Cecille D. Browne (cbrowne@burnham.org)
Graduate Program: Molecular Pathology
Lab PI: Robert C. Rickert, Ph.D.
Undergraduate Institution: San Diego State University
Med-into-Grad clinical training area: Rheumatology, Allergy and Immunology
Clinical mentors: Gary S.Firestein, M.D: gfirestein@ucsd.edu
Susan E. Sweeney, M.D., ssweeney@ucsd.edu
Quote: “The training definitely broadened my perspective. I learned in depth about immunopathologies such as SLE, RA, gout and scleroderma and others that I have never heard before. The research lab that I hope to someday direct will be based on clinically relevant research insights derived from this experience. I truly believe that this experience will make me a stronger scientist.”
Rational for Med-into-Grad training:
My thesis research is centered on B cell development, differentiation and function. The specific research questions that I am pursuing are: 1) Does the tumor suppressor PTEN play a role in B cell tolerance, and if so, what is the mechanism by which PTEN maintains B cell tolerance? 2) Does the adaptor molecule SHEP1 play a role in B cell homing, and if so, what specific chemotactic responses are mediated by SHEP1? 3) How do integrins promote B cell proliferation? and 4) What is the immunological consequence of a B cell-specific loss of CD98? Dysregulation of B cell subcompartments had been linked to autoimmune and immunodeficiency diseases. Therefore, understanding the cellular and molecular signals that orchestrate the development of these compartments would lead to a better understanding of immunopathologies.
I am interested in the Med-Into-Grad program because I would like to see the clinical presentations of immunopathologies and their management. I wanted to see how clinicians assess and monitor the progress of patients and to learn about current immunotherapeutics. This program would strengthen by ability someday to contribute to a better understanding of immunoregulation and to offer novel immunotherapeutics or immunodiagnostics. I believe that exposure in a clinical setting would give me a unique perspective that will help me become a stronger scientist. The experience would stimulate clinically relevant scientific questions, which could serve as foundations to the translational research laboratory that I hope to direct someday.
Medical training and identification of medically relevant research issues:
In outpatient clinics, I accompanied physicians in seeing outpatients in Rheumatology Clinics at the VA Hospital and at UCSD Medical Center Ambulatory Care Center at Hillcrest. Here I saw patients with various rheumatologic disorders such as rheumatoid arthritis, ankylosing spondylitis, scleroderma and osteoarthritis. I also observed how physicians take patient history, perform physical exam, and perform clinical procedures, such as steroid injections to alleviate pain in joints. After taking a careful history, the physician (typically a ‘fellow’ or a resident) would present the information to an attending physician. A discussion of the clinical problem and the therapeutic plan would follow and then the attending physician, the fellow and I would go back to the patient to talk about the problem and the plan of action.
During inpatient hospital rounds at the VA Hospital, UCSD Medical Center at Thornton and Hillcrest, I joined the Rheumatology Team that saw hospitalized patients with rheumatologic diseases. In this setting, a resident would give an overview of the clinical situation and then a discussion would follow. Afterwards, as a team, we would all go to the hospital room and talk with the patient about the situation and the plan of action.
In case conferences, physicians presented intriguing clinical cases and the therapeutic plan that they had adopted. The presentation was followed by a discussion and validation of the plan.
In the Rheumatology, Allergy, Immunology Seminar Series, faculty members present basic and clinical research in the field of rheumatology, allergy and immunology.
The Rheumatology Grand Rounds is another venue for clinical training in a classroom format. Topics ranged from newest approaches to disease management, cutting-edge diagnostics, perspectives on a particular disease and clinical trials. In addition to the above activities, I’ve also attended a monthly Immunology Journal Club, as well as the weekly Medicine Grand Rounds.
Insight to research areas:
It was remarkable to see varying severity of a rheumatologic disease. For example, one patient with rheumatoid arthritis may come in for stiffness and pain in one or two joints, with minimal interference with daily activities. Another patient (often a new patient to the clinic) with the same disease may come in a wheelchair and with severely deformed fingers. Furthermore, it is interesting why patients tolerate medications differently. Fortunately, there are therapeutic alternatives when one medication does not appear to work. It would be helpful if there is a way to predict responses to widely used therapeutics such as methotrexate or prednisone. Overall, the molecular mechanisms behind rheumatologic diseases such as scleroderma, systemic lupus erythematosus, ankylosing spondylitis are not completely understood. Therefore, each one of these diseases calls for continued investigative research.
