Philip Fleckman

University of Washington Seattle, Seattle, Washington, United States

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Publications (118)562.42 Total impact

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    ABSTRACT: Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2016 · Wound Repair and Regeneration
  • Lauren K. Biesbroeck · Philip Fleckman
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    ABSTRACT: Nail disorders are commonly seen by both primary practitioners and dermatologists. Nail disease spans a broad variety of diagnoses, including infectious, inflammatory, and neoplastic disorders. Dermatophyte onychomycosis can have clinical findings that overlap with other disorders, mandating adequate diagnosis before treatment is initiated. Periungual verrucae typically respond to keratolytics or cryotherapy, but may require topical immunotherapy or intralesional injections. Any verruca recalcitrant to treatment should undergo biopsy to rule out squamous cell carcinoma. Patients with longitudinal melanonychia or erythronychia involving a single digit should be evaluated by a dermatologist, with biopsy of nail matrix considered to rule out underlying malignancy.
    No preview · Article · Sep 2015 · The Medical clinics of North America
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    ABSTRACT: Secondary infections and impaired desquamation complicate certain inherited ichthyoses, but their cellular basis remains unknown. In healthy human epidermis, the antimicrobial peptides cathelicidin (LL-37) and human β-defensin 2 (HBD2), as well as the desquamatory protease kallikrein-related peptidase 7 (KLK7), are delivered to the stratum corneum (SC) interstices by lamellar body (LB) exocytosis. To assess whether abnormalities in the LB secretory system could account for increased risk of infections and impaired desquamation in inherited ichthyoses with known abnormalities in LB assembly (Harlequin ichthyosis [HI]), secretion (epidermolytic ichthyosis [EI]), or postsecretory proteolysis (Netherton syndrome [NS]). Samples from library material were taken from patients with HI, EI, NS, and other ichthyoses, but with a normal LB secretory system, and in healthy controls and were evaluated by electron microscopy and immunohistochemical analysis from July 1, 2010, through March 31, 2013. Changes in LB secretion and in the fate of LB-derived enzymes and antimicrobial peptides in ichthyotic patients vs healthy controls. In healthy controls and patients with X-linked ichthyosis, neutral lipid storage disease with ichthyosis, and Gaucher disease, LB secretion is normal, and delivery of LB-derived proteins and LL-37 immunostaining persists high into the SC. In contrast, proteins loaded into nascent LBs and their delivery to the SC interstices decrease markedly in patients with HI, paralleled by reduced immunostaining for LL-37, HBD2, and KLK7 in the SC. In patients with EI, the cytoskeletal abnormality impairs the exocytosis of LB contents and thus results in decreased LL-37, HBD2, and KLK7 secretion, causing substantial entombment of these proteins within the corneocyte cytosol. Finally, in patients with NS, although abundant enzyme proteins loaded in parallel with accelerated LB production, LL-37 disappears, whereas KLK7 levels increase markedly in the SC. Together, these results suggest that diverse abnormalities in the LB secretory system account for the increased risk of secondary infections and impaired desquamation in patients with HI, EI, and NS.
    No preview · Article · Jan 2015 · JAMA Dermatology
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    ABSTRACT: H&E staining (A1 and B1), trichohyalin staining (TCHH) (A2 and B2) and Ki-67 staining (A3 and B3) in organotypic raft cultures expressing empty vector (pLZRS) (Mock) or HPV16 oncogenes E6/E7 are shown in the upper group of figures. Note many suprabasal mitotic cells in B1 (inset). In the lower group of figures, staining for cell cycle regulatory proteins, p16ink4a (C1 and D1), p21waf (C2 and D2) and pRB (C3 and D3) in Mock rafts and HPV16 rafts are shown. Some (secondary antibody) nonspecific staining of the cornified layer was present in all materials tested in this study. The dermoepidermal junction is indicated by dotted lines. Bar depicts 100 µm.
    Full-text · Dataset · Oct 2014
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    ABSTRACT: Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16ink4a, p21waf, pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16ink4a and p21waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.
    Full-text · Article · Oct 2014 · PLoS ONE
  • Nathan W Coleman · Philip Fleckman · Jerry I Huang

