L K Ashman

University of Newcastle, Newcastle, New South Wales, Australia

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Publications (109)539.91 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Familial gastrointestinal stromal tumours (GISTs) are rare but otherwise well-characterized tumour syndromes, most commonly occurring on a background of germline-activating mutations in the tyrosine kinase receptor c-KIT. The associated clinical spectrum reflects the constitutive activation of this gene product across a number of cell lines, generating gain-of-function phenotypes in interstitial cells of Cajal (GIST and dysphagia), mast cells (mastocytosis) and melanocytes (hyperpigmentation). We report a three-generation kindred harbouring a c-KIT germline-activating mutation resulting in multifocal GISTs, dysphagia and a complex melanocyte hyperpigmentation and hypopigmentation disorder, the latter with features typical of those observed in Waardenburg type 2 syndrome (WS2F). Sequencing of genes known to be causative for WS [microphthalmia transcription factor (MITF), Pax3, Sox10, SNAI2 ] failed to show any candidate mutations to explain this complex cutaneous depigmentation phenotype. Our case report conclusively expands the clinical spectrum of familial GISTs and shows a hitherto unrecognized link to WS. Possible mechanisms responsible for this novel cause of WS2F will be discussed.
    Clinical Genetics 06/2011; 79(6):554-60. · 4.25 Impact Factor
  • Leonie Ashman, Renate Griffith
    Clinical Biochemistry - CLIN BIOCHEM. 01/2011; 44(13).
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    ABSTRACT: This study was designed to determine the role of CD151 in platelet thrombus formation in vivo and define the contribution of platelet vs. endothelial CD151 in regulating platelet thrombus formation in vivo. Using intravital microscopy and ferric chloride (FeCl(3)) injury of mesenteric arterioles, we found that thrombi formed in CD151(+/-) and CD151(-/-) mice were smaller and less stable, than those formed in CD151(+/+) mice, with a tendency for embolization. Similarly, in Folt's FeCl(3)-induced carotid injury model, both CD151(+/-) and CD151(-/-) mice showed more prolonged times to 95% vessel occlusion than CD151(+/+) mice. In addition, laser-induced injury of cremaster muscle arterioles showed that thrombi formed in CD151(+/-) and CD151(-/-) mice were smaller and less stable than those formed in CD151(+/+) mice. Following platelet depletion/reconstitution with ex vivo-labeled donor platelets, platelet-depleted CD151(+/+) mice that received reconstitution with CD151(-/-) platelets had smaller thrombi that were unstable and embolized. In contrast, platelet-depleted CD151(-/-) mice that received reconstitution with CD151(+/+) platelets had normal thrombi that were stable. These data provide evidence that platelet CD151 is required for regulating thrombus formation in vivo.
    Journal of Thrombosis and Haemostasis 10/2009; 7(12):2074-84. · 6.08 Impact Factor
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    ABSTRACT: A major question in immunology is how DC can display limited amounts of individual peptide-MHC complexes and still induce cross-linking of T-cell receptors to initiate cellular responses. One suggested mechanism is that MHC exists at the cell surface in high avidity multimers, and tetraspanin proteins, known to laterally associate with both MHC classes I and II, promote MHC multimerisation. To validate this theory, we tested the ability of DC deficient in either one of two typical tetraspanin molecules: CD37 or CD151 to present peptide to Ag-specific T cells. Surprisingly, although they exhibited no developmental or maturation defects, DC lacking either CD37 or CD151 expression were hyper-stimulatory to T cells. We demonstrate that CD37 and CD151 control DC-mediated T-cell activation by two different mechanisms: CD151 regulates co-stimulation whereas CD37 regulates peptide/MHC presentation. The implications of these results on the model suggesting that tetraspanins promote MHC multimerisation are discussed.
