XBP1 Protein Expression In Colon Adenocarcinoma
Pls: Jonathan Lin, Michael Peterson
XBP1 (X-box binding protein 1) is upregulated as a consequence of activation of the unfolded protein response (UPR), which is in turn activated by cellular stresses, such as hypoxia. Initial studies have shown that XBP-1 mRNA levels are increased in microarray studies on human breast, colon, and lung cancer cell lines in vitro. A causal link has been demonstrated between XBP1 production and viability of tumors in hypoxic condition in mice. We hypothesize that XBP1 will be expressed preferentially in tumors of high histologic grade and pathologic stage. In very limited studies (<5 cases), Dr. J. Lin has demonstrated XBP1 immunostaining in colon adenocarcinoma.
An XBP1 antibody is commercially available that has been shown to work on formalin-fixed paraffin embedded tissue. We plan on obtaining this antibody and staining a series of colon adenocarcinomas, with the plan to correlate the protein expression of XBP1 with the tumor grade, stage, and other clinicopathologic data. IRB approval has already been obtained for this study.
Pathogenesis of Salmonella Colitis
Pl: Josh Fierer
We have a mouse model of Salmonella colitis that we are using to study the pathogenesis of Salmonella induced diarrhea. I would be interested in working with a resident to develop a scoring system for colonic inflammation and perhaps doing some immuno-histology for cytokines and immune cells.
Immunohistochemical Analysis of Human Carcinomas to detect the incidence of the presence of the nonhuman N-glycolylneuraminic acid (Neu5Gc), to determine efficacy of use as a cancer biomarker.
Pl: Nissi Varki
Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. This project is to identify the incidence of Neu5Gc within human carcinomas and to determine its usefulness as a carcinoma biomarker.
Protocol: Each assay (involving frozen sections) will take 2-3 consecutive days, with at least 3-4 hours per day.
Reference: SL Diaz et al. Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid in Human Tissues and Biotherapeutic Products. PLoS One 2009; 4(1): e4241.
Expression of Siglec-11 / 16 in the Ovary
Pls: Nissi Varki,
Siglecs (Sialic acid binding Immunoglobulin-like lectins) are cell surface signaling receptors that recognize sialylated glycans. Expression of the subset of CD33-related Siglecs, is mostly restricted to various leukocyte types, with relevance to immune regulatory functions. We earlier reported a human specific gene conversion of the genomic regions encoding the extracellular domain of Siglec-11, associated with human-specific expression in human brain microglia. Review of gene microarray data showed a surprisingly high expression of SIGLEC11 mRNA in human ovaries. We confirmed this expression by examining ovary sections by immunohistochemistry analyses using 4C4, a mouse monoclonal antibody directed against the extracellular domain of Siglec-11, and expression was mainly observed on human ovarian stroma. In contrast, in chimpanzee ovaries, expression was higher along the ovarian surface tunica. Interestingly, this Siglec-11 expression appeared enhanced in ovarian samples from post-menopausal women, and in those samples from patients with abnormal follicular development, polycystic ovarian syndrome. Recently, a relatively rare functional allele was identified for a previously defined human Siglec pseudogene, Siglec-16. Due to high sequence similarity between the extra-cellular domains of human Siglec-11 and Siglec-16, the 4C4 antibody can cross-react with Siglec-16. We are currently pursuing the possibility that SIGLEC16 genotype may be associated with individual physiological and pathological variations of ovarian follicle growth, ovulation and menopause in humans, and perhaps play a role in the growth of ovarian stromal tumors.
Protocol: This project involves extracting DNA from paraffin sections of ovarian stromal tumors and performing PCR. This project is near completion, needing a few experiments to tie up some loose ends before writing the paper.
Role of the inducible nitric oxide synthase in mammary tumor progression
Hypothesis: Mammary tumor cells that have undergone epithelial to mesenchymal transition (EMT) can promote tumorigenesis through the release of inducible nitric oxide synthase (iNOS) mediated NO release.
Aim: To determine whether iNOS-mediate NO release by mammary tumor cells with an EMT phenotype plays a role in mammary tumor progression.
Methods :Mammary tumor cell lines with a well differentiated or EMT phenotype will be characterized in vitro for iNOS-mediated NO production. Well-differentiated cell lines will be injected orthotopically into the mammary fat pad with and without various concentrations of EMT tumor cells. Mice will be treated with and without specific iNOS inhibitors and tumorigenesis monitored over a 6 to 10 week period. Tumors will be prepared for histological analysis as well as gene expression analysis by real time PCR. Lungs will be examined for tumor metastasis.
