American Society for Investigative Pathology Awards & Honors
1999 Postdoctoral Merit Award

      In humans, IL-8 appears to be the most important CXC chemokine involved with neutrophil recruitment. Consequently, studies of in vivo inflammation in animal models need to consider the effects of chemokines such as IL-8 if the results are going to be extrapolated to the human condition. This can be complicated in murine models of inflammation because no exact homologue has been identified for human IL-8. Two murine chemokines, KC and MIP2, are considered likely functional equivalents to human IL-8, but they are structurally most homologous to human Gro-alpha and Gro-beta, respectively. We designed this study as an initial test of the hypothesis that either KC or MIP2 serves as the functional equivalent to IL-8 for in vivo models of inflammation.

      We developed recombinant KC and MIP2 proteins, neutralizing polyclonal antibodies and sandwich ELISAs that were suitable for quantifying both chemokines with a high degree of sensitivity and specificity (10-30 pg/ml). Our initial study involved two models of mild peritonitis for which BALB/c mice were given either 3% thioglycollate (2 ml, IP, n = 14) or 1% glycogen (2.5 ml, n = 6) or normal saline (2.5 ml, n = 6). After 4 h, plasma and residual peritoneal fluid were collected and total neutrophils quantified. Thioglycollate and glycogen elicited a similar number of neutrophils in the peritoneum (~2.3 X 106 cells). Total KC in the peritoneum was greater than total MIP2 for both thioglycollate (mean ng/ml ± SE; 7.1 ± 1.6 vs 4.5 ± 1.0; P < 0.05) and glycogen models (2.5 ± 1.2 vs 0.3 ± 0.2; P < 0.05). Plasma KC (18 ± 2 ng/ml) in the thioglycollate model was 49 times greater than plasma MIP2 (P < 0.05) while plasma KC (24 ± 5) in the glycogen model was 68 times greater than plasma MIP2 (P < 0.05). Total neutrophils were highly correlated with plasma KC (r = 0.75; P < 0.05) and peritoneal KC (r = 0.62; P < 0.05). BALB/c mice were then passively immunized with polyclonal anti-KC or anti-MIP2 or both or given control serum (3 mice per treatment, 3 independent experiments). Two hours later these mice received an IP thioglycollate treatment and they were processed 4 h later. Anti-MIP2 was associated with a 71% decrease in total peritoneal neutrophils while anti-KC produced an 82% decrease. The combination of antibodies resulted in a 90% decline in total neutrophils recruited to the peritoneum compared with control animals (P < 0.05). Total neutrophils were highly correlated with plasma KC (r = 0.75; P < 0.05) and peritoneal MIP2 (r = 0.47; P < 0.05). A multiple regression model including plasma KC and peritoneal MIP2 explained 69% of the variance for neutrophil recruitment for these latter experiments.This is the first study to use neutralizing antibodies to directly evaluate the roles of murine KC and MIP2 in an inflammatory model. We found that passive immunization against either chemokine produced a significant reduction in peritoneal neutrophils (> 70%). Neutrophil counts in the peripheral blood of antibody treated animals were not significantly different, which indicates that the reduced number of peritoneal neutrophils was due to a lack of recruitment rather than due to a reduction in the peripheral pool of neutrophils. While we have not identified the exact mechanisms by which these two chemokines mediate an inflammatory response, it is clear that with 90% of neutrophil recruitment explained by KC and MIP2 there is little reason to expect the presence of an exact IL-8 homologue in the murine repertoire of chemokines. In addition, it is clear that neutralization studies that fail to consider multiple chemokines may overestimate the importance of any single chemokine during an inflammatory response. Finally, our study suggests that KC may play both a local and systemic role during inflammation while MIP2 appears to be mostly restricted to local inflammatory compartments. Further work is needed to identify the link between changes in chemokine levels and subsequent inflammatory response and wound healing. Additional studies are underway to correlate the severity of inflammation with KC and MIP2 for several other models of inflammation.

