Jane J. Smythe, M. D. Molecular Medicine icon

Jane J. Smythe, M. D. Molecular Medicine


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APPLICATION FOR THE USE OF ANIMALS IN RESEARCH

Submit an electronic version to CompMed@research.usf.edu for veterinary pre-review prior to

submission to the University of South Florida, Institutional Animal Care & Use Committee,

MDC 35, phone 974-7106, fax 974-7091, email IACUC@research.usf.edu

PHS #A4100-01; USDA #58-15; AAALAC #434

(type all responses) IACUC#: R________________

  1. TITLE OF PROJECT

A Non-Invasive Immune Therapy for Pediatric Cancers Resistant to Conventional Treatment

(Note: The title of this IACUC application must match the title of the grant that supports this research. If the described activity involving animals is supported by multiple grants with different titles, or a grant is awarded later, during the 3-year approval period of this protocol, that supports this research, inform the IACUC by completing and submitting a Request to Amend an Animal Use Protocol form.)

2
Professor
. PERSONNEL

^


Jane J. Smythe, M.D.

Molecular Medicine

Principal Investigator (including degrees) Position (Academic Appointment) Department



MDC 200009

555-1234

jjsmythe@health.usf.edu

Campus Address PI Email Address PI Phone



Carline Someone

csomeone@health.usf.edu

555-2345

Secondary Study Contact Secondary Study Contact Email Address Secondary Study Contact Phone

  1. QUALIFICATIONS

^ Name & Degree

Role

Years Experience/Species

Cert#

Jane J. Smythe, M.D.

PI

15/Mice

456

Carline Someone, B.S., M.S.

T

8/Mice

789

Raymond Notsmart

T

*0/Mice

1005

James J. Julienne, Ph.D.

SI

10/Mice

1112

*PI and Comparative Medicine Training Coordinator will train in all techniques and animal handling procedures.













(Note: Indicate role of involved personnel as either Principal (PI), or Secondary (SI) Investigators, or technicians/assistants (T). Indicate each individual's years of experience with species described herein (e.g., 6 yrs/mice, 4 yrs/rabbits). Certification of orientation, training, and experience regarding the regulatory, occupational health and safety, and care and use aspects of the species requested herein is required prior to IACUC review of this protocol, and is available from the IACUC c/o the Division of Comparative Medicine, 974-9796. Certification numbers for each involved individual must be listed.)


Agency: Pediatric Oncology Foundation, Sarasota Grant #: 777888999


^ 4. FUNDING SOURCE

(Indicate funding agency, grant #, and account # if known.)


5. LOCATION This protocol will be conducted at (please check location(s)): NOTE – Animal removal/relocation from a facility requires PRIOR APPROVAL.

ALZ

COM

CPH

CRI

IDRB

KRC

LSA

NCF

PSY

SCA

SRB

VAH




X


































  1. CERTIFICATION

A search for alternatives and alternative methods, including a search of the following relevant database(s) indicated below, covering the indicated years (at least the last 10 yrs.), and using the indicated search term(s), demonstrates that suitable alternatives to these procedures, and to aspects of these procedures which may cause pain or discomfort to animals, or to this animal use are not available or applicable. The methods described will be used and continuously refined so as to reduce animal discomfort and use. Conduct will be in accordance with the PHS policy, AWR, Guide, AAALAC guidelines, DEA regulations, and IACUC Policies. This project was designed with the consultation of a veterinarian. The described animal use does not duplicate previous or existing studies. This description is complete and accurate. Implementing changes to this description requires prior written IACUC approval. Current biomedical supplies will be used. Complete animal procedural/surgical/testing records will be maintained. Personnel are certified as adequately trained and experienced, and have complied with IACUC occupational health & safety policies. Comparative Medicine will be notified whenever data from any pre-clinical study involving animals is submitted to the Food & Drug Administration.


See Attached List

Database: Years: Search Terms:


See Attached List

1990 - 2011

__________________________________________________________________________________________

Signature of Principal Investigator Date

Note: A single addendum page of response is permissible, if it is identified as a response to which item # and page #.


