Douglas B Cines

University of Pennsylvania, Philadelphia, Pennsylvania, United States

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Publications (257)2047.92 Total impact

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    ABSTRACT: Existing approaches for measuring hemostasis parameters require multiple platforms, can take hours to provide results, and generally require 1-25 mL of sample. We developed a diagnostic platform that allows comprehensive assessment of hemostatic parameters on a single instrument and provides results within 15 min using 0.04 mL of blood with minimal sample handling.
    Clinical chemistry. 06/2014;
  • Douglas B Cines, Adam Cuker, John W Semple
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    ABSTRACT: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by antibody-mediated platelet destruction. The platelet, as an accessible target, has made ITP an attractive disorder in the study of autoimmunity. However, the pathogenesis of ITP has proven complex with diverse pre-existing challenges to the immune system in the form of infection, genetic predisposition, underlying autoimmune repertoire, inhibition of platelet production, perturbations of cell mediated affector and effector pathways, sequestered harbors within lymphoid organs, and responsiveness to intervention. This chapter surveys key new insights into the pathogenesis of ITP and attempts to integrate them into a model that may serve as a template for future investigation.
    La Presse Médicale 03/2014; · 1.17 Impact Factor
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    ABSTRACT: Contraction of blood clots is necessary for hemostasis, wound healing and to restore flow past obstructive thrombi, but little has been known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.
    Blood 12/2013; · 9.78 Impact Factor
  • Lubica Rauova, Douglas B Cines
    Blood 11/2013; 122(23):3707-8. · 9.78 Impact Factor
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    ABSTRACT: Babies are frequently exposed to cerebral hypoxia and ischemia (H/I) during the perinatal period as a result of stroke, problems with delivery or post delivery respiratory management. The sole FDA approved treatment for acute stroke is tissue-type plasminogen activator (tPA). Endogenous tPA is upregulated and potentiates impairment of pial artery dilation in response to hypotension after H/I in pigs. Mitogen-activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after H/I, with ERK contributing to impaired vasodilation. This study examined the hypothesis that H/I aggravates the vascular response to two important procontractile mediators released during CNS ischemia, endothelin-1 (ET-1) and thromboxane, which is further enhanced by tPA and ERK MAPK. Cerebral hypoxia (pO2 35 mmHg for 10 min via inhalation of N2) followed immediately by ischemia (global intracranial pressure elevation for 20 min) was produced in chloralose anesthetized piglets equipped with a closed cranial window. H/I aggravated pial artery vasconstriction induced by ET-1 and the thromboxane mimetic U 46619. Potentiated vasoconstrictor responses were blocked by EEIIMD, an inhibitor of tPA's signaling and vascular activities, but unchanged by its inactive analogue EEIIMR. The cerebrospinal fluid concentration of ERK MAPK determined by ELISA was increased by H/I, potentiated by tPA, but blocked by EEIIMD. The ERK MAPK antagonist U 0126 blocked H/I augmented enhancement of ET-1 and U 46619 vasoconstriction. These data indicate that H/I aggravates ET-1 and thromboxane mediated cerebral vasoconstriction by upregulating endogenous tPA and ERK MAPK.
    Neurocritical Care 11/2013; · 3.04 Impact Factor
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    ABSTRACT: Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans. However, only a fraction of patients with anti-PF4/heparin antibodies develop HIT, implying that only a subset of these antibodies is pathogenic. The basis for the pathogenic potential of anti-PF4/heparin antibodies remains unclear. To elucidate the intrinsic PF4-binding properties of HIT-like monoclonal antibody (KKO) vs. non-pathogenic antibody (RTO) at the single-molecule level, we utilized optical trap-based force spectroscopy to measure the strength and probability of binding of surface-attached antibodies with oligomeric PF4 to simulate interactions on cells. To mimic the effect of heparin in bringing PF4 complexes into proximity, we chemically cross-linked PF4 tetramers using glutaraldehyde. Analysis of the force histograms revealed that KKO-PF4 interactions had approximately 10-fold faster on-rates than RTO-PF4 and apparent equilibrium dissociation constants differed ≈10-fold with similar force-free off-rates (koff=0.0031 and koff=0.0029 s(-1)). Qualitatively similar results were obtained for KKO and RTO interacting with PF4/heparin complexes. In contrast to WT PF4, KKO and RTO showed lower and similar binding probabilities to cross-linked PF4K50E, which forms few if any oligomers. Thus, formation of stable PF4 polymers results in much stronger interactions with the pathogenic Ab without a significant effect on the binding of the non-pathogenic Ab. These results suggest a fundamental difference in the antigen-binding mechanisms between model pathogenic and non-pathogenic anti-PF4 antibodies that might underlie their distinct pathophysiological behaviors.