Current address
University of Michigan                                734-764-4593 (day)
Department of Pathology                              734-480-1334 (eve)
1301 Catherine St., Med. Sci. I, M2210         FAX 734-763-6476
Ann Arbor, MI 48109
drcall@umich.edu

Education
PhD, 1997, Zoology, Washington State University, Pullman, WA. GPA 3.95.
MS, 1990, Wildlife Management, Humboldt State University, Arcata, CA. GPA 3.8.
BS, 1987, Wildlife Management, Washington State University, Pullman, WA. GPA 3.9.
Fall 1991, School of the Environment, Duke University, Durham, NC. GPA 4.0.
Spring 1983, Elma High School, Elma, WA. GPA 4.0.

Research and teaching experience
April 1997 - Present
Postdoctoral Fellow, Univ. Michigan, Dept. Pathology. Our lab is studying the role of cytokines in sepsis, asthma, and other immunopathogenic disease in humans and murine models. My primary contribution involves studies of the relative expression of the murine chemokines KC and MIP2 in several models of acute and chronic inflammation. I have also published a study on the effects of anticoagulants on cytokine production in human blood. This training has added substantially to my skills for the study of coding and noncoding regions of DNA that are applicable to molecular ecology and ecological epidemiology. These skills combined with previous training are also applicable to studies of transcription, translation and biological activity of proteins that are important for toxicological and evolutionary investigations. Advisor: Dr. D. Remick.
June 1998.
BioSci 420/524, General Ecology, 4 semester credits, Eastern Michigan Univ., Ypsilanti, MI. I taught a 4-week intensive field ecology course at the Kresge Environmental Center, Lapeer, MI. The course focused on basic field and analytical techniques and included two field projects and an independent project. Lectures focused on development and application of scientific reasoning skills and covered applied and theoretical aspects of evolutionary ecology, population ecology, and conservation biology.
April 1993 - April 1997
Doctoral research, Washington State Univ., Dept. Zoology. I studied genetic structure of amphibian populations relative to patterns of forest fragmentation and my research involved both field and lab work. I characterized novel microsatellite markers for two frog species in addition to producing multilocus fingerprint profiles to study population differentiation. I also designed computer simulations to test the reliability of inferences drawn from genetics data for small, nonequilibrium populations. I presented several papers at professional meetings, and competed successfully for several grants and have completed two publications from this work. Advisor: Dr. J. Hallett.
April 1992 - April 1997
Research assistant for Dr. J. Hallett, WSU. I assisted with a study of forest fragmentation and with a study of forest harvest practices in riparian management zones. My initial responsibilities included development of a habitat classification system based on Landsat imagery and development and management of several datalayers with ARC-INFO. Additional responsibilities included study site selection and preparation, data collection (small mammal, avian and botanical), supervision of as many as 20 employees, and data analysis.
September 1994 - May 1995
Teaching Assistant for Introductory Biology (Bio Sci 103, fall semester) and Honors Biology (Bio Sci 298, spring semester) at WSU. For the latter course I designed most of the lab exercises and assembled several lectures on ecological and conservation topics.
August 1991 - December 1991
Research assistant for Dr. L. Maguire, School of the Environment, Duke Univ. I assisted with a workshop on population viability analysis of Northern Goshawks on the Kaibab Plateau, July 1992, Tucson, AZ.
May 1987 - June 1991
Research Assistant/Associate for Dr. R. Gutiérrez, Humboldt State Univ. I was project leader for my Masters research on foraging ecology of California Spotted Owls and a crew leader for a demography study in southern California and Utah. Duties included study design, data collection (habitat and demographic) and analysis, writing reports, preparing manuscripts, and some budgetary planning. I was also responsible for supervising and training field crews for three research projects involved with studies of foraging ecology, nesting habitat, home-range and demographics of Spotted Owls. I was an author/coauthor on four papers from this work.