    No preview · Article · May 2014 · The Journal of hand surgery
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    ABSTRACT: The incidence, cost, morbidity, and mortality associated with non-healing of chronic skin wounds are dramatic. With the increasing numbers of people with obesity, chronic medical conditions, and an increasing life expectancy, the healthcare cost of non-healing ulcers has recently been estimated at $25 billion annually in the United States. The role played by bacterial biofilm in chronic wounds has been emphasized in recent years, particularly in the context of the prolongation of the inflammatory phase of repair. Rapid high-throughput genomic approaches have revolutionized the ability to identify and quantify microbial organisms from wounds. Defining bacterial genomes and using genetic approaches to knock out specific bacterial functions, then studying bacterial survival on cutaneous wounds is a promising strategy for understanding which genes are essential for pathogenicity. When an animal sustains a cutaneous wound, understanding mechanisms involved in adaptations by bacteria and adaptations by the host in the struggle for survival is central to development of interventions that favor the host. Characterization of microbiomes of clinically well characterized chronic human wounds is now under way. The use of in vivo models of biofilm-infected cutaneous wounds will permit the study of the mechanisms needed for biofilm formation, persistence, and potential synergistic interactions among bacteria. A more complete understanding of bacterial survival mechanisms and how microbes influence host repair mechanisms are likely to provide targets for chronic wound therapy.
    No preview · Article · Sep 2013
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    ABSTRACT: This statement, focused on melanonychia and nail plate dermoscopy, is intended to guide medical professionals working with melanonychia and to assist choosing appropriate management for melanonychia patients. The International Study Group on Melanonychia was founded in 2007 and currently has 30 members, including nail experts and dermatopathologists with special expertise in nails. The need for common definitions of nail plate dermoscopy was addressed during the Second Meeting of this Group held in February 2008. Prior to this meeting and to date (2010) there have been no evidence-based guidelines on the use of dermoscopy in the management of nail pigmentation.
    Full-text · Article · Apr 2013 · Anais brasileiros de dermatologia
  • Philip Fleckman · Karin Jaeger · Kathleen A Silva · John P Sundberg
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    ABSTRACT: Recent studies of mice with hair defects have resulted in major contributions to the understanding of hair disorders. To use mouse models as a tool to study nail diseases, a basic understanding of the similarities and differences between the human and mouse nail unit is required. In this study we compare the human and mouse nail unit at the macroscopic and microscopic level and use immunohistochemistry to determine the keratin expression patterns in the mouse nail unit. Both species have a proximal nail fold, cuticle, nail matrix, nail bed, nail plate, and hyponychium. Distinguishing features are the shape of the nail and the presence of an extended hyponychium in the mouse. Expression patterns of most keratins are similar. These findings indicate that the mouse nail unit shares major characteristics with the human nail unit and overall represents a very similar structure, useful for the investigation of nail diseases and nail biology. Anat Rec, 296:521-532, 2013. © 2013 Wiley Periodicals, Inc.
    No preview · Article · Mar 2013 · The Anatomical Record Advances in Integrative Anatomy and Evolutionary Biology
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    ABSTRACT: Management of ichthyoses is a complex and continuously dynamic process. Primary treatments of ichthyosis are by means of topical moisturizers and topical medications. Patients and families need to have reasonable and realistic expectations when it comes to topical therapy. Topical medications cannot cure the scaling, but can gradually reduce it and thus improve their condition. No one treatment regimen works for everyone, and the best topical therapy for each patient may be the result of months (or years) of painstaking effort on both the physician's and the patient's behalf. As patients get older and their activities and lifestyles change, so should their topical treatment regimen. Bear in mind that the more complex the skin care regimen and costly the topical treatments, the less likely a patient and their family will be compliant.
    No preview · Article · Feb 2013 · Dermatologic Therapy