    European Journal of Immunology 01/2009; 39(1):50-5. · 4.97 Impact Factor
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    ABSTRACT: The tetraspanin CD151 forms a stoichiometric complex with integrin α3β1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of α3β1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. α6β1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucα1–2Gal and bisecting GlcNAc-β(1→4) linkage on N-glycans of the α3 integrin subunit. The modulatory activity of CD151 toward α3β1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of α3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward α3β1. We also found that glycosylation of CD151 is also critical; Asn159 → Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or α3β1 but negated its modulatory function. Changes in the glycosylation pattern of α3β1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of α3β1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.
    Journal of Biological Chemistry 12/2008; 283(51):35445-35454. · 4.65 Impact Factor
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    ABSTRACT: The tetraspanin CD151 forms a stoichiometric complex with integrin alpha3beta1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of alpha3beta1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. alpha6beta1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucalpha1-2Gal and bisecting GlcNAc-beta(1-->4) linkage on N-glycans of the alpha3 integrin subunit. The modulatory activity of CD151 toward alpha3beta1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of alpha3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward alpha3beta1. We also found that glycosylation of CD151 is also critical; Asn(159) --> Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or alpha3beta1 but negated its modulatory function. Changes in the glycosylation pattern of alpha3beta1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of alpha3beta1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.
    Journal of Biological Chemistry 11/2008; 283(51):35445-54. · 4.65 Impact Factor
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    ABSTRACT: The receptor tyrosine kinase c-KIT plays a key role in normal mast cell development. Point mutations in c-KIT have been associated with sporadic or familial mastocytosis. Two unrelated pairs of apparently identical twins affected by cutaneous mastocytosis attending the Mastocytosis Clinic at the Royal Children's Hospital, Melbourne, provided an opportunity to assess the possible contribution of c-KIT germline mutations or polymorphisms in this disease. Tissue biopsy, blood and/or buccal swab specimens were collected from 10 children with mastocytosis. To detect germline mutations/polymorphisms in c-KIT, we studied all coding exons by denaturing high pressure liquid chromatography. Exons showing mismatches were examined by direct sequencing. The influence of the substitution identified was further examined by expressing the variant form of c-KIT in factor-dependent FDC-P1 cells. In both pairs of twins, a heterozygous ATG to CTG transition in codon 541 was observed, resulting in the substitution of a methionine residue in the transmembrane domain by leucine (M541L). In each case, one parent was also heterozygous for this allele. Expression of M541L KIT in FDC-P1 cells enabled them to grow in human KIT ligand (stem cell factor, SCF) but did not confer factor independence. Compared with cells expressing wild-type KIT at a similar level, M541L KIT-expressing cells displayed enhanced growth at low levels of SCF, and heightened sensitivity to the KIT inhibitor, imatinib mesylate. The data suggest that the single nucleotide polymorphism resulting in the substitution M541L may predispose to paediatric mastocytosis.
    British Journal of Dermatology 10/2008; 159(5):1160-9. · 3.76 Impact Factor
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    ABSTRACT: Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.
    Biochemical Journal 10/2008; 417(1):391-400. · 4.65 Impact Factor
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    ABSTRACT: CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin alpha 3 beta 1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin alpha 3 beta 1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin alpha 3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin alpha 3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin alpha 3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin alpha 3 beta 1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin alpha 3 beta 1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly(176)/Gly(177), Leu(191) and Gln(194) comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin alpha 3 beta 1 demonstrated that the segment from Cys(185) to Cys(192), including Leu(191), was involved in the stable association of CD151 with integrin alpha 3 beta 1, as was the Gln(194)-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu(191) and Gln(194), comprise an interface between CD151 and integrin alpha 3 beta 1, and, along with the epitope including Gly(176)/Gly(177), are concealed by bound integrin.
    Biochemical Journal 08/2008; 415(3):417-27. · 4.65 Impact Factor
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    ABSTRACT: Small molecule protein kinase inhibitors show great promise as anti-cancer agents, however, de novo and acquired resistance present problems. These are reviewed and illustrated using the receptor tyrosine kinase, KIT, as an example. Emerging solutions are presented, such as targeting active kinase conformations.
    Mini Reviews in Medicinal Chemistry 11/2006; 6(10):1101-10. · 2.87 Impact Factor
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    ABSTRACT: The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.