This project is expected to take 150-200 h.
Examining the expression of the novel cytokine IL-17D in low versus high grade astrocytomas
Hypothesis: Based on mouse data, the expression of IL-17D is expected to be increased in highly immunogenic tumors that undergo immune rejection. Preliminary microarray data suggest that IL-17D is increased in low grade pilocytic astrocytomas compared to high grade gliomas. The major goal of this project is to use immunohistochemistry to confirm that IL-17D protein is more abundant in low versus high grade astrocytomas.
Aim 1: Develop a protocol for reliably detecting human IL-17D protein in paraffin tissue sections by immunohistochemistry.
Aim 2: Obtain tissue blocks of low and high grade astrocytomas and stain for IL-17D protein. Correlate protein expression with the grade of the tumor.
Aim 3: Determine whether IL-17D is expressed in tumor cells or infiltrating immune cells by immunohistochemistry. Use adjunct markers to identify tumor cells versus immune cells. Determine whether IL-17D protein expression correlates with the quantity of immune cell infiltrate.
Time commitment: Highly flexible. This project must be driven completely by the resident. There is no required timeline for this project. The rate limiting step is the development of a reliable protocol to detect IL-17D protein (Aim 1). This may take 1-3 months. After that, the correlation studies (Aims 2-3) will take approximately 1 month, depending on the amount of time spent per week.
Expertise: The resident must be able to identify tumor cells and immune cells using morphology or special stains in tumor sections. The resident will gain expertise in developing a staining protocol to detect IL-17D protein.
Publication potential. If all aims are completed and support the primary hypothesis, the potential to publish is very high.
Examining the infiltration of neutrophils into mouse tumors that overexpress IL-17D.
Pl: Jack Bui
Hypothesis: IL-17D is thought to mediate tumor rejection, but the mechanism of rejection is not known. The hypothesis of this study is that IL-17D recruits neutrophils to mediate anti-tumor activity.
Aim 1: Develop a protocol for detecting neutrophils in mouse tumor sections.
Aim 2: Stain mouse tumors that express high or low levels of IL-17D for neutrophils, and correlate the infiltration of neutrophils with IL-17D expression.
Aim 3: (Optional). Harvest tumor masses from mice and generate tumor cross sections for studies.
Time commitment: Highly flexible. This project must be driven completely by the resident. There is no required timeline for this project. The rate limiting step is the development of a reliable protocol to detect neutrophils in mouse tumor sections (Aim 1). This should not take longer than 1 month since published protocols are available. After that, the correlation studies (Aims 2&3) will take approximately 6 months, since the availability of tumor masses will depend on how fast the tumors grow in mice. A graduate student will assist with or perform all of Aim 3.
Expertise: The resident must be able to identify mouse neutrophils using immunohistochemistry. The resident will gain expertise in the surgical removal and subsequent study of tumor masses from mice.
Publication potential. If all aims are completed and support the primary hypothesis, the potential to publish is very high.
Clinical research projects in GYN (Pap smear), Breast and Thyroid cytology.
GYN cytology: A quality improvement study on negative cervical biopsies in comparison to the concurrent Pap smears with diagnosis of HSIL or AIS. We propose that concurrent negative surgical specimens of pap smears showing HSIL should be further evaluated with consensus review, deeper-level sections and/or p16 immunostaining. Routine incorporation of these practices in the described clinical scenario may ultimately prevent underdiagnoses of HSIL on cervical biopsies.
Breast: Interdepartmental investigation on the pathological outcomes of non-mass enhancement lesions in breast biopsies. This term is used in approximately 1/5 of rush breast biopsies without specific differential diagnosis. This will be a retrospective quality control study to look at the statistical analysis of this non-specific entity.
Thyroid cytology: An on-going quality control study on the outcome of category 3 Bethesda diagnosis of thyroid FNA diagnosis. The rate of FLUS or atypical diagnosis is controlled and our goal is to have low number of atypical diagnosis.