^ 6. Certification (Continued)


(1) List of search terms:


“animal models for cancer” : “animal models for cancer vaccine development” : “Alternatives to animal models”

“Neuroblastoma in animal models” : “Alternatives to animal experimentation” : “Alternatives to Animals for human cancer models” : “Alternatives to painful procedures in animals”


(2) The following literature searches on unnecessary duplication were carried out on PubMed at :

http://www.ncbi.nlm.gov from 2007 through 1990 using the following searches


“animal models for cancer” (42 abstracts, on hand, 1989-2010)

“animal models for cancer vaccine development”

“Alternatives to animal models”

“Neuroblastoma in animal models” (30 abstracts on hand, 1975-2010)

“Alternatives to animal experimentation”

“Alternatives to Animals for human cancer models” (16 abstracts on hand, 1991-2010)

“Alternatives to painful procedures in animals” (3 abstracts)


I did not find any duplication of the work proposed in this application. The only alternatives to animal models in cancer dealt primarily with the assessment of carcinogenic compounds.


(3) In searches for Non-Animal alternatives, the following web sites were examined:


http://www.shef.ac.uk/b/bioethics-today/Lancaster/animal/experiment.htm


http://europa.eu.int/comm/research/info/conferences/rrr/rrr_en.html


http://sis.nlm.nih.gov/animalalt/aa02.html


http://www.nih.gov/science/models/


http://dels.nas.edu/ilar/jour_online/43_1/Ethical.asp


http://www.nal.usda.gov/awic/


http://altweb.jhsph.edu/


^ PROTOCOL CLASSIFICATION

7. Please check one.

a


. frame15 New Project. frame16 3rd Yr renewal replacing previous approved IACUC Protocol #


b. Please provide a brief summary (a few sentences) describing work accomplished during the last approval period and how the work proposed in this renewal extends the previous studies.

^ JUSTIFICATION FOR THE USE OF ANIMALS

8. Briefly state in lay terms the purpose of this request (i.e., research hypothesis or teaching objectives).

(Note: Your response must be in language that a high school senior can understand.)


The data obtained in previous studies supported the hypothesis that the insertion of a bacterial gene into tumor cells would alter the ability of the tumor cells to form tumors when injected into mice. In those studies, mice that were given various concentrations of tumor cells alone all produced tumors within 10-14 days. However, of those mice receiving the equivalent concentrations of tumor cell with the inserted bacterial gene, only 2 out of 18 (of the highest concentration) developed tumors. Furthermore, at 40 days following injection of these modified tumor cells, these were the only mice to exhibit tumors. The studies on the immune response (IR) in those mice suggest that this type of gene therapy will induce a significant IR that is able to prevent the tumor cells from replicating and forming tumors at the site of inoculation. This application is to build upon these very successful studies to find out if this form of immunotherapy is capable of inducing an IR that would be successful in 1) preventing tumors from forming in mice previously “immunized with the modified tumor cells when challenged with non-transfected cells (normal tumor cells) and 2) whether or not the IR generated by giving killed modified tumor cells would be capable of inducing tumor regression in tumor bearing mice.




9. Briefly outline or describe in lay terms the procedures in which animals will be used (i.e., the general sequence and schedule of what will be done to the animals). Section 9 or Section 11 of the application should contain an explanation/rationale for assigning the specific number of animals to the different Pain Categories in Section 10.

(Note: For complicated experimental designs, it may be helpful to the IACUC if this response includes a flow chart, diagram, timeline, or table which depicts the experiments or sequence of events. Your response must be in language that a high school senior can understand.)


A/J mice will be used in all the experiments. Mice will be injected with predetermined numbers of tumor cell directly under the skin on the left flank, between the front and hind legs. The total volume injected into each mouse will be 0.1 ml. Two experimental protocols are planned:


Protocol 1: (Challenge Experiment). In these experiments the mice will be injected with 3x106 of the gene modified tumor cell (N2/55). At 14-to-17 days following injection with the N2/55 cells, the mice will be challenged with 3x106 parental tumor cell (N-2a). This protocol will require three groups of animals (control group receiving 3x106 N2a cells alone and two ‘vaccinated’ groups receiving 3x106 and 1x106 N2/55 and challenged with 3x106 N2a cells). As stated in Section 11, 18 mice per group will be used.

Protocol 2: (Tumor Regression Experiment). In these experiments, seven groups of mice (18 mice/group) will be injected with 1x106 N-2a. At 5, 10 and 15 days after injection, mice will be given 3x106 or 1x106 UV irradiated N2/55 tumor cells (please see attachment for schedule).


