ASIP Summer Research Opportunity Program in Pathology (SROPP)
The ASIP Summer Research Opportunity Program in Pathology (SROPP) promotes the entry of under-represented scientists into the mainstream of the basic, translational, and clinical research communities. ASIP has partnered with the Intersociety Council for Pathology Information (ICPI) to provide summer research opportunities in pathobiology mentored by ASIP members.
Students participate in the lab activities of an ASIP member at a host university for 10 weeks during the summer. Housing is provided by the host university, which is paid directly to the university by ASIP. ASIP provides a stipend to the student to cover travel costs, living expenses, and a book allowance. The student also receives complimentary ASIP membership for one year.
Below are the reports submitted by the 2016 SROPP students.
Liliana Espinoza, undergraduate student from St. Mary's University in Phoenix, participated in the ASIP SROPP in the laboratory of Dr. Cecelia Yates at the School of Nursing, University of Pittsburgh, and is now pursuing a PhD at the University of Texas Health Science Center at San Antonio.
I was fortunate to be a part of the Summer Research Opportunity Program in Pathology (SROPP) - sponsored by the American Society for Investigative Pathology (ASIP) and the Intersociety Council for Pathology Information (ICPI) - for two consecutive summers. This internship provided me with the opportunity to conduct research under the tutelage of my previous mentor, Dr. Cecelia Yates, and my co-mentor, Dr. Monte Willis. I can undoubtedly say that the experience I have gained will continue to influence my professional career as a future medical researcher for years to come. Not only did I learn more about conducting high impact research and in so doing add to my library of methodologies, but I also benefited from the knowledge and expertise of my research mentor, as she provided me with valuable advice related to pursuing a doctoral degree, being an effective and efficient researcher, and exploring post-doctoral career options following graduate school.
My project was conducted in myocarditis, which is an inflammatory disease of the heart muscle that commonly results in cardiac dysfunction and death. The severe cardiac inflammation that characterizes the pathogenesis of myocarditis is mediated by leukocytes entering the cardiac tissue and is often accompanied by remodeling and cardiomyocyte apoptosis. Due to the fact that myocarditis is caused by viral infection, possible cell sources for viral replication are cardiac fibroblasts (CF) and cardiomyocytes (CM), which are the two primary cell types found in the heart. It has been recently observed that CXC chemokine ligand 10 (CXCL10) is unregulated in the myocardium during the early stages of infection. CXCL10 is thought to be the master regulator of myocardial interactions between cardiac and immune cell migration, perhaps affecting cardiac damage and the clinical progression of myocarditis.
In my previous summer project, we demonstrated that IP-10p (developed in our lab), a CXCL10 agonist via its receptor CXCR3, inhibits growth factor-induced motility in both CF and CM. Currently, clinical therapies for myocarditis are predominantly based on symptoms and diagnosis of the underlying infection but lack a targeted approach to inhibiting the cardiac damage induced by infections. As a first step toward this approach, we designed, developed, and tested in vitro peptides with the potential to inhibit the cardiac damage induced by infections. In the study, we tested two fifth-generation small peptide antagonists of CXCL10-CXCR3, CR3-5-3 and CR3-5-5, developed by in silico prediction-based functional peptide design to directly bind to CXCL10.
To test the hypothesis that CR3-5-3 and CR3-5-5 peptides similarly block CXCL10-CXCR3 signaling and function on cardiac fibroblasts and cardiomyocytes, CM and CF were isolated from wild-type and CXCR3-null FVB neonatal mice. Following isolation, CM, CF, or CM/CF in both direct and indirect co-cultures were challenged with CM and FB activating growth factor, TGF-ß. Antagonist peptide treatments, CR3-5-3 and CR3-5-5, were given alone or in combination with CXCL10. In vitro cellular function assays were assessed for migration, apoptosis, and ECM secretion. Real time quantitative polymerase chain reaction (RTPCR) was employed in order to determine alterations in relative gene expression of both CM and FB, following treatment. Our data showed that CR3-5-5 directly blocked CXCL10 inhibition of motility and apoptosis. Additionally, CR3-5-3 and CR3-5-5 altered CXCL10-induced gene expression on both FB and CM. Finally, it was also found that CR3-5-5 interfered with CXCL10 inhibition of TGF-ß-induced matrix production.
Taken as a whole, this summer internship was of paramount importance to my continued development as a student and researcher. I consider that the SROPP is the perfect stepping stone for students pursuing a career in the biomedical sciences. The collaborative nature of the projects, intrinsic gratification that comes from conducting a meaningful research project, helpful advice from MD PhD and PhD students, and weekly seminars on current research being conducted at the University of Pittsburgh allowed me to further substantiate my decision to pursue a graduate degree. I am especially thankful for the mentorship I continue to receive from Dr. Yates; her guidance helped foster my critical thinking skills, encouraged me to look at other research studies related to my project, and gave me the confidence to find ways to improve my experimental design. I am thankful for the instrumental skills I have gained during this summer internship because I will carry them forward in life. Dr. Yates genuinely cares about her lab and its constituents, and my experience would not have been as gratifying had it not been for her unfaltering support.
