American Society for Investigative Pathology

American Society for Investigative Pathology Awards & Honors
2003 Predoctoral Merit Award

Ryan M. Gill

University of Kansas Medical Center

Characterization of the Novel TNF Superfamily Member, LIGHT, in Human Placenta
Ryan M. Gill^* and Joan S. Hunt^{*†

*}Department of Pathology and Laboratory Medicine
^†Department of Anatomy and Cell Biology
University of Kansas Medical Center
3901 Rainbow Blvd.
Kansas City, KS 66160-7400

Messages encoding virtually all members of the tumor necrosis factor (TNF) superfamily are detected in human placentas, including LIGHT/TNFSF14 (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes). Although LIGHT reportedly activates T-cells and kills tumor cells, its expression and function in placenta have yet to be explored. By RT-PCR, immunoblotting and immunohistochemistry, we demonstrated mRNA, protein expression and differential cellular localization of LIGHT and its receptor proteins [herpes virus entry mediator (HVEM), lymphotoxin beta receptor (LTβR) and decoy receptor 3 (DcR3/TR6)] in term human placentas and fetal membranes. Since LIGHT has been reported to induce apoptosis in tumor cells expressing both HVEM and LTβR, and since we observed that placental cells express these receptors, we investigated LIGHT’s in vitro effect on cytotrophoblast cells purified from human placentas. By using a metabolic enzyme activity assay (MTT) and immunoblotting for active caspase-3, we observed that LIGHT and interferon-γ (IFNγ) alone could not kill cytotrophoblast cells yet LIGHT plus IFNγ triggered apoptosis. This effect could be blocked by LIGHT’s soluble receptor, DcR3. Following this observation we focused our studies on determining how IFNγ permitted LIGHT to kill cytotrophoblast cells. We investigated two members of the inhibitor of apoptosis (IAP) family known to interact with an adaptor for HVEM (cIAP-1 and cIAP-2). We found that cIAP-2 expression was significantly induced by LIGHT but not by IFNγ or LIGHT/IFNγ. Since cIAP-2 can inhibit activation of caspase-3, and subsequent apoptosis, we investigated if this molecule regulated LIGHT/IFNγ-mediated apoptosis. Primary human cytotrophoblast cells did not tolerate transfection well. Therefore, we turned to a model cell line (HT-29) reported to be highly susceptible to LIGHT/IFNγ-mediated apoptosis. We found that HT-29 cells transfected with a control morpholino were not susceptible to LIGHT-mediated apoptosis but that when cIAP-2 expression was knocked down by an antisense morpholino, LIGHT could kill these cells. These data strongly suggest that LIGHT may be an important placental cytokine and indicate that regulation of cIAP-2 is critical for LIGHT/IFNγ-mediated apoptosis, a process that is essential for both placental pathology and normal development.

CURRICULUM VITAE
Ryan M. Gill, Ph.D.
University of Kansas Medical Center
3901 Rainbow Blvd.
Kansas City, KS 66160-7400
Tel: 913-588-2734 Fax: 913-588-7180 email: rgill2@kumc.edu

Education:

Year	Degree	Institution
1999	B.S. magna cum laude (Microbiology)	Colorado State University, Ft. Collins, CO
2002	Frontiers In Reproduction Course	Marine Biological Laboratory, Wood’s Hole, MA
2003	Ph.D., Pathology	University of Kansas Medical Center, Kansas City, KS Mentor: Joan S. Hunt, Ph.D. Thesis title: “Characterization of the Novel TNF Superfamily Member, LIGHT, in Human Placenta”
2005	M.D. in progress	University of Kansas Medical Center, Kansas City, KS