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    ABSTRACT: This chapter discusses skin as the largest organ of the body based on surface area, although muscles and bones have more mass. It functions as a barrier to protect against desiccation and pathogenic microorganisms. Often, mammals are considered to have identical or very similar anatomical, histological, and physiological structures and features based on a high degree of genetic homology. Translational research focuses on features that allow comparison of specific features of interest for studies that are not possible in humans. One obvious difference in the structure of humans is the presence of cutis reticularis, which tethers the skin tightly to the dermis. Another obvious difference is that humans do not have the same relative distribution or amount of hair or the hair cycle seen in rodents. These differences underlie how human skin controls the epidermal barrier for protection against desiccation, infection, trauma, and ultraviolet radiation.
    No preview · Article · Dec 2012
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    ABSTRACT: [This corrects the article on p. e40973 in vol. 7.].
    No preview · Article · Oct 2012 · PLoS ONE
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    ABSTRACT: Abbreviations: SC, stratum corneum
    Full-text · Article · Sep 2012 · Journal of Investigative Dermatology
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    ABSTRACT: Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.
    Full-text · Article · Jul 2012 · PLoS ONE
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    Philip Fleckman
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    ABSTRACT: The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
    Preview · Article · Jul 2012 · Journal of Investigative Dermatology
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    ABSTRACT: Pityriasis rubra pilaris (PRP) is a papulosquamous disorder phenotypically related to psoriasis. The disease has been occasionally shown to be inherited in an autosomal-dominant fashion. To identify the genetic cause of familial PRP, we ascertained four unrelated families affected by autosomal-dominant PRP. We initially mapped PRP to 17q25.3, a region overlapping with psoriasis susceptibility locus 2 (PSORS2 [MIM 602723]). Using a combination of linkage analysis followed by targeted whole-exome sequencing and candidate-gene screening, we identified three different heterozygous mutations in CARD14, which encodes caspase recruitment domain family, member 14. CARD14 was found to be specifically expressed in the skin. CARD14 is a known activator of nuclear factor kappa B signaling, which has been implicated in inflammatory disorders. Accordingly, CARD14 levels were increased, and p65 was found to be activated in the skin of PRP-affected individuals. The present data demonstrate that autosomal-dominant PRP is allelic to familial psoriasis, which was recently shown to also be caused by mutations in CARD14.
    Full-text · Article · Jun 2012 · The American Journal of Human Genetics
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    ABSTRACT: Bacterial biofilm has been shown to play a role in delaying wound healing of chronic wounds, a major medical problem that results in significant health care burden. A reproducible animal model could be very valuable for studying the mechanism and management of chronic wounds. Our previous work showed that Pseudomonas aeruginosa (PAO1) biofilm challenge on wounds in diabetic (db/db) mice significantly delayed wound healing. In this wound time course study, we further characterize the bacterial burden, delayed wound healing, and certain aspects of the host inflammatory response in the PAO1 biofilm-challenged db/db mouse model. PAO1 biofilms were transferred onto 2-day-old wounds created on the dorsal surface of db/db mice. Control wounds without biofilm challenge healed by 4 weeks, consistent with previous studies; none of the biofilm-challenged wounds healed by 4 weeks. Of the biofilm-challenged wounds, 64% healed by 6 weeks, and all of the biofilm-challenged wounds healed by 8 weeks. During the wound-healing process, P. aeruginosa was gradually cleared from the wounds while the presence of Staphylococcus aureus (part of the normal mouse skin flora) increased. Scabs from all unhealed wounds contained 10(7) P. aeruginosa, which was 100-fold higher than the counts isolated from wound beds (i.e., 99% of the P. aeruginosa was in the scab). Histology and genetic analysis showed proliferative epidermis, deficient vascularization, and increased inflammatory cytokines. Hypoxia inducible factor expression increased threefold in 4-week wounds. In summary, our study shows that biofilm-challenged wounds typically heal in approximately 6 weeks, at least 2 weeks longer than nonbiofilm-challenged normal wounds. These data suggest that this delayed wound healing model enables the in vivo study of bacterial biofilm responses to host defenses and the effects of biofilms on host wound healing pathways. It may also be used to test antibiofilm strategies for treating chronic wounds.