Potential Research collaborations:
During my clinical experience, I found the following open questions in Rheumatology:
Gout is an autoinflammatory arthritis that can be caused by the sedimentation of monosodium urate crystals in articular joints. Although hyperuricemia can promote the development of gout by increasing the likelihood of urate crystal deposition, hyperurecemia per se does not automatically lead to gout. This suggests a variable ability among humans to react to crystal load. Macrophages are responsible for the production of potent proinflammatory cytokines, IL-1b and IL-6, as well as chemokines that promote the infiltration of neutrophils during crystal-induced inflammation. The molecular pathway by which macrophages are induced to produce IL-1b by crystals is not completely understood. Recent studies have described the role of inflammasomes in mediating crystal-induced inflammatory responses. A patient with familial periodic fever syndrome with a high level of serum IL-1b carry compound SNPs in the NALP3 (Q750K) and the CARD8 (C10X). Since crystal-induced IL-1b production also utilizes the NALP3 inflammasome, it is possible that these activating mutations also play a role in the development of gout. Furthermore, it has been suggested that lysosomal instability leads to the leakage of cathepsin B into the cytosol and cathepsin B is ‘sensed’ by the components of the inflammasome leading to its immediate assembly. The mechanistic role of the lysosomal exopeptidase, cathepsin B, in the activation of the NALP3 inflammasome is not known. In the NALP3 mutations, does the need for cathepsin B no longer exists? Revealing the direct or indirect action of cathepsin B on the inflammasome assembly or components thereof is important in elucidating how the danger signals of crystals are communicated to the inflammasome and in understanding the mechanisms of inflammasomal dysfunction, which could lead to gout.
I propose the following specific questions: 1) Would the Q750K mutation in NALP3 result in crystal-induced hyper-inflammatory responses in vitro and in vivo? 2) Would the loss of CARD8 result in elevated IL-1b secretion and 3) Does cathepsin B directly cleave components of the inflammasome leading to inflammasone activation?
I propose to 1) Prepare a NALP3 (Q750K) mutation in a GFP vector, introduce this in a monocytic cell line (THP1) and then treat cells with uric acid crystals 2) Obtain siRNA to CARD8 and transfect THP1 cells with siRNA targeting the CARD8 domain. For both 1) and 2) assess the production of caspase 1 and IL-1b. 3) Treat THP1 cells with cathepsin B inhibitor or vehicle control. Subject cell lysates to 2D gel electrophoresis. Compare spots between gel profiles and determine which of these protein or protein fragments are derived from components of the NALP3 inflammasome.
Training in diagnostics & therapeutics, and identification of unmet diagnostic & therapeutic needs:
Through my experience in the program, I have acquired an understanding of diagnostics and therapeutics used in Rheumatology. I learned that for Rheumatology patients, laboratory tests are critical in the management of diseases. Among the laboratory test results, a rheumatologist looks at markers of inflammation, such as ESR (erythrocyte sedimentation rate), autoimmune antibodies, such as anti-CCP, RF and ANA and indications of liver (liver enzymes) and kidney (GFR) damage. In addition, MRI and X-rays may also be used to assess the extent of joint damage.
For lupus, I learned that in general, NSAIDs are prescribed for pain. Plaquenil may be used and for this drug, vision tests are prescribed due to potential damage this drug can cause to the eyes. Prednisone is typically used for severe pain and it is important to ‘taper’ the use of this drug rather than abruptly stop it. In more aggressive lupus, higher doses of corticosteroids are used and in addition, cyclophosphamide, or mycophenolic acid may be used.
For gout, aside from NSAIDS that are prescribed for pain, allopurinol, which is an isomer of hypoxantine may be prescribed. Allopurinol inhibits the oxidation of hypoxantine and therefore prevents the formation of purine and uric acid. Colchicine (a microtubule inhibitor) prevents the deposition of uric acid crystals. Patients with gout are also prescribed a diet low in purines.