XBP1 PROTEIN EXPRESSION IN LUNG CARCINOMA
XBP1 (X-box binding protein 1) is upregulated as a consequence of activation of the unfolded protein response (UPR), which is in turn activated by cellular stresses, such as hypoxia. Initial studies have shown that XBP-1 mRNA levels are increased in microarray studies on human breast, colon, and lung cancer cell lines in vitro. A causal link has been demonstrated between XBP1 production and viability of tumors in hypoxic condition in mice. We hypothesize that XBP1 will be expressed preferentially in tumors of high histologic grade and pathologic stage.
An XBP1 antibody is commercially available that has been shown to work on formalin-fixed, paraffin-embedded tissue. We plan on obtaining this antibody and staining a series of lung carcinomas, with the plan to correlate the protein expression of XBP1 with the histologic type, tumor grade, stage, and other clinicopathologic data. IRB approval has already been obtained for this study.
Research Ethics in Pathology
Michael Kalichman, Director, UCSD Research Ethics Program
Brief Description: The collection of tissues and diagnostic testing for patients and research subjects are a routine part of the practice of Pathology, but the ethical dimensions of this work are often missed. This is important not only to the practice of medicine and science, but to the individual who may be harmed on the one hand because of a breech in confidentiality or on the other by a failure to be notified of significant findings. Examples of projects of possible interest are varied, but include assessments of: (1) awareness of Pathologists of the ethical dimensions of their practice; (2) patient and/or research subject understanding of informed consent documents; and (3) comparative effectiveness of methods to increase understanding in trainees about the ethical dimensions of the practice of Pathology.
Time required: Varies depending on the scope of the project selected, but minimally 5 hrs/wk for 10 weeks.
Development of a Serum PCR Assay for Disseminated Histoplasmosis
Disseminated histoplasmosis is a major problem in severely immunocompromised patients, particularly with AIDS. Symptoms may be nonspecific, and rapid diagnosis is critical for treatment. The most useful test to diagnose disseminated histoplasmosis to date is a urine-based EIA for a glycosylated antigen. This assay is only available in one commercial laboratory with the inherent delays in send-out testing. A Histoplasma Genome Project has confirmed a number of specific genes, which could be used diagnostically. We hypothesize that a qualitative (and potentially quantitative) PCR assay in serum will be more sensitive than a urine antigen test. A serum bank from patients with histoplasmosis will be available with a goal of validating a new test for clinical use.
Grp78/BiP,CHOP, and XBP-1 IN GLIAL TUMORS
GRP78/BiP (Glucose-Regulated Protein molecular weight 78/Immunoglobulin Binding Protein), CHOP (C/EBP Homologous Protein), and XBP1 (X-box binding protein 1) are upregulated by cellular stresses, such as hypoxia, protein misfolding, and endoplasmic reticulum. Initial studies have shown that mRNA levels of these genes are increased in microarray studies on human brain, breast, colon, and lung cancer cell lines
in vitro. A causal link has been demonstrated between Grp78/BiP and XBP1 production and viability of tumors in hypoxic condition in mice. We hypothesize that Grp78/BiP, CHOP, and XBP-1 will be expressed preferentially in tumors of high histologic grade and pathologic stage.
Antibodies against these proteins are available that have been shown to work on formalin-fixed paraffin embedded tissue. We plan on obtaining these antibodies and staining a series of glial tumors, with the plan to correlate the protein expression of Grp78/BiP, CHOP, and XBP-1 with the tumor grade, stage, and other clinicopathologic data. IRB approval has already been obtained for this study.
Bacterial Cardiac Endothelial Biofilms
Lynette B Corbeil
It has been shown in 2009 that
Histophilus somni forms biofilms after septicemia, the hallmark of H. somni disease. Biofilms are most prominent in cardiac capillaries and venules in the left papillary muscle as well as on cardiac valves in the natural host. We have developed a mouse model of the septicemic disease and intend to use this model to study cardiac biofilms by histopathology, immunohistochemistry and perhaps transmission electron microscopy. We also have immunized mice against one of the key virulence factors involved in biofilm formation (IbpA) and against an IbpA mutant lacking virulence functions. These mice will be challenged with the virulent pathogen to determine if the immunizations not only protect against septicemia but also against biofilms. This project will involve study of the cardiac lesions in the mouse model as well as in fixed cardiac tissue of natural cases, to determine the role of the Ibpa virulence factor in biofilm formation and cardiac disease. Longer projects would involve in vitro assays as well.
If you have any immediate questions, please contact our Residency Program Manager, Alexandra Murtha, at
firstname.lastname@example.org or (858) 249-1096.