    Full-text · Article · May 2012 · Wound Repair and Regeneration
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    ABSTRACT: This study investigates mouse cutaneous responses to long-term percutaneously implanted rods surrounded by sphere-templated porous biomaterials engineered to mimic medical devices surrounded by a porous cuff. We hypothesized that keratinocytes would migrate through the pores and stop, permigrate, or marsupialize along the porous/solid interface. Porous/solid-core poly(2-hydroxyethyl methacrylate) [poly(HEMA)] and silicone rods were implanted in mice for 14 days, and for 1, 3, and 6 months. Implants with surrounding tissue were analyzed (immuno)histochemically by light microscopy. Poly(HEMA)/skin implants yielded better morphologic data than silicone implants. Keratinocytes at the poly(HEMA) interface migrated in two different directions. "Ventral" keratinocytes contiguous with the dermal-epidermal junction migrated into the outermost pores, forming an integrated collar surrounding the rods. "Dorsal" keratinocytes appearing to emanate from the differentiated epithelial layer, extended upward along and into the exterior portion of the rod, forming an integrated sheath. Leukocytes persisted in poly(HEMA) and silicone pores for the duration of the study. Vascular and collagen networks within the poly(HEMA) pores matured as a function of time up to 3-months implantation. Nerves were not observed within the pores. Poly(HEMA) underwent morphological changes by 6 months of implantation. Marsupialization, foreign body encapsulation, and infection were not observed in any implants.
    Full-text · Article · May 2012 · Journal of Biomedical Materials Research Part A
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    ABSTRACT: Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.
    Preview · Article · Mar 2012 · Wound Repair and Regeneration
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    ABSTRACT: Recently a new polyomavirus was identified in a patient with trichodysplasia spinulosa (TS), a rare follicular skin disease of immunocompromised patients characterized by facial spines and overgrowth of inner root sheath cells. Seroepidemiological studies indicate that TSPyV is ubiquitous and latently infects 70% of the healthy individuals. To corroborate the relationship between active TSPyV infection and TS disease by analyzing the presence, load, and precise localization of TSPyV infection in TS patients and in controls. TS lesional and non-lesional skin samples were retrieved from TS patients through a PubMed search. Samples were analyzed for the presence and load of TSPyV DNA with quantitative PCR, and for expression and localization of viral protein with immunofluorescence. Findings obtained in TS patients (n=11) were compared to those obtained in healthy controls (n=249). TSPyV DNA detection was significantly associated with disease (P<0.001), with 100% positivity of the lesional and 2% of the control samples. Quantification revealed high TSPyV DNA loads in the lesional samples (∼10(6)copies/cell), and low viral loads in the occasionally TSPyV-positive non-lesional and control samples (<10(2)copies/cell). TSPyV VP1 protein expression was detected only in lesional TS samples, restricted to the nuclei of inner root sheath cells over-expressing trichohyalin. The high prevalence and load of TSPyV DNA only in TS lesions, and the abundant expression of TSPyV protein in the affected hair follicle cells demonstrate a tight relation between TSPyV infection and TS disease, and indicate involvement of active TSPyV infection in TS pathogenesis.
    Full-text · Article · Dec 2011 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology

Publication Stats

6k Citations
562.42 Total Impact Points

Institutions

  • 1984-2016
    • University of Washington Seattle
      • • Division of Dermatology
      • • Department of Orthopaedics and Sports Medicine
      • • Department of Laboratory Medicine
      • • Department of Pathology
      • • Department of Medicine
      Seattle, Washington, United States
  • 2013
    • American University Washington D.C.
      Washington, Washington, D.C., United States
  • 2005
    • Seattle University
      Seattle, Washington, United States
  • 2000
    • University of Toronto
      Toronto, Ontario, Canada
  • 1999
    • Massachusetts General Hospital
      • Department of Dermatology
      Boston, Massachusetts, United States
  • 1992
    • University of Texas Health Science Center at Houston
      • Department of Pediatric Dentistry
      Houston, Texas, United States
  • 1990
    • Children's Hospital & Medical Center
      Omaha, Nebraska, United States