    Journal of Biological Chemistry 10/2013; · 4.60 Impact Factor
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    ABSTRACT: Thrombopoietin-receptor agonists (TPOra) are the only treatments for immune thrombocytopenia (ITP) for which evidence of efficacy and safety from randomized, placebo-controlled trials is available. We sought to determine the long-term tolerability of the TPOra romiplostim, with a particular focus on thrombosis, bleeding, bone marrow reticulin, neoplasms/haematological malignancies and fatal events. Data from 13 romiplostim clinical trials in which 653 ITP patients received romiplostim for up to 5 years (921.5 patient-years) were pooled; subject incidence rates and exposure adjusted event rates (per 100 patient-years) were calculated. The rate of thrombotic events (6% of patients; 7.5 events/100 patient-years) did not appear to increase over time; 7 events were associated with platelet counts >400 x 10(9) /L and 10 with romiplostim doses exceeding current recommendations. Serious and grade ≥3 bleeding each occurred in approximately 8% of patients (~11 events/100 patient-years). Adverse events of bone marrow reticulin were recorded for 12 patients (1.8%; 1.3 events/100 patient-years; confirmed by bone biopsy in 10 patients), and bone marrow collagen for 1 patient (0.2%; 0.1 events/100 patient-years; confirmed by trichrome staining). Neoplasms and haematological malignancies occurred in 2.1% and 0.8% of patients, respectively (2.2 and 0.7 events/100 patient-years). Fatal events occurred in 3.7% of patients (2.6 events/100 patient-years; 4 events treatment-related). Romiplostim is the TPOra for which the longest duration of safety data is available. Our data demonstrate that long-term romiplostim treatment is well tolerated, with no new safety signals, even in patients treated for up to 5 years. This article is protected by copyright. All rights reserved.
    European Journal Of Haematology 08/2013; · 2.41 Impact Factor
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    ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is almost lethal. One of the underlying reasons for this lethality is thought to be the presence of cancer stem cells (CSC), which impart chemo-resistance and promote recurrence, but the mechanisms responsible for this biology are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). Therefore, in the present study we examined the role of uPA in the generation of PDAC-CSC. We observed a subset of cells identifiable as a side-population (SP) when sorted by flow-cytrometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possessed the properties of CSC. A large fraction of these SP cells were CD44- and CD24-positive, were gemcitabine resistant, possess sphere forming ability and exhibit increased tumorogenisity, which are known characteristic of cancer stemness. Increased tumorogenisity and gemcitabine resistance was decreased after suppression of uPA. We observed that uPA interacts directly with transcription factors Lhx-2, HOXA5 and Hey to possibly promote cancer stemness. uPA regulates Lhx-2 expression by suppressing expression of miR-124, and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality.
    Molecular biology of the cell 07/2013; · 5.98 Impact Factor
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    ABSTRACT: Despite continued achievements in anti-thrombotic pharmacotherapy, difficulties remain in managing patients at high risk for both thrombosis and hemorrhage. Utility of anti-thrombotic agents (ATAs) in these settings is restricted by inadequate pharmacokinetics and narrow therapeutic indices. Use of advanced drug delivery systems (ADDS) may help to circumvent these problems. Various nanocarriers, affinity ligands, and polymer coatings provide ADDS that have the potential to help optimize ATA pharmacokinetics, target drug delivery to sites of thrombosis, and sense pathologic changes in the vascular microenvironment, such as altered hemodynamic forces, expression of inflammatory markers, and structural differences between mature hemostatic and growing pathological clots. Delivery of ATA using biomimetic synthetic carriers, host blood cells, and recombinant fusion proteins that are activated preferentially at sites of thrombus development has shown promising outcomes in preclinical models. Further development and translation of ADDS that spare hemostatic fibrin clots hold promise for extending the utility of ATAs in the management of acute thrombotic disorders through rapid, transient, and targeted thromboprophylaxis. If the potential benefit of this technology is to be realized, a systematic and concerted effort is required to develop clinical trials and translate the use of ADDS to the clinical arena.
