types from HIV infected humans and/or animals mandates the strict 
adherence to laboratory safety standards and worker protection protocols.

Annual meeting: Grantees are expected to meet annually to share results, to 
ensure that NIAAA has a comprehensive view of the technological advances in 
the field, to expedite the dissemination of new technology, and to afford an 
opportunity for collaborative problem solving among investigators.  
Applicants may request travel funds in their budgets for the principal 
investigator to attend these meetings.

INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of the NIH that women and members of minority groups and 
their sub-populations must be included in all NIH-supported biomedical and 
behavioral research projects involving human subjects, unless a clear and 
compelling rationale and justification are provided indicating that inclusion 
is inappropriate with respect to the health of the subjects or the purpose of 
the research.  This policy results from the NIH Revitalization Act of 1993 
(Section 492B of Public Law 103-43).

All investigators proposing research involving human subjects should read the 
UPDATED "NIH Guidelines for Inclusion of Women and Minorities as Subjects in 
Clinical Research," published in the NIH Guide for Grants and Contracts on 
August 2, 2000 
(http://grants.nih.gov/grants/guide/notice-files/NOT-OD-00-048.html), 
a complete copy of the updated Guidelines are available at  
http://grants.nih.gov/grants/funding/women_min/guidelines_update.htm:  The 
revisions relate to NIH defined Phase III clinical trials and require: a) all 
applications or proposals and/or protocols to provide a description of plans 
to conduct analyses, as appropriate, to address differences by sex/gender 
and/or racial/ethnic groups, including subgroups if applicable, and b) all 
investigators to report accrual, and to conduct and report analyses, as 
appropriate, by sex/gender and/or racial/ethnic group differences.

INCLUSION OF CHILDREN AS PARTICIPANTS IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of NIH that children (i.e., individuals under the age of 21) 
must be included in all human subjects research, conducted or supported by 
the NIH, unless there are scientific and ethical reasons not to include them.  
This policy applies to all initial (Type 1) applications submitted for 
receipt dates after October 1, 1998.

All investigators proposing research involving human subjects should read the 
"NIH Policy and Guidelines on the Inclusion of Children as Participants in 
Research Involving Human Subjects" that was published in the NIH Guide for 
Grants and Contracts, March 6, 1998, and is available at the following URL 
address: http://grants.nih.gov/grants/guide/notice-files/not98-024.html. 

Investigators also may obtain copies of these policies from the program staff 
listed under INQUIRIES.  Program staff may also provide additional relevant 
information concerning the policy.

REQUIRED EDUCATION ON THE PROTECTION OF HUMAN SUBJECT PARTICIPANTS

NIH policy requires education on the protection of human subject participants 
for all investigators submitting NIH proposals for research involving human 
subjects.  This policy announcement is found in the NIH Guide for Grants and 
Contracts Announcement dated June 5, 2000, at the following website: 
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-00-039.html.

DATA AND SAFETY MONITORING PLAN

As of the October 2000 receipt date, applicants must supply a general 
description of the Data and Safety Monitoring Plan for ALL clinical trials, 
this must be included in the application 
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-00-038.html
The degree of monitoring should be commensurate with risk. NIH Policy for 
Data and Safety Monitoring requires establishment of formal Data and Safety 
Monitoring Boards for multi-site clinical trials involving interventions that 
entail potential risk to the participants. The absence of this information 
will negatively affect your priority score.

PUBLIC ACCESS TO RESEARCH DATA THROUGH THE FREEDOM OF INFORMATION ACT

The Office of Management and Budget (OMB) Circular A-110 has been revised to 
provide public access to research data through the Freedom of Information Act 
(FOIA) under some circumstances.  Data that are (1) first produced in a 
project that is supported in whole or in part with Federal funds and (2) 
cited publicly and officially by a Federal agency in support of an action 
that has the force and effect of law (i.e., a regulation) may be accessed 
through FOIA.  It is important for applicants to understand the basic scope 
of this amendment.  NIH has provided guidance at: 
http://grants.nih.gov/grants/policy/a110/a110_guidance_dec1999.htm.