Grants and awards

American Society for Investigative Pathology Merit Award, Dec 1998.
Brislawn Memorial Scholarship, Spring 1996.
WSU College of Sciences student minigrant ($300), Spring 1996.
EPA STAR Fellowship ($43,300), Fall 1995-Fall 1996.
WSU Graduate School Fellowship, summer 1995.
James R. King Fellowship ($2,500), Fall 1994.
Mazamas ($2,960), Spring 1994.
The Nature Conservancy ($1,000), Spring 1993.
Sigma Xi ($390), Spring 1993.
Northwest Scientific Society ($350), Spring 1993.
WSU Graduate School Travel Grant ($500), Spring 1993.
Wildlife Student Achievement Scholarship, 1985, 1986, 1987.
Phi Beta Kappa Scholarship, 1986.
Associated Western University Internship, INEL, Idaho Falls, ID, 1986.
Mark E. Reed Memorial Scholarship, 1983, 1986.
Elk's Scholarship, county, state, national levels, 1983.

Professional/Volunteer activities

Current society memberships:
American Society for Investigative Pathology
Society for Conservation Biology
Ecological Society of America
The Wildlife Society
Phi Kappa Phi National Honor Society.

Positions and activities:
Participant in Walk to Cure Diabetes, Ypsilanti, MI, September 1998.
Volunteer judge for Forsythe Middle School Science Fair, Ann Arbor, MI, February 1998.
Department of Zoology Invited Speakers Committee, 1995-96.
Member of University Graduate Studies Committee, WSU, Fall 1996.
Graduate and Professional Student Association Senator, WSU, 1995-1996.
Women's Transit driver, WSU, fall 1993.
Program Committee Co-chair, WSU Chapter of Society for Conservation Biology, 1992-93.
President of Washington State University Student Chapter of The Wildlife Society, 1986-87.
Volunteer for Shasta National Forest spotted owl survey crew, summer 1986.

Publications
     Call, D.R., J.G. Hallett, S.G. Mech, and M. Evans. 1998. Considerations for measuring genetic variation and population structure with multilocus fingerprinting. Molecular Ecology 7:1337-1346.
     Call, D.R. and J.G. Hallett. (1998). PCR primers for microsatellite loci in the anurans Rana luteiventris and Hyla regilla. Molecular Ecology 7:1085-1087.
     Call, D.R. and D.G. Remick. (1998). Low molecular weight heparin is associated with greater cytokine production in a stimulated whole blood model. SHOCK 10:192-197.
     Call, D.R., R.J. Gutiérrez and J. Verner. (1992). Habitat use and home-range size of California Spotted Owls in the Sierra Nevada. Condor 94:880-888.
     Gutiérrez, R.J., J. Verner, D.R. Call, G.S. Steger, W.S. LaHaye, and K. McKelvey. (1993). Habitat relationships of the California spotted owl. In Verner, J. and R.J. Gutiérrez, (eds). The California Spotted Owl: a technical assessment. Pacific Southwest Forest and Range Experiment Station. Gen. Tech. Rept. Berkeley, CA.
     Hennein, H.A. et al. (in press). Veno-venous modified ultrafiltration and circulating cytokines: A prospective randomized study. Journal of Thoracic and Cardiovascular Surgery.
     LaHaye, W.S., R.J. Gutiérrez, and D.R. Call. (1997). Nest-site selection and reproductive success of California Spotted Owls. Wilson Bulletin 109:42-51.
     LaHaye, W.S., Gutiérrez, R.J. and D.R. Call. (1992). Demography of an insular population of Spotted Owls (Strix occidentalis occidentalis). Pages 803-814 In D. McCullough and R. Barrett, editors. Wildlife 2001: Populations. Elsevier Press.

Other documents
     Call, D.R. 1997. Microsatellite characteristics and population structure for two anurans (Rana luteiventris and Hyla regilla). PhD Dissertation, Washington State Univ., Pullman, WA.
     Call, D.R. 1997. Microsatellite characteristics and population genetic structure for Rana leuteiventris and Hyla regilla. Appendix A in East-side studies: research results. Volume 3 of Wildlife use of managed forests: a landscape perspective. Final report TFW-WL4-98-002 to the Timber, Fish and Wildlife Cooperative Monitoring, Evaluation, and Research Committee, Washington Department of Natural Resources. Olympia, Washington.
     Call, D.R. and J.G. Hallett. 1997. Detecting early differentiation between small, nonequilibrium populations. Appendix B in East-side studies: research results. Volume 3 of Wildlife use of managed forests: a landscape perspective. Final report TFW-WL4-98-002 to the Timber, Fish and Wildlife Cooperative Monitoring, Evaluation, and Research Committee, Washington Department of Natural Resources. Olympia, Washington.
     Call, D.R. 1990. Home range and habitat use by California Spotted Owls in the central Sierra Nevada. MS Thesis, Humboldt State Univ., Arcata, CA.