    Blood 06/2013; · 9.78 Impact Factor
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    ABSTRACT: Traumatic brain injury (TBI) is associated with loss of cerebrovascular autoregulation, which leads to cerebral hypoperfusion. Mitogen activated protein kinase (MAPK) isoforms ERK, p38, and JNK and endothelin-1 (ET-1) are mediators of impaired cerebral hemodynamics after TBI. Excessive tPA released after TBI may cause loss of cerebrovascular autoregulation either by over-activating N-methyl-D-aspartate receptors (NMDA-Rs) or by predisposing to intracranial hemorrhage. Our recent work shows that a catalytically inactive tPA variant (tPA-S481A) that competes with endogenous wild type (wt) tPA for binding to NMDA-R through its receptor docking site but that cannot activate it, prevents activation of ERK by wt tPA and impairment of autoregulation when administered 30 min after FPI. We investigated the ability of variants that lack proteolytic activity but bind/block activation of NMDA-Rs by wt tPA (tPA-S481A), don't bind/block activation of NMDA-Rs but are proteolytic (tPA-A296-299), or neither bind/block NMDA-Rs nor are proteolytic (tPA-A296-299S481A) to prevent impairment of autoregulation after TBI and the role of MAPK and ET-1 in such effects. Results show that tPA-S481A given 3h post TBI, but not tPA-A296-299 or tPA-A29-299S481A prevents impaired autoregulation by upregulating p38 and inhibiting ET-1, suggesting that tPA-S481A has a realistic therapeutic window and focuses intervention on NMDA-Rs to improve outcome.
    Journal of neurotrauma 06/2013; · 4.25 Impact Factor
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    ABSTRACT: How single chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (>4 nM) induces pERK1/2 (MAPK44 and 42) and pAkt (S(473)) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD98059 or U0126 and pAktS(473) activation is inhibited by Wortmannin or LY294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAktS(473), in SIN1(-/-) cells indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high molecular weight kininogen (HK) compete with ScuPA for induction of pERK1/2 and pAktS473. A peptide to the integrin binding site on uPAR, a β1 integrin peptide that binds uPAR, antibody 6S6 to β1 integrin, the tyrosine kinase inhibitors AG1478 or PP3, and siRNA knockdown of VEGFR2, but not HER1-4, blocks ScuPA-induced pERK1/2 and pAktS(473). ScuPA-induced endothelial cell proliferation is blocked by inhibitors of pERK1/2 and pAktS(473), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiates aortic sprouts and matrigel plug angiogenesis in normal, but not uPAR deficient, mouse aortae or mice, respectively, but they are blocked by PD98059, LY294002, AG1478 or cleaved HK. In sum, this investigation indicates a novel, a non-proteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, β1 integrins, and VEGFR2 leading to angiogenesis. Kininogens or peptides from it down regulate this pathway.
    AJP Heart and Circulatory Physiology 05/2013; · 4.01 Impact Factor
  • Adam Cuker, Douglas B Cines
    Blood 04/2013; 121(15):2818-9. · 9.78 Impact Factor
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    ABSTRACT: Laboratory testing for heparin-induced thrombocytopenia (HIT) has important shortcomings. Immunoassays fail to discriminate platelet-activating from non-pathogenic antibodies. Specific functional assays are often impracticable due to the need for platelets and radioisotope. We describe two assays that may overcome these limitations. The KKO-inhibition test (KKO-I) measures the effect of plasma on binding of the HIT-like monoclonal antibody KKO to PF4/heparin. DT40-luciferase (DT40-luc) is a functional test comprised of a B-cell line expressing FcγRIIa coupled to a luciferase reporter. We compared these assays to polyspecific and IgG-specific PF4/heparin ELISAs in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin antibodies. HIT was defined as a 4Ts score ≥4 and positive 14C-serotonin release assay. HIT-positive plasma demonstrated greater mean inhibition of KKO binding than HIT-negative plasma (78.9% vs. 26.0%, p<0.0001) and induced greater luciferase activity (3.14-fold basal vs. 0.96-fold basal, p<0.0001). The area under the ROC curve (AUC) was greater for KKO-I (0.93) than for the polyspecific (0.82, p=0.020) and IgG-specific ELISA (0.76, p=0.0044) and for DT40-luc (0.89) than for the IgG-specific ELISA (p=0.046). KKO-I and DT40-luc showed better discrimination than two commercially available immunoassays, are simple to perform, and hold promise for improving the specificity and feasibility of HIT laboratory testing.