After injection, all the mice will be observed on a daily basis for either tumor development (Protocol 1), tumor regression (Protocol 2), for the time it takes for the first visible signs of tumor production/regression, and/or for the size of the tumors and the growth/regression rate of the tumors. Once the tumors reach 1.5 cm in diameter, the mice will be euthanatized. At the time of euthanasia, blood will be collected and the spleen and liver will be harvested along with the tumor. The purpose for taking these samples (spleen and blood) is to measure the immune response (humoral and Cell mediated), if any, to both the tumor cell and the bacterial antigen. The liver and tumor will be used for histopathology.




^ CHARACTERISTICS OF ANIMALS AND PAIN CATEGORY OF RESEARCH

10. List and describe the animals to be studied. Indicate the anticipated number of animals to be used in each Pain Category of Research and the total number of animals involved during the 3-year approval period of this protocol. Indicate strain or line designation if rodents are requested. Indicate the number of each species/strains that will be involved in planned procedures that are anticipated to produce momentary, slight, or no pain, discomfort or distress (Pain Category A); more than momentary or slight pain, discomfort or distress which is alleviated by the use of appropriate anesthetics and/or analgesics (Pain Category B); or pain, discomfort, or distress, which cannot, or is not alleviated by the administration of appropriate anesthetics and/or analgesics (Pain Category C). (Note: Refer to IACUC Principles and Procedures XII. The same number of animals requested here must be justified in response to item #11, below.)

Species, Strain, or Line

^ Murine, A/J strain










Characteristics

(age, sex, weight)

^ 7-10 week old females










#used in Pain Category A procedures (no or slight discomfort).

-0-










#used in Pain Category B procedures (discomfort alleviated).

-0-










#used in Pain Category C procedures

(discomfort not alleviated).

180










Total Number Requested

180











11. Briefly describe the rationale using statistical analysis, whenever possible, used to determine the total number of each species of animals declared above in response to item #10 that will be needed for use during the 3-year approval period of this protocol. Section 9 or Section 11 of the application should contain an explanation/rationale for assigning the specific number of animals to the different Pain Categories in Section 10.

(Note: For complicated experimental designs, it may be helpful to the IACUC if this explanation indicates the # of animals needed for each experimental group, the # of groups required, and the analyses conducted using each group. Alternatively, it may be helpful to the IACUC if a flow chart depicting the sequence of events and the number of animals required for each step is shown.)


In consultation with a statistician, the sample size was derived from the following:


The purpose of these studies is to investigate the differences in tumorogenicity between control tumor cells and a cancer vaccine in which the cells have been modified. The measurements to be studied are (i) the development/regression of the tumor at the site of inoculation, (ii) the time it takes for the first measurable tumors to develop, (iii) the growth/regression of the tumor during the study period and (iv) growth/regression rate of the tumor.


The research design in these studies will be a “True Experiment,” as the independent variable will be manipulated and randomly assigned to groups. The statistical procedure to be used will be a Multivariate Analysis of Variance (MANOVA).


Statistical power analysis was conducted in order to determine the sample size for each group. Statistical power analysis is a procedure for studying the likelihood that a particular test of statistical significance will lead to rejection of a false null hypothesis. The following factors were taken into consideration:

  1. Sample size: It is a fact that the power increases with sample size,

  2. Level of significance: The second determinant of statistical power is the p value at which the null hypothesis is to be rejected. The research has set the level of significance in this study to p<0.05

  3. Effect size: This determinant of statistical power is an estimate of the magnitude of the difference, relationship, or effect in the population


In anticipation of a significant effect and using the table for “Sample size for a four-group MANOVA” and a power of 0.80 will mean that we should still use a sample size of 18 mice per group.



12. Briefly describe the characteristics of the animals requested that justify its use in this protocol.


The clone Neuro2A cell line was established from a spontaneous tumor in A/J mice and has been carried in this strain since. This murine tumor cell line closely resembles human neuroblastoma, a pediatric brain tumor. This cell line and mouse strain has been used by many other investigators to develop a neuroblastoma model. Females are specified because references indicate prior work with this murine tumor cells was accomplished in female mice.