Academic Honors and Awards:
1999    National Science Foundation (NSF) Graduate Research Award, Honorable Mention
1999    University of Kansas Medical Center (KUMC) M.D./Ph.D. Scholarship
2000    American Academy of Allergy, Asthma, and Immunology (AAAAI) Research Fellowship
2000    KUMC Lawson-Mann Research Fellowship
2001    KUMC Student Leadership Award, Nominee
2002    KUMC Department of Pathology and Laboratory Medicine Research Grant
2002    KUMC Biomedical Training Program Scholar
2003 American Society for Investigative Pathology Merit Award

Travel awards:
2001    KUMC Graduate Student Travel Award
2001    KUMC Medical Student Travel Award
2003    KUMC Reproductive Science Center Travel Award
2003    FIR Alumni Travel Award

Professional Societies and Affiliations:
Year               Organization
1999-pres.       Phi Kappa Phi (elected)
1999-pres.       Sigma Xi (elected)
1999-pres.       American Medical Association
1999-pres.       American Medical Student Association
2002-pres.       American Society for Investigative Pathology

Recent Service:
1999-2000             Duchesne Clinic, Kansas City, Kansas - Volunteer
2000-2002             Science Pioneers “Meet the Mentors” day – Volunteer Mentor
2002                     Kansas premedical advisor conference – Invited Speaker

Administration:
1999-2000             KUMC Social Basis for the Practice of Medicine Course, Student Advisor
1999-2001             Medical Genetics Journal Club, Organizer
1999-2002             KUMC M.D./Ph.D. webpage design and maintenance
2000                     KUMC Medical Microbiology Course, Student Advisor
2001-pres.             Kansas Biomedical Research Infrastructure Network (BRIN) Grant, Student Representative.
2001-2002             KUMC M.D./Ph.D. Student Council Vice President.
2002-2003             KUMC M.D./Ph.D. Student Council President

2002-pres Pathology Student Interest Group, Organizer

Teaching Activities:
2001-2002       KUMC Medical Immunology Course, Small Group Instructor
2002-2003       KUMC Medical Immunology Course, PBL Facilitator
2002-2003       Research Supervision and Training for a 4^th year KUMC Medical Student

Meeting Participation:

Participant, NIH 15^th Annual National M.D./Ph.D. Student Meeting, Aspen CO 2000
Participant, NIH 16^th Annual National M.D./Ph.D. Student Meeting, Aspen CO 2001
Participant, 49^th Annual Society for Gynecologic Investigation (SGI) Meeting, Los Angeles, CA 2002
Participant, NIH 4^th Annual Frontiers in Reproduction Symposium, Boston, MA 2002
Participant, 1^st Annual Kansas City Life Sciences Meeting, Kansas City, KS 2003
Participant, Experimental Biology, San Diego, CA 2003
Invited Speaker, Chugai Symposium for Young Investigators (FASEB), San Diego, CA 2003
Participant, NIH 5^th Annual Frontiers in Reproduction Symposium, Woods Hole, MA 2003
Invited Speaker, KUMC Biomedical Scholars Research Day, Kansas City, KS 2003

Recent Research Presentations:
“Localization of the TNF superfamily member, LIGHT, in the human placenta.”
Ryan M. Gill, Teresa A. Phillips, and Joan S. Hunt
KUMC Student Research Forum (2001)
Kansas City, KS.
Oral Presentation

“Localization of the TNF superfamily member, LIGHT, in the human placenta.”
Ryan M. Gill, Teresa A. Phillips, Jian Ni, and Joan S. Hunt
NIH 16^th annual MD/PhD student conference (2001)
Aspen, CO.
Poster Presentation

“Localization of the TNF superfamily member, LIGHT, and its TNFR superfamily member receptors: HVEM, LTβR, and DcR3/TR6, in human placenta.”
Ryan M. Gill, Jian Ni and Joan S. Hunt
Society for Gynecologic Investigation (2002)
Los Angeles, CA.
Selected Oral Presentation

“Differential expression of LIGHT and its receptors in human placentas and amniochorion membranes.”
Ryan M. Gill, Jian Ni and Joan S. Hunt
KUMC Student Research Forum (2002)
Kansas City, KS.
Oral Presentation

“The TNF supergene family: potential functions in human placentas.”
Joan S. Hunt, Teresa A. Phillips, Hakhyun Ka and Ryan M. Gill.