Ellah Nzikoba, undergraduate student from Tufts University, participated in the ASIP SROPP in the laboratory of Dr. Diane Bielenberg at Harvard Medical School.
Ellah Nzikoba, ASIP SROPP student, 2016
Over the past 3 months I have been honored to study and work in a contemporary laboratory at Harvard Medical School thanks to the generosity of the Summer Research Opportunity Program in Pathology (SROPP) sponsored by the American Society for Investigative Pathology (ASIP). I was mentored and supervised daily by Dr. Diane Bielenberg, PhD, a cancer biologist in the Vascular Biology Program at Boston Children's Hospital. My summer experience was enriched by participating in the Continuing Umbrella of Research Experiences (CURE) Program at the Dana Farber Harvard Cancer Center where I was immersed in a diverse community of nearly forty summer students. Weekly activities included Monday seminars focused on cancer research and cancer treatment given by local experts, a journal club where we discussed cancer-related published articles from basic research to clinical trials, evening seminars that included a panel discussion on career opportunities in biomedical sciences, weekly lab meetings in which we took turns sharing our data, and a book club where we read and discussed Pandora's DNA: Tracing the Breast Cancer Genes Through History, Science, and One Family Tree by Lizzie Stark.
However, the majority of my time was spent in the laboratory. My project focused on the role of the cell surface receptors called neuropilins in renal function. Two neuropilin receptors called NRP1 and NRP2 have disparate roles in angiogenesis and cancer progression depending on which ligand they bind. The VEGF family of ligands bind to NRPs to promote angiogenesis, the sprouting of new blood vessels, and thereby tumor growth. In contrast, the semaphorin 3 (SEMA3) family of ligands bind to NRPs to inhibit neovasculariza-tion and therefore are potent anticancer drugs in preclinical trials. A recent Phase Ib clinical trial using anti-NRP1 antibodies in combination with anti-VEGF antibodies and chemotherapy (Cancer Chemother Pharmacol 73(5):951-60, 2014) was discontinued due to the high level of proteinuria seen in human cancer patients. Based on this human trial, we hypothesized that NRP1 may be highly expressed in podocytes, specialized epithelial cells in the glomeruli of the kidney that are involved in protein filtration from the blood into the urine. Normally, high molecular weight proteins are not found in the urine, but if a drug causes renal toxicity then the podocyte-endothelial barrier may become damaged and release proteins into the urine, a conditioned called proteinuria.
Using immunoblotting I examined the expression of Nrp1* and Nrp2* in various mouse organs. I determined that Nrp1 was highly expressed in kidney protein lysates, whereas Nrp2 was only weakly expressed. To localize the expression of the Nrp receptors within the kidney, I used immunohistochemistry in paraffin and frozen tissue sections. Nrp1 was highly expressed in podocytes in the glomeruli and also found in epithelial cells of the renal tubules and in endothelial cells of blood vessels. Nrp2, on the other hand, was only found in endothelial cells, particularly in veins and lymphatic vessels in the kidney. Our immunohistochemistry results were confirmed by staining kidneys from transgenic Nrp2+/lacZ mice with X-gal reagent to locate cells with a beta-galactosidase gene insert (denoting Nrp2 expression). To further examine the function of the Nrp receptors in vivo in the kidney, I performed protein assays to quantify the amount of protein in the urine collected from mice housed in metabolic cages. Mouse urine is much more concentrated than human urine. When I compared urine from wildtype mice (Nrp2+/+) and mice lacking Nrp2 expression (Nrp2-/-), I found low levels of protein in both conditions suggesting that targeting the NRP2 receptor, unlike the NRP1 receptor, may not cause renal toxicity. However, when we injected mice systemically with adenoviral constructs encoding SEMA3 proteins, we did see an increase in urine protein levels indicating that these inhibitory proteins may cause podocyte damage or interfere with filtration function. Taken together, our data suggest that anti-cancer therapies should target the NRP2 receptor rather than the NRP1 receptor to limit toxicity while maintaining efficacy.
Personally, this summer's internship provided by ASIP gave me the unique opportunity to gain hands-on laboratory experience in a fast-paced and energetic environment. I learned numerous laboratory techniques and interacted daily with students, postdoctoral fellows, clinical fellows, and professors at every level. Most importantly this experience taught me to think carefully, to analyze my data critically, and to prepare my results in a timely manner. At the end of the summer, I prepared an abstract and presented my research in the form of an E-poster to the Harvard Community. I plan to present my studies at the upcoming ABRCMS meeting in 2016 and at Experimental Biology in 2107. My future goals are to continue a career in the biomedical sciences and to apply to an MD/PhD program following my graduation from Tufts University in 2019.
*Editor's Note: Nrp1 and Nrp2 are the mouse analogs of human NRP1 and NRP2.