^ LABORATORY POLICY CONSIDERATIONS

13. Will animals be involved in procedures that are anticipated to have the potential to produce more than momentary or slight pain, discomfort, or distress, which cannot, or will not be alleviated by the administration of appropriate anesthetics and/or analgesics?


No: frame22 Proceed to Item #14. Yes: frame23 Within the space below, define the clinical criteria that will be used to ensure timely intervention and treatment, or removal of the animals from the study, either in advance of, or immediately after recognition of the discomfort. The earliest possible clinical end point, which will contribute to the resolution of the hypothesis, must be identified and utilized. If avoidance or alleviation of animal pain or discomfort adversely affects the protocol, provide a detailed justification of why treatments cannot be initiated.


Mice exhibiting one or more of the following signs of severe or chronic pain or distress will be euthanatized (see Section 24):


(i) lethargy, (ii) loss of inquisitiveness, (iii) dehydration, (iv) soiled hair coat, (v) closed eye lids, (vi) muscle wasting, (vii) distended stomach, (viii) hunched posture, (ix) ataxia (x) soiled anogenital area, (xi) circling and (xii) exhibiting tumors >/= 1.5 cm in diameter .


Furthermore, any ulceration of subcutaneous tumors, or growth of a tumor that interferes with the animal's locomotor ability or ability to obtain food and/or water will also be used as a criterion for intervention (euthanasia)





14. Is animal death (excluding death from euthanasia) an intentional endpoint in this protocol (e.g., survival analysis, radiation, toxicity, or carcinogenesis testing)?


No: frame25 Proceed to Item #15. Yes: frame26 Within the space, explain why an earlier endpoint is not possible.

15. Will any animal research/procedural/testing areas outside of the animal facility be used for this protocol?

No: frame28 Proceed to Item #16. Yes: frame29 Within the space, provide a scientific or logistical justification for why animals must be removed from the facility, indicate the location, the approximate number of hours animals will be held at this site, and whether the animals will need to be returned to housing after the procedure, or that the procedure will be terminal.

16. Will this study be performed in accordance with 21 CFR 58 as a GLP study?


No: frame31 Proceed to item #17. Yes: frame32 Attach a copy of the GLP Study Protocol.


17. Will scheduled substances controlled by the Drug Enforcement Administration be used in the protocol?


No: frame33 Proceed to item #18. Yes: frame34 Within the space, list the controlled substances to be used.

^ EXPERIMENTAL PROCEDURES


18. Are other than standard and routine husbandry and handling practices required for this protocol (e.g., food, fluid, or caloric restriction, unique diets or nutritional supplements, specialized caging or environments, or non-standard health monitoring)?

No: frame36 Proceed to Item #19. Yes: frame37 Attach "Appendix A: Special Husbandry."


19. Are test substances administered to animals as part of this protocol (e.g., radioisotopes, toxic, immunogenic, pharmacologic, infectious, or carcinogenic agents, biomaterials, or cells)?


No: frame38 Proceed to Item #20. Yes: frame39 Attach "Appendix B: Test Substances."


20. Are specimens collected from animals prior to euthanasia as part of this protocol (e.g., tissues, blood, lymph, or other body fluids)?


No: frame40 Proceed to Item #21. Yes: frame41 Attach "Appendix C: Specimen Collection, Ante Mortem.”


21. Will surgery be performed on animals as part of this protocol?


No: frame42 Proceed to Item #22. Yes: frame43 Attach "Appendix D: Surgery."


22. Will animals be subject to experimental procedures other than those described above (e.g., behavioral manipulations, noxious stimuli, forced exercise, or physical restraint)?


No: frame44 Proceed to Item #23. Yes: frame45 Attach "Appendix E: Other Experimental Procedures.”


23. Will a human patient procedural area be required for this protocol?


No: frame46 Proceed to item #24. Yes: frame47 Attach "Appendix F: Patient Procedural Area."


  1. Will Veterans Affairs resources (e.g., VA lab space, VA vivarium, VA equipment, VA funding, VA personnel, VA time) be used in any aspect of this research?


X


No: Proceed to item #25. Yes: Attach "Appendix G: VA Hospital Resources Information."

EUTHANASIA



25. Are animals euthanatized for postmortem tissue collection, or are euthanatized at the completion of this study?

No: frame50 Within the space below indicate the final disposition of the involved animals.