Manuscripts in preparation/review
     Ebong S., Call D.R., Bolgos G., Newcomb D.E., Granger J., O'Reilly M. and D.G. Remick. (in review). Immunopathologic alterations in non-lethal sepsis.
     Granger, J, M. O'Reilly, D.R. Call, S. Ebong, A. Taur, B. Williams, M. Nauss, J. Millican and      D.G. Remick. (in review). A sandwich ELISA for measurement of picogram quantities of murine granulocyte colony stimulating fractor.
     Nemzek, J.A., Newcomb, D., Call, D.R., and D.G. Remick. (in review). Optimization of enzyme-linked immunosorbant assays using commercially matched antibody pairs: removal of plasma inhibition.
     Remick, D.G., Newcomb, D.E., Bolgos, G.L., and D.R. Call (in review). Comparison of the mortality and inflammatory response of two models of sepsis: lipopolysaccharide vs. cecal ligation and puncture.
     Remick, D.G., Call, D.R., Ebong, S., Newcomb, D.E., Nemzek, J. and G. Bolgos. (in review). Combination immunotherapy with TNF-SR pluse IL-1RA fails to decrease sepsis mortality.
     Remick, D.G., Green L.B., Newcomb D.E., Garg S.J., Bolgos G.L. and D.R. Call. (in review). Chemokine redundancy ensures an appropriate biologic response.
     Call, D.R., Bolgos G., Wollenberg G. and D.G. Remick. (in prep). Differential expression of KC and MIP2 chemokines in two models of inflammation.
     Call, D.R. and J.G. Hallett. (in prep). Detecting early differentiation between small, nonequilibrium populations.
     Hallett, J.G., D.R. Call, L.A. Nordstrom and M.A. O'Connell. (in prep). Quantifying habitat fragmentation in forested landscapes.