    Molecular Biology of the Cell 07/2006; 17(6):2707-21. · 4.60 Impact Factor
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    ABSTRACT: Patients with pancreatic adenocarcinoma have a poor prognosis due to the extraordinary high invasive capacity of this tumor. Altered integrin and tetraspanin expression is suggested to be an important factor. We recently reported that after protein kinase C activation, colocalization of alpha6beta4 with the tetraspanin CO-029 strongly supports migration of a rat pancreatic adenocarcinoma. The finding led us to explore whether and which integrin-tetraspanin complexes influence the motility of human pancreatic tumors. Integrin and tetraspanin expression of pancreatic and colorectal adenocarcinoma was evaluated with emphasis on colocalization and the impact of integrin-tetraspanin associations on tumor cell motility. The majority of pancreatic and colorectal tumors expressed the alpha2, alpha3, alpha6, beta1, and beta4 integrins and the tetraspanins CD9, CD63, CD81, CD151, and CO-029. Expression of alpha6beta4 and CO-029 was restricted to tumor cells, whereas alpha1, alpha2, alpha3, alpha6, beta1, and CD9, CD81, CD151 were also expressed by the surrounding stroma. CD63, CD81, and beta1 expression was observed at comparably high levels in healthy pancreatic tissue. alpha3beta1 frequently colocalized and coimmunoprecipitated with CD9, CD81, and CD151, whereas alpha6beta4 colocalized and coimmunoprecipitated mostly with CD151 and CO-029. Notably, protein kinase C activation strengthened only the colocalization of CD151 and CO-029 with beta4 and was accompanied by internalization of the integrin-tetraspanin complex, decreased laminin 5 adhesion, and increased cell migration. alpha6beta4 is selectively up-regulated in pancreatic and colorectal cancer. The association of alpha6beta4 with CD151 and CO-029 correlates with increased tumor cell motility.
    Clinical Cancer Research 05/2005; 11(8):2840-52. · 7.84 Impact Factor
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    ABSTRACT: The receptor tyrosine kinase, KIT, displays activating mutations in the kinase domain, which are associated with various cancers. We have used homology modelling based on the crystal structures of the insulin receptor kinase in active and inactive conformations to predict the corresponding structures of the KIT kinase domain. We have prepared four KIT models, one each for the active and inactive conformations of the wild-type and of the Asp816Val mutant proteins. We have also placed ATP into the active conformations and the inhibitor, STI571, into the inactive conformations. All models have been fully energy minimised. The molecular modelling studies described here explain (i) why Asp816Val KIT is constitutively active, (ii) why the nature of the substituting amino acid at residue 816 is relatively unimportant, and (iii) why the Asp816Val substitution confers resistance to the KIT-inhibitory drug STI571. The models will be valuable for predicting other kinase inhibitory drugs that may be active on wild-type and mutant forms of KIT. During the course of this work, a crystal structure of the active conformation of the KIT kinase domain has been published. Our model of the active conformation of the Asp816Val mutant is strikingly similar to this crystal structure, whereas our model of the active conformation of the wild-type kinase domain of KIT differs from the crystal structure in some respects. The reasons for this apparent discrepancy are discussed.
    Journal of Molecular Graphics and Modelling 11/2004; 23(2):139-52. · 2.33 Impact Factor
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    ABSTRACT: The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.
    The Journal of Cell Biology 01/2004; 163(6):1351-62. · 10.82 Impact Factor
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    ABSTRACT: The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.