Yes: frame51 Does the method of euthanasia and means of assuring death following euthanasia comply with IACUC Policy XX which describes acceptable methods of animal euthanasia within these laboratories?


No: frame52 Within the space below indicate why a deviation from policy is necessary. Yes: frame53 Within the space below describe the method of euthanasia used for each species requested, indicating dose and route if a chemical agent, or provide a justification if a physical method.


In accordance to the recommendations set forth in the AVMA Guidelines on Euthanasia – June 2007, we will use carbon dioxide delivered from gas cylinders (compressed gas). Animals will be placed, individually, in an empty chamber and an optimal flow rate of 20% displacement of the chamber volume per minute will be used. As stated in the IACUC “Policies of Animal Care and Use,” increasing the concentration of CO2 (33% displacement) after one minute will be used, as this may help to avoid or minimize discomfort and/or distress. Following CO2 exposure, assurance that the animal is dead will be made (verification of cessation of respiratory and cardiac activity/movement), followed by cervical dislocation. Every time the chamber is to be used, it will be cleaned and dried prior to use. Furthermore, the chamber will be cleaned and dried between each mouse to be euthanatized.



STOP here. Complete and attach any required appendixes as indicated above. This application is complete.

^ APPENDIX B: TEST SUBSTANCES

(Complete & Submit for Review Only If Response to Item #19 was "Yes")


1. Complete the table below. Name the test substance(s) that will be administered to animals.


Indicate the class of each substance as either an: (A) infectious agent, (B) primary human explant, uncharacterized human blood, lymph or tissue specimen, (C) recombinant DNA, (D) radioisotope, (E) carcinogen, (F) hazardous or toxic chemical, (G) biological toxin, (H) Cell line, (I) Adjuvant, (J) Antigenic Substance, (K) Pharmacologic Agent, or (L) Other class of substance by using the appropriate capital letter. Indicate to which species each substance will be administered. Indicate the dose (e.g., μg/gm bwt), volume per administration, route (e.g., i.v., i.p., s.c.), interval (e.g. 1x, daily, eod, every 3rd day), and duration of administrations (e.g., 5 wks).


Substance

(list all substances including vehicle/control)

Class

(above)

Species

(administered to)

Dose/Volume/Route

(3.0mg/kg bw / 0.1ml / i.p.)

Interval

(1x, eod, ev 3rd day)

Duration

(5 weeks)

1. Tumor cells (Neuro2A)

H/C

Mouse

3x106, 1x106/0.1 ml/SC

1 X

N/A

2. Tumor cells (Neuro2A + Emm55)

H/C

Mouse

3x106, 1x106/0.1 ml/SC

Every 5 da

15 da (3X)

3.
















4.
















5.
















6.
















7.
















8.
















9.
















10.

















2. In the same order in which substances to be administered are listed in the table above, very briefly indicate below the purpose of substance administration in the relation to the hypothesis, and the expected effect to the animal(s). If none, so state.


Purpose and Expected Effect of Substance Administration to the Animal

  1. Challenge experiments (protocol 1)

The investigators will be looking for either a total abrogation of tumor development or a delay in tumor formation.

2. Tumor regression experiments (protocol 2)

The investigators anticipate that the killed, modified tumor cell will induce a significant IR that will be able to induce regression of pre-existing tumors.




4.


5.


6.


7.


8.


9.


10.




APPENDIX B: TEST SUBSTANCES (cont.)

3. Is there a possibility that any of the test substance(s) could cause more than momentary or slight pain, discomfort, or distress to the animals, either immediately following substance administration, or as a consequence long after administering the test substance(s)?


No: frame55 Proceed to item #4. Yes: frame56 List those substances in the space below. For each such substance describe the consequences of administration that have a potential to cause animal discomfort, pain, or distress, and how discomfort, pain or distress will be anticipated, minimized or alleviated (e.g., nude mice administered tumor cells will be humanely euthanatized if induced tumors become >1.0 cm in diameter, or if tumors ulcerate, or if tumors interfere with posture, locomotion or feeding). If discomfort, pain, or distress will not be alleviated, then justify how treatments interfere with the procedures or the interpretation of results.