Presentations and posters
     Pacific Northwest Laboratories, Richland, WA. Call, "Using genetic markers to assess interpopulation migration following habitat disturbance," February 1999.
     Northwest Fisheries Science Center, Seattle, WA. Call, "Using single and multilocus microsatellites to assess genetic structure of anuran populations," October 1998.
     University of Michigan Cancer Center, Ann Arbor, Michigan. Woodford, Evans, Call, Remick and Rochford, "Chemokine and chemokine receptor mRNA expression in Epstein-Barr virus-associated B-cell lymphomas," poster, September 1998.
     Michigan Forum on Inflammation, Ann Arbor, Michigan. Ebong, Call, Bolgos, Newcomb, Granger, O'Reilly and Remick, "Immunopathologic alterations in non-lethal sepsis," June 1998.
     Michigan Forum on Inflammation, Ann Arbor, Michigan. Call and Remick, "Low molecular weight heparin is associated with greater cytokine production in a stimulated whole blood model," June 1998.
     Michigan Forum on Inflammation, Ann Arbor, Michigan. Nemzek, Call, Ebong, Bolgos, Newcomb and Remick, "Immunopathology of a 2 hit model of pulmonary injury," June 1998.
     SHOCK Society meetings, San Antonio, Texas. Nemzek, Call, Ebong, Bolgos, Newcomb and Remick, "Immunopathology of a 2 hit model of pulmonary injury," June 1998.
     Exp. Biol., San Francisco, CA. Call and Remick. "Low molecular weight heparin is associated with greater cytokine production in a stimulated whole blood model," poster, April 1998.
     Exp. Biol., San Francisco, CA. Granger, O'Reilly, Call, and Remick. "Development of a sandwich ELISA for measurement of picogram quantities of murine granulocyte colony-stimulating factor," poster, April 1998.
     Exp. Biol., San Francisco, CA. Bolgos, Call, Ebong, Newcomb and Remick. "IL-1ra + TNF-SR fail to improve sepsis survival," poster, April 1998.
     Exp. Biol., San Francisco, CA. Ebong, Bolgos, Call, Nybom and Remick. "Inflammatory changes in non-lethal sepsis," poster, April 1998.
     Society for Conservation Biology, Victoria, B.C. Call and Hallett. "Habitat fragmentation and molecular data: The lower limits of genetic inference," May 1997.
     Northwest Scientific Association, Cheney, WA. Call, Hallett, and Mech. "Using genetic differentiation to assess local population dynamics: An alternative to demographic studies?" March 1997.
     Department of Zoology, Washington State University, Pullman, WA. Call. " Using population genetics as a predictive tool in conservation biology: an evaluation," December 1996.
     Conference on Declining and Sensitive Amphibians, Boise, ID. Call. "The limits of genetic inference," November 1996.
     Timber Fish and Wildlife Workshop, Olympia and Cheney WA. Call and Mech. "Analyzing movement patterns of vertebrates on fragmented landscapes," October 1996.
     Northwest Scientific Association and Wildlife Society, Tacoma, WA. Call, Mech, and Hallett. "Detecting population differentiation using multilocus DNA fingerprinting," March 1996.
     Department of Botany, Washington State Univ., Pullman, WA. Call, "Global Amphibian Decline - is it real?" November 1994.
     American Mammalogists' Society, Bellingham, WA. Call, Nordstrom, Hallett, Campbell, and O'Connell, "Quantifying habitat fragmentation in forested landscapes," June 1993.
     Northwest Scientific Association, La Grande, OR. Call, Nordstrom, Hallett, Campbell, and O'Connell, "Quantifying habitat fragmentation in forested landscapes," March 1993.
     Cooper Ornithological Society, Seattle, WA. LaHaye, Gutiérrez and Call, "Nest site selection in an insular population of California Spotted Owls," June 1992.
     Wildlife 2001 conference, Oakland, CA. LaHaye, Gutiérrez and Call, "Demography of an insular population of Spotted Owls," July 1991.
     American Ornithologists' Union and Cooper Ornithological Society, Los Angeles, CA. Call, "Habitat use by foraging California Spotted Owls in the central Sierra Nevada," June 1990.
     Local chapters of Audubon Society and American Forestry Association, Mt. Shasta, CA. Call, "Habitat use by California Spotted Owls," February 1990.

The following abstracts will be presented during the April '99 Experimental Biology meeting:
Bolgos, Call, Newcomb and Remick. Two models of septic shock: similarities and differences. Call, Bolgos, Wollenberg, Newcomb, Ebong, Nemzek and Remick. Defining the relative roles of two IL-8 homologues (murine KC and MIP2) during inflammation (accepted for oral presentation).
Ebong, Call, Bolgos, Nemzek, Newcomb and Remick. Immunopathologic alterations in murine sepsis models of increasing lethality.
Nemzek, Call, Ebong, Bolgos, Newcomb and Remick. Immunopathology of a "2-hit" model of lung injury.

The following abstracts will be presented during the June '99 SHOCK meeting:
Remick, Green, Newcomb, Garg, Bolgos and Call. Chemokine redundancy ensures an appropriate biologic response.
Nemzek, Call, Ebong, Bolgos, Newcomb and Remick. Immunopathology of a 2 hit model of pulmonary injury.

     The Department of Pathology at the University of Michigan is composed of nearly 60 faculty members. The department is very committed to fulfilling its roles in education, clinical service, and research. There are active education programs for training medical students, graduate students, house officers in pathology, and post doctoral fellows. Clinical services are provided to the University of Michigan health system and includes the full range of anatomic and clinical pathology. There ares several active investigators at University of Michigan with extensive grant support from industry, NIH, and the NCI. The University of Michigan has enjoyed a strong reputation under leadership of our department chair, Peter A. Ward M.D. For additional information visit the home page, http://www.pathology.med.umich.edu/.

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