Applicants may wish to place data collected under this RFA in a public 
archive, which can provide protections for the data and manage the 
distribution for an indefinite period of time.  If so, the application should 
include a description of the archiving plan in the study design and include 
information about this in the budget justification section of the 
application. In addition, applicants should think about how to structure 
informed consent statements and other human subjects procedures given the 
potential for wider use of data collected under this award.

URLS IN NIH GRANT APPLICATIONS OR APPENDICES 

All applications and proposals for NIH funding must be self-contained within 
specified page limitations.  Unless otherwise specified in an NIH 
solicitation, internet addresses (URLs) should not be used to provide 
information necessary to the review because reviewers are under no obligation 
to view the Internet sites.  Reviewers are cautioned that their anonymity may 
be compromised when they directly access an Internet site.

LETTER OF INTENT

Prospective applicants are asked to submit 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 other key personnel 
and participating institutions, and the number and title of the RFA in 
response to which the application may be submitted.  Although a letter of 
intent is not required, is not binding, and does not enter into the review of 
a subsequent application, the information that it contains allows NIAAA staff 
to estimate the potential review workload and plan the review.

The letter of intent is to be sent to:

RFA-AA-02-008
Extramural Project Review Branch
National Institute on Alcohol Abuse and Alcoholism
6000 Executive Boulevard, Suite 409, MSC 7003
Bethesda, MD  20892-7003
Rockville, MD  20852 (for express/courier service) 
Telephone:  (301) 443-4375  FAX:  (301) 443-6077

by the letter of intent receipt date indicated.

APPLICATION PROCEDURES

The PHS 398 research grant application instructions and forms (rev. 5/2001) 
at http://grants.nih.gov/grants/funding/phs398/phs398.html must be used in 
applying for these grants. This version of the PHS 398 is available in an 
interactive, searchable format. For further assistance contact GrantsInfo, 
Telephone 301/710-0267, Email:  GrantsInfo@nih.gov.

SPECIFIC INSTRUCTIONS FOR MODULAR GRANT APPLICATIONS 

The modular grant concept establishes specific modules in which direct costs 
may be requested as well as a maximum level for requested budgets. Only 
limited budgetary information is required under this approach.  The 
just-in-time concept allows applicants to submit certain information only 
when there is a possibility for an award. It is anticipated that these 
changes will reduce the administrative burden for the applicants, reviewers 
and NIH staff.  The research grant application form PHS 398 (rev. 5/2001) at 
http://grants.nih.gov/grants/funding/phs398/phs398.html is to be used in 
applying for these grants, with modular budget instructions provided in 
Section C of the application instructions.

The RFA label available in the PHS 398 (rev. 5/2001) application form must be 
affixed to the bottom of the face page of the application.  Type the RFA 
number on the label.  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.  In addition, the RFA title and number must be typed on 
line 2 of the face page of the application form and the YES box must be 
marked. The RFA label is also available at: 
http://grants.nih.gov/grants/funding/phs398/label-bk.pdf.

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

CENTER FOR SCIENTIFIC REVIEW
NATIONAL INSTITUTES OF HEALTH
6701 ROCKLEDGE DRIVE, ROOM 1040, MSC 7710
BETHESDA, MD  20892-7710
BETHESDA, MD  20817 (for express/courier service)

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

RFA AA-02-008
Extramural Project Review Branch
National Institute on Alcohol Abuse and Alcoholism
Willco Bldg, Suite 409
6000 Executive Blvd, MSC 7003
Bethesda, MD  20892-7003
Rockville, MD  20852 (for express/courier service)

Applications must be received by the application receipt date listed in the 
heading of this RFA.  If an application is received after that date, it will 
be returned to the applicant without review.

The Center for Scientific Review (CSR) 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.  The 
CSR will not accept any application that is essentially 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 completeness by the CSR and 
responsiveness by the (IC).  Incomplete and/or non-responsive applications 
will be returned to the applicant without further consideration.

Applications that are complete and responsive to the RFA will be evaluated 
for scientific and technical merit by an appropriate peer review group 
convened by the NIAAA in accordance with the review criteria stated below.  
As part of the initial merit review, all applications will receive a written 
critique and undergo a process in which only those applications deemed to 
have the highest scientific merit, generally the top half of the applications 
under review, will be discussed, assigned a priority score, and receive a 
second level review by the (IC) National Advisory Council or Board.