(Note: Log entries describing health concerns or complications that develop as a consequence of substance administration to nonrodent mammals, and their treatment and resolution, or when animals are euthanatized must be kept by the PI in the animal facility on forms provided by Comparative Medicine.)


The administration of the Neuro2A cells subcutaneously will potentially result in tumors under the skin at the site of inoculation. In later stages of the disease, this may result in the metastatic spread of the malignancy. Comparisons of tumor size and burden between groups will be made. If tumor growth is in excess of 1.5 cm in diameter or that treatment has no significant effect on tumor size, progression and/or metastasis, the study will be terminated. Mice that exhibit any tumors that ulcerate or have tumors that impede the mice from grooming and/or motion, or that interfere with the animal's ability to obtain food and/or water, will also be euthanatized.


Because of the possibility that the tumor will metastasize, the mice will be examined for possible signs that are indicative of tumor spread. Previous studies have shown that micrometastasis can occur and histological studies have shown evidence of microscopic foci of tumor cells in the liver and lung but not in other tissues examined such as bone marrow and spleen. The signs indicative of metastasis include: (i) lethargy, (ii) loss of inquisitiveness, (iii) dehydration, (iv) soiled hair coat, (v) closed eye lids, (vi) muscle wasting, (vii) Distended stomach, (viii) hunched posture, (ix) ataxia (x) soiled anogenital area, (xi) respiratory distress and/or (xii) circling. Animals exhibiting any of the above listed signs will be euthanatized.



4. Are any of the test substances listed above in response to item 1, page 7, included in any of the classes A through G, and considered a regulated or potentially hazardous material to research or animal care personnel?


No: frame58 STOP here. This appendix is complete. Yes: frame59 List those substances in the space below and describe procedures to ensure these substances and/or animals to which these substances have been administered will be handled safely (e.g., BSL-2, USF Chemical Hygiene Plan, Universal Precautions, etc.):



The mice will be administered tumor cells that have been modified/changed. Adhering to normal husbandry and handling practices (gloves, lab coat) should be sufficient for safe handling of the animals.





Test substance(s) of any of the classes A-G listed above under item 1, page 7 are either regulated or potentially hazardous and require prior authorization of use by the appropriate Safety Committee. Signature of the Radiation Safety, Biohazard/Recombinant DNA Safety, or Chemical Safety Compliance Officer below indicates that the applicant has consulted with the appropriate Safety Committee, and that that committee has approved the use of the test substance(s). Signature of the applicant PI on page 1 ensures that research personnel will abide by all relevant, universal precautions regarding blood-borne pathogens, appropriate biosafety level precautions, radiation safety procedures, and the chemical hygiene plan.


(Note: Approval of an application involving hazardous materials is often contingent on a pre-performance meeting involving staff that represents the applicant’s laboratory, the Division of Comparative Medicine, the IACUC, and the appropriate Safety Committee(s). This meeting is required in order to ensure that all involved personnel are aware of the precautions, containment practices, facilities, protective devices, disposal and decontamination procedures, and other necessary safety procedures that must be followed to protect personnel, and prevent accidental animal exposure to the hazardous material.)


___________________________________________________________________________________________

Signature of Safety Officer(s) Date


Description of Appendix B is complete.


^ Protocol 1 : Challenge Experiment


Groups

Primary Inoculation : Cell Type/Conc

# Mice/group

^ Day Following Primary Inoc

Challenge Cell Type/Conc

1 (Control)

3x106 N-2a

18

-

None

^ 2 ( Experimental)

3x106 N-2/55

18

14-17

3x106 N-2a

3 (Experimental)

3x106 N-2/55

18

14-17

3x106 N-2a



Protocol 2: Regression of Pre-existing N-2a Tumors in A/J Mice














^ Vaccine Dose (N2/55)




Groups

Challenge Dose (N-2a)

# Mice/group

Day 5

Day 10

Day 15

1

1x106

18

-

-

-

2

1x106

18

3x106

-

-

3

1x106

18

3x106

3x106

-

4

1x106

18

3x106

3x106

3x106

5

1x106

18

1x106

-

-

6

1x106

18

1x106

1x106

-

7

1x106

18

1x106

1x106

1x106



At day 20 all surviving mice in the above groups will be euthanatized and samples (blood, spleen liver and tumor) will be taken as previously described.


CMDC #001.13

Effective 12/10





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