    Blood 02/2013; · 9.78 Impact Factor
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    ABSTRACT: In a previous publication on new terminology, definitions and outcome criteria for immune thrombocytopenia (ITP), the International Working Group (IWG) on ITP acknowledged that response to treatment should consist of clinically meaningful endpoints, like bleeding manifestations, and that platelet count may not be the ideal parameter to capture the benefits of therapy. The IWG now proposes a consensus based ITP-specific bleeding assessment tool (ITP-BAT) with definitions and terminology consistent with those adopted for other bleeding disorders. Bleeding manifestations were grouped into three major domains: Skin (S), visible Mucosae (M) and Organs (O) with Gradation of severity (SMOG). Each bleeding manifestation is assessed at the time of examination. Severity is graded from 0 to 3 or 4, with grade 5 for any fatal bleeding. Bleeding reported by the patient without medical documentation is graded 1. Within each domain, the same grade is assigned to bleeding manifestations of similar clinical impact. The "worst bleeding manifestation since the last visit" (observation period) is graded (a suitable data-base collection form is provided). Then, the highest grade within each domain is recorded. The SMOG system provides a consistent description of the bleeding phenotype in ITP and the IWG unanimously supports its adoption and validation in future clinical studies.
    Blood 01/2013; · 9.78 Impact Factor
  • Alan H Lazarus, John W Semple, Douglas B Cines
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    ABSTRACT: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by both accelerated clearance of autoantibody-sensitized platelets and suboptimal platelet production. A number of studies have provided evidence of disturbed innate and adaptive immune responses in patients with ITP. This brief review will highlight some of the more recent work in this field and highlight other findings that provide a potential link between ITP, systemic lupus erythematosus (SLE), and autoimmune hemolytic anemia (AHA).
    Seminars in Hematology 01/2013; 50 Suppl 1:S68-70. · 2.46 Impact Factor
  • Abd Al-Roof Higazi, Douglas B Cines
    Blood 11/2012; 120(19):3870-1. · 9.78 Impact Factor
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    ABSTRACT: Tissue-type plasminogen activator (tPA) remains the sole thrombolytic approved by the FDA for the treatment of pulmonary embolism (PE). tPA has not been replaced by third generation plasminogen activators, e.g. Reteplase (Ret) and Tenecteplase (TNK) that circulate with longer life-spans and in theory should have more extended potency in vivo. One reason for this paradox is the inability to assign units of activity to plasminogen activators based on specific biologically relevant standards, which impairs objective comparison. Here, we compare clot permeation, retention and fibrinolytic activities of tPA, TNK and Ret in vitro and clot composition over time with outcome in a mouse model of disseminated pulmonary microembolism (ME). When clots were incubated in the continuous presence of drug, tPA, TNK and Ret lysed fibrin clots identically in the absence of PA inhibitor-1 (e.g. PAI-1). Ret, which has lower fibrin affinity and greater susceptibility to inhibition by PAI-1 than tPA, was less effective in lysing plasma clots, while TNK was less effective when the fibrin content of the clots was enhanced. However, when clots were afforded only brief exposure to drug, as occurs in vivo, Ret showed more extensive clot permeation, greater retention and lysis than tPA or TNK. These results were reproduced in vivo in a mouse model of ME. These studies indicate the need for more relevant tests of plasminogen activator activity in vitro and in vivo and they show that clot permeation and retention are important potential predictors of clinical utility.
    Biochemical pharmacology 10/2012; · 4.25 Impact Factor
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    ABSTRACT: Prevailing approaches to manage autoimmune thrombotic disorders, such as heparin-induced thrombocytopenia (HIT), antiphospholipid syndrome (APS) and thrombotic thrombocytopenic purpura (TTP) include immunosuppression and systemic anticoagulation, though neither provides optimal outcome for many patients. A different approach is suggested by the concurrence of autoantibodies and their antigenic targets in the absence of clinical disease, such as platelet factor 4 (PF4) in HIT and β(2)-glycoprotein-I (β(2)GPI) in APS. The presence of autoantibodies in the absence of disease suggests that conformational changes or other alterations in endogenous protein autoantigens are required for recognition by pathogenic autoantibodies. In TTP, the clinical impact of ADAMTS13 deficiency caused by autoantibodies likely depends on the balance between residual antigen, i.e. enzyme activity, and demand imposed by local genesis of ultralarge multimers of von Willebrand factor (ULvWF). A corollary of these concepts is that disrupting PF4 and β(2)GPI conformation (or ULvWF oligomerization or function) might provide a disease-targeted approach to prevent thrombosis without systemic anticoagulation or immunosuppression. Validation of this approach requires a deeper understanding of how seemingly normal host proteins become antigenic or undergo changes that increase antibody avidity, and how they can be altered to retain adaptive functions while shedding epitopes prone to elicit harmful autoimmunity.