Review Criteria

The goals of NIH-supported research are to advance our understanding of 
biological systems, improve the control of disease, and enhance health.  In 
the written comments reviewers will be asked to discuss the following aspects 
of the application in order to judge the likelihood that the proposed 
research will have a substantial impact on the pursuit of these goals.  Each 
of these criteria will be addressed and considered in assigning the overall 
score, weighting them as appropriate for each application.  Note that the 
application does not need to be strong in all categories to be judged likely 
to have major scientific impact and thus deserve a high priority score.  For 
example, an investigator may propose to carry out important work that by its 
nature is not innovative but is essential to move a field forward.

(1) Significance:  Does this study address an important problem? If the aims 
of the application are achieved, how will scientific knowledge be advanced?  
What will be the effect of these studies on the concepts or methods that 
drive this field?

(2) Approach:  Are the conceptual framework, design, methods, and analyses 
adequately developed, well-integrated, and appropriate to the aims of the 
project?  Does the applicant acknowledge potential problem areas and consider 
alternative tactics?

(3) Innovation:  Does the project employ novel concepts, approaches or 
method? Are the aims original and innovative?  Does the project challenge 
existing paradigms or develop new methodologies or technologies?

(4) Investigator:  Is the investigator appropriately trained and well suited 
to carry out this work?  Is the work proposed appropriate to the experience 
level of the principal investigator and other researchers (if any)?

(5) Environment:  Does the scientific environment in which the work will be 
done contribute to the probability of success?  Do the proposed experiments 
take advantage of unique features of the scientific environment or employ 
useful collaborative arrangements?  Is there evidence of institutional 
support?

In addition to the above criteria, in accordance with NIH policy, all 
applications will also be reviewed with respect to the following:

o  The adequacy of plans to include both genders, minorities and their 
subgroups, and children as appropriate for the scientific goals of the 
research.  Plans for the recruitment and retention of subjects will also be 
evaluated.

o  The reasonableness of the proposed budget and duration in relation to the 
proposed research.

o  The adequacy of the proposed protection for humans, animals or the 
environment, to the extent they may be adversely affected by the project  
proposed in the application.

o  The adequacy of the proposed plan to share data, if appropriate.

Schedule

Letter of Intent Receipt Date:    December 28, 2001
Application Receipt Date:         January 23, 2002
Peer Review Date:                 March-April 2002
Council Review:                   May 2002
Earliest Anticipated Start Date:  July 1, 2002

AWARD CRITERIA

Award criteria that will be used to make award decisions include:

o  scientific merit (as determined by peer review)
o  availability of funds
o  programmatic priorities.

INQUIRIES

Inquiries concerning this RFA are encouraged.  The opportunity to clarify any 
issues or answer questions from potential applicants is welcome.

Direct inquiries regarding programmatic issues to:

Laurie Fleischman, Ph.D.
Program Officer
Collaborative and Special Health Programs Branch - CSHPB
Office of Collaborative Research, NIAAA
6000 Executive Blvd, Room 302
Bethesda, MD  20892
Telephone:  (301) 402-9402
FAX:  (301) 480-2358
Email:  lfleisch@mail.nih.gov

Direct inquiries regarding fiscal matters to:

Judy Simons
Office of Planning and Resource Management
National Institute on Alcohol Abuse and Alcoholism
6000 Executive Boulevard MSC 7003
Bethesda, MD  20892-7003
Telephone:  (301) 443-2434
FAX:  (301) 443-3891
Email:  jsimons@niaaa.nih.gov

AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic Assistance No. 
93.273.  Awards are made under authorization of Sections 301 and 405 of the 
Public Health Service Act as amended (42 USC 241 and 284) and administered 
under NIH grants policies and Federal Regulations 42 CFR 52 and 45 CFR Parts 
74 and 92.  This program is not subject to the intergovernmental review 
requirements of Executive Order 12372 or Health Systems Agency review.

The PHS strongly encourages all grant recipients to provide a smoke-free 
workplace and promote the non-use of all tobacco products.  In addition, 
Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in 
certain facilities (or in some cases, any portion of a facility) in which 
regular or routine education, library, day care, health care, or early 
childhood development services are provided to children.  This is consistent 
with the PHS mission to protect and advance the physical and mental health of 
the American people.

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