SSR (2002)
Baltimore, MD.

“LIGHT ligand-receptor signaling and potential functions in human placentas.”
Ryan M. Gill and Joan S. Hunt
Frontiers in Reproduction Course (2002)
Wood’s Hole, MA
Boston, MA
Oral Presentation & Poster Presentation

“The TNF superfamily and pregnancy: support for a Th2 bias.”
Teresa A. Phillips, Ryan M. Gill, Hakhyun Ka, and Joan S. Hunt
TNF superfamily conference, 9^th international congress (2002)
La Jolla, CA

“Human placental cytotrophoblast cells: resistance and susceptibility to killing by LIGHT and interferon-γ.”
Ryan M. Gill and Joan S. Hunt
Experimental Biology (2003)
San Diego, CA
Poster Presentation

“Human placental cytotrophoblast cells: resistance and susceptibility to killing by LIGHT and IFN-γ” Ryan M. Gill and Joan S. Hunt
Chugai Symposium for Young Investigators, FASEB (2003)
San Diego, CA
Invited Speaker

“Human placental cytotrophoblast cells; resistance and susceptibility to killing by LIGHT and IFNγ, a role for HIAP-1”
Ryan M. Gill and Joan S. Hunt
KUMC Student Research Forum (2003)
Kansas City, KS
Oral Presentation

“HIAP-1 regulates LIGHT-mediated apoptosis in human placental cells.”
Ryan M. Gill and Joan S. Hunt
KUMC Biomedical Scholars Research Day (2003)
Kansas City, KS
Invited Speaker

Peer-Reviewed Original Research Papers:

1. Gill RM, Ni J, Hunt JS. (2002) Differential expression of LIGHT and its receptors in human placentas and amniochorion membranes. American Journal of Pathology, 161(6) 2011-2017.

Peer-Reviewed Review Papers:

1. Gill RM, Ka H, Hunt JS. (2003) TRAIL, LIGHT, and the expanding tumor necrosis factor superfamily in human placentas. PINSA, B69(2) 41-56.

Published Abstracts

Gill RM, Ni J, Hunt JS. (2002) Localization of the TNF superfamily member, LIGHT, and its TNFR superfamily member receptors: HVEM, LTβR, and DcR3/TR6, in the human placenta. Journal of the Society for Gynecologic Investigation, 9(1), Supplement, 74A-75A.

Hunt JS, Phillips TA, Ka H, Gill RM. (2002) The TNF supergene family: potential functions in human placentas. Biology of Reproduction, 66, Supplement 1, 87.

Gill RM, Hunt JS. (2003) Human placental cytotrophoblast cells: resistance and susceptibility to killing by LIGHT and interferon-γ. FASEB, 17(4) A669.

University of Kansas
Department of Pathology and Laboratory Medicine

The Department of Pathology and Laboratory Medicine at the University of Kansas bridges the basic sciences with clinical care of patients. The department’s goals are (1) to provide the highest quality anatomic and clinical pathology services to Kansas and the Kansas City area (2) to maintain innovative research programs at the vanguard of medicine and science (3) to provide the best possible learning experience for medical and graduate students and (4) to incorporate all of these ideals into training the next generation of pathologists that have entered our residency program.

The Department of Pathology and Laboratory Medicine at the University of Kansas Medical Center provides diagnostic and consultative services in anatomic pathology and laboratory medicine. Continuing expansion of our faculty and technical staff will provide an update of our clinical, diagnostic, and research expertise, with full laboratory support in multiple disciplines. We are also affiliated with the Kansas City VA Medical Center. Incorporation of all our services into the University of Kansas Pathology Residency program provides outstanding service to our staff physicians, and also strengthens our services as a tertiary care center for the State of Kansas.

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