    Biochemical and Biophysical Research Communications 05/2003; 304(1):107-12. · 2.28 Impact Factor
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    ABSTRACT: In both mice and humans alternate splicing results in isoforms of c-Kit characterized by the presence or the absence of a tetrapeptide sequence, GNNK, in the juxtamembrane region of the extracellular domain. Dramatic differences in the kinetics and magnitude of activation of the intrinsic tyrosine kinase activity of c-Kit between the GNNK- and GNNK+ isoforms has previously been shown. Here we report the analysis of downstream targets of receptor signaling, which revealed that the signaling was differentially regulated in the two splice forms. The kinetics of phosphorylation of Shc, previously demonstrated to be phosphorylated by Src downstream of c-Kit, was stronger and more rapid in the GNNK- form, whereas it showed slower kinetics in the GNNK+ form. Inhibition of Src family kinases with the specific Src family kinase inhibitor SU6656 altered the kinetics of activation of the GNNK- form of c-Kit so that it resembled that of the GNNK+ form. In cells expressing the GNNK- form, SCF was rapidly degraded, whereas in cells expressing the GNNK+ form only showed a very slow rate of degradation of SCF. In the GNNK+ form the Src inhibitor SU6656 only had a weak effect on degradation, whereas in the GNNK- form it dramatically inhibited degradation. In summary, the two splice forms show, despite only a four-amino acid sequence difference, remarkable differences in their signaling capabilities.
    Journal of Biological Chemistry 04/2003; 278(11):9159-66. · 4.65 Impact Factor
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    ABSTRACT: This study assessed the ability of recombinant human stem cell factor (rHuSCF) to mobilize stem cells in 44 patients who had failed a prior mobilization (CD34(+) yield 0.5-1.9 x 10(6)/kg BW) with filgrastim-alone or chemotherapy-plus-filgrastim. The same mobilization regimen was used with the addition of rHuSCF. In the filgrastim-alone group (n=13), rHuSCF 20 microg/kg was started 3 days before filgrastim and continued for the duration of filgrastim. In the chemotherapy-plus-filgrastim group (n=31), rHuSCF 20 microg/kg/day plus filgrastim 5-10 microg/kg/day were administered concurrently. Leukaphereses were continued to a maximum of four procedures or a target of >or=3 x 10(6) CD34(+) cells/kg. In both groups, CD34(+) yield (x 10(6)/kg BW) of the study mobilization was higher than that of the prior mobilization (median: 2.42 vs 0.84 P=0.002 and 1.64 vs 0.99 P=<0.001, respectively). In all 54 and 45% of patients in the filgrastim-alone group and chemotherapy-plus-filgrastim group, respectively, reached the threshold yield of 2 x 10(6)/kg. The probability of a successful mobilization was the same in those with a CD34+ yield of 0.5-0.75 x 10(6)/kg BW in the prior mobilization as in those with 0.76-1.99 x 10(6)/kg BW. Downmodulation of c-kit expression and a lower percentage of Thy-1 positivity in the mobilized CD34(+) cells were noted in the successful mobilizers compared with those in the poor mobilizers. This study shows that rhuSCF is effective in approximately half the patients who had failed a prior mobilization and allows them to proceed to transplant. It also points to the likely role of the SCF/c-kit ligand pair in mobilization.
    Bone Marrow Transplantation 03/2003; 31(5):371-8. · 3.54 Impact Factor
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    Journal of Biological Chemistry 01/2003; 278(29):9159-9166. · 4.65 Impact Factor
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    American Journal Of Pathology 09/2002; 161(2):737-8; author reply 738-9. · 4.60 Impact Factor
  • American Journal of Pathology - AMER J PATHOL. 01/2002; 161(2):737-739.

Publication Stats

4k Citations
539.91 Total Impact Points

Institutions

  • 2003–2011
    • University of Newcastle
      • • Department of Biological Sciences
      • • School of Environmental and Life Sciences
      • • Faculty of Health and Medicine
      Newcastle, New South Wales, Australia
  • 1994–2001
    • Adelaide Cancer Centre
      Tarndarnya, South Australia, Australia
    • Cancer Research Center of Lyon
      Lyons, Rhône-Alpes, France
  • 1998–2000
    • IMVS Pathology
      • Haematology Division
      Adelaide, South Australia, Australia
  • 1984–1999
    • University of Adelaide
      • • Discipline of Anatomy and Pathology
      • • Discipline of Microbiology and Immunology
      Adelaide, South Australia, Australia
  • 1993–1996
    • Virginia Commonwealth University
      • • Department of Internal Medicine
      • • School of Medicine
      Richmond, VA, United States