For rheumatoid arthritis, aside from NSAIDs, prednisone may be prescribed. Methotrexate (to block T cell activation) is widely used. This drug inhibits folic acid formation, which is essential in DNA synthesis but at low concentration, this drug has been shown to block immune cell activation. Patients taking methotrexate are also taking folic acid supplements. In severe forms of RA, TNFa blockers, such as Enbrel (etanercept), Remicade (Infliximab) or Humira (adalimumab) or the B cell blocker, Rituxan, may be used.
There are issues associated with current therapeutics. The obvious one being immunosuppression, which can suppress autoimmune attacks, but can render a patient susceptible to infections. Sometimes if a patient has an ongoing systemic infection, use of immunosuppresants may be postponed until the infection is resolved. This could lead to prolonged discomfort on the part of the patient. Another issue is that some of these drugs may work in some patients and not in others. Also, the ESR test result is one of the parameters used to assess inflammation, however, this test is only an indirect test. A test directly quantifying inflammatory cytokines is very costly.
Diagnostic & Therapeutic collaborations:
It would be helpful if a diagnostic kit that is not cost prohibitive can be developed to quantify TNFa in patient serum. Since a quantitative ELISA is costly, perhaps a semi-quantitative test for inflammatory cytokines can be developed. Or, if not too costly, a diagnostic kit can be developed consisting of a culture plate of cells expressing TNF receptors conjugated to different fluorescent proteins, which can induce FRET upon trimerization that will occur upon engagement with TNF in patient serum. The cell line will have to be engineered so that signal is not transduced downstream of trimerization. The FRET signals can easily be read by a fluorescent reader.
Long term impact:
The training definitely broadened my perspective. I learned in depth about immunopathologies such as SLE, RA, gout and scleroderma and others that I have never heard before. The research lab that I hope to someday direct will be based on clinically relevant research insights derived from this experience. I truly believe that this experience will make me a stronger scientist.
Student-specific experiences:
The clinical experience definitely had an impact in my life. First, the experience gave me a strengthened respect for clinicians. The rheumatologists at UCSD School of medicine are the best in the field. They all demonstrated professionalism and outstanding bedside manner, no matter how difficult patients can sometimes be. Also, meeting patients with illnesses that are not completely understood compels an urgency of discovery by continued investigative research. Though my work will be in the laboratory, my career objectives will be guided by a strong concern for improving human health. My experience gave me a renewed sense of purpose.
Advice for new trainees--Autumn preparatory quarter:
During the Fall quarter, the Student-Meets-the-Patient course is an excellent opportunity to meet people with different diseases and to listen to lectures about these diseases.
My advice to new trainees is to start reading about the diseases that interest them and read about the therapeutics available. Make a list of questions you may have and keep this list handy for the Winter clinical experience where you can realize the answers to these questions yourself or ask rheumatologists you are working with. Check the website of the clinical division you are interested in. For example, the Rheumatology division website directed me to pages containing information on dates, times, locations, organizers, lecture titles, etc. E-mail the organizers of various lecture series and asked to be placed in their e-mail reminder list. If possible meet with your clinical advisor during the Fall quarter and get a head start on setting up your badge, getting your white coat, and knowing the locations of your clinical activities.
Advice for new trainees—Winter clinical training quarter:
1. Keep a calendar of activities provided by the clinical program.
2. Introduce yourself to everyone in the clinic. Display your badge at all times. Get to know most of the attending physicians/faculty members in your division. Read about their research interests for potential future interactions.
3. Be respectful of the physician you are shadowing. Be aware of how you address them in the presence of a patient. Feel free to ask the physician a question but know when is a good time to interrupt.
4. Read a review article of a disease once a week. Keep a folder of articles and bring this folder with you in the clinic to read when you have time.
5. Keep a diary of your experience to include notes from talks, notes from clinical encounters, notes from case conferences. Print out flyers of seminars/conferences you attended. These may come in handy someday when you want to recall the clinical manifestations of diseases you have seen and the clinically relevant research questions you have discovered.
6. Have fun.
Take home perspective on Med-into-Grad at UCSD:
The Med-Into Grad program is an outstanding program that provides a rare opportunity to connect with the clinical environment and stimulate the generation of research questions that will help human health. I enthusiastically endorse this program to other graduate students.