    Blood 09/2012; · 9.78 Impact Factor
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    ABSTRACT: Thrombomodulin-bound thrombin cleaves protein C (PC) zymogen in blood plasma producing activated protein C (APC), which exerts anti-coagulant, anti-inflammatory, anti-apoptotic and CNS-protective effects. Recombinant APC and thrombomodulin (TM) are both in clinical studies for management of acute conditions including sepsis. Methods that permit accurate measurement of APC in plasma are needed for clinical monitoring and mechanistic studies in animal models. However, the two existing methods require either long incubation periods with substrate, resulting in high background or they also recognize protein C inhibitor (PCI) complexed with APC (APC:PCI), which convolutes analysis of the amount of APC generated. Here we describe a robust quantitative in vivo assay that measures APC generation at both low levels of human protein C seen in chronic inflammatory disease and at physiological levels that shows a >99% fit with in vitro data.
    Journal of immunological methods 06/2012; 384(1-2):21-4. · 2.35 Impact Factor

Publication Stats

10k Citations
2,047.92 Total Impact Points


  • 1989–2014
    • University of Pennsylvania
      • • Department of Medicine
      • • Department of Pathology and Laboratory Medicine
      • • Department of Anesthesiology and Critical Care
      • • Division of Translational Medicine and Human Genetics
      • • Division of Hematology/Oncology
      • • Department of Pathology
      Philadelphia, Pennsylvania, United States
  • 1983–2014
    • Hospital of the University of Pennsylvania
      • • Department of Pathology and Laboratory Medicine
      • • Department of Pharmacology
      • • Department of Cell and Development Biology
      • • Department of Obstetrics and Gynecology
      • • Division of Hematology/Oncology
      Philadelphia, Pennsylvania, United States
  • 2012
    • Weill Cornell Medical College
      New York City, New York, United States
  • 2011
    • McMaster University
      Hamilton, Ontario, Canada
  • 2003–2011
    • The Children's Hospital of Philadelphia
      • • Department of Pediatrics
      • • Division of Hematology
      • • Department of Pathology and Laboratory Medicine
      Philadelphia, PA, United States
    • The Scripps Research Institute
      • Department of Cell and Molecular Biology
      La Jolla, CA, United States
  • 2010
    • Stanford University
      • Department of Pathology
      Stanford, CA, United States
    • University of Alabama at Birmingham
      • Department of Medicine
      Birmingham, AL, United States
  • 1998–2010
    • Hebrew University of Jerusalem
      • Department of Biological Chemistry
      Jerusalem, Jerusalem District, Israel
    • Tel Aviv University
      Tell Afif, Tel Aviv, Israel
  • 2009
    • Spanish National Centre for Cardiovascular Research
      Madrid, Madrid, Spain
  • 2006
    • University of Texas Health Science Center at Tyler
      Tyler, Texas, United States
  • 2005–2006
    • University of Colorado
      • Division of Pulmonary Sciences and Critical Care Medicine
      Denver, CO, United States
    • Northeast Institute of Geography and Agroecology
      • State Key Laboratory of Structural Chemistry
      Beijing, Beijing Shi, China
  • 2002
    • Duke University Medical Center
      • Division of Hematology
      Durham, NC, United States
  • 2001
    • Case Western Reserve University
      • Division of Hematology and Oncology
      Cleveland, OH, United States
  • 2000
    • University of New Mexico
      • Cancer Research and Treatment Center
      Albuquerque, NM, United States
  • 1995–1998
    • University of Michigan
      • Department of Internal Medicine
      Ann Arbor, MI, United States
  • 1997
    • University of California, Los Angeles
      Los Angeles, California, United States
  • 1988–1989
    • Temple University
      • Department of Medicine
      Philadelphia, PA, United States