Thomas N Darling

Uniformed Services University of the Health Sciences, Maryland, United States

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Publications (72)454.07 Total impact

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    ABSTRACT: The 2012 International Tuberous Sclerosis Complex Clinical Consensus Conference was convened to update the last consensus statement in 1998. Skin and dental lesions are common in tuberous sclerosis complex (TSC) and are a frequent concern for patients. Recognition of these lesions is imperative for early diagnosis, given the treatment advances that may improve patient outcomes.
    JAMA dermatology. 07/2014;
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    Rajesh L Thangapazham, Thomas N Darling, Jon Meyerle
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    ABSTRACT: Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices.
    International Journal of Molecular Sciences 05/2014; 15(5):8407-27. · 2.46 Impact Factor
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    ABSTRACT: Somatic mutations in the phosphatidylinositol/AKT/mTOR pathway cause segmental overgrowth disorders. Diagnostic descriptors associated with PIK3CA mutations include fibroadipose overgrowth (FAO), Hemihyperplasia multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi, Scoliosis/skeletal and spinal (CLOVES) syndrome, macrodactyly, and the megalencephaly syndrome, Megalencephaly-Capillary malformation (MCAP) syndrome. We set out to refine the understanding of the clinical spectrum and natural history of these phenotypes, and now describe 35 patients with segmental overgrowth and somatic PIK3CA mutations. The phenotypic data show that these previously described disease entities have considerable overlap, and represent a spectrum. While this spectrum overlaps with Proteus syndrome (sporadic, mosaic, and progressive) it can be distinguished by the absence of cerebriform connective tissue nevi and a distinct natural history. Vascular malformations were found in 15/35 (43%) and epidermal nevi in 4/35 (11%) patients, lower than in Proteus syndrome. Unlike Proteus syndrome, 31/35 (89%) patients with PIK3CA mutations had congenital overgrowth, and in 35/35 patients this was asymmetric and disproportionate. Overgrowth was mild with little postnatal progression in most, while in others it was severe and progressive requiring multiple surgeries. Novel findings include: adipose dysregulation present in all patients, unilateral overgrowth that is predominantly left-sided, overgrowth that affects the lower extremities more than the upper extremities and progresses in a distal to proximal pattern, and in the most severely affected patients is associated with marked paucity of adipose tissue in unaffected areas. While the current data are consistent with some genotype-phenotype correlation, this cannot yet be confirmed. © 2014 Wiley Periodicals, Inc.
    American Journal of Medical Genetics Part A 04/2014; · 2.30 Impact Factor
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    ABSTRACT: Earlier studies showed that dermal cells lose trichogenic capacity with passage, but studies on the effect of keratinocyte passage on human hair follicle neogenesis and graft quality has been hampered by the lack of a suitable model system. We recently documented human hair follicle neogenesis in grafted dermal-epidermal composites and in the present study we determined the effects of keratinocyte passage on hair follicle neogenesis. Dermal equivalents were made with cultured human dermal papilla cells and were overlaid with either primary or passaged human keratinocytes to form dermal-epidermal composites; these were then grafted onto immunodeficient mice. Superior hair follicle neogenesis was observed using early keratinocyte cultures. Characteristics such as formation of hair shafts and sebaceous glands, presence of hair follicles with features of anagen or telogen follicles, and reproducible hair and skin function parameters make this model a tool to study human hair follicle neogenesis and development.This article is protected by copyright. All rights reserved.
    Experimental Dermatology 04/2014; · 3.58 Impact Factor
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    ABSTRACT: We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) vs 53% (39/73)]. Both had a very high proportion of UV-type mutations (occurring at adjacent pyrimidines) [91% (10/11) vs 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62) and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho-S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus the clinical and histological appearances and the molecular pathology of these UV related XP nevi and melanomas were different from nevi and melanomas in the general population. This article is protected by copyright. All rights reserved.
    Pigment Cell & Melanoma Research 01/2014; · 5.84 Impact Factor
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    ABSTRACT: Tuberous Sclerosis Complex (TSC) is characterized by the formation of tumors in multiple organs and is due to germline mutation in one of two tumor suppressor genes, TSC1 and TSC2. As for other tumor suppressor gene syndromes, the mechanism of somatic second-hit events in TSC tumors is unknown. We grew fibroblast-like cells from 29 TSC skin tumors from 22 TSC subjects, and identified germline and second-hit mutations in TSC1/TSC2 using next-generation sequencing. Eighteen of 22 (82%) subjects had a mutation identified, and 8 of the 18 (44%) subjects were mosaic with mutant allele frequencies of 0 to 19% in normal tissue DNA. Multiple tumors were available from 4 patients, and in each case second-hit mutations in TSC2 were distinct indicating they arose independently. Most remarkably, 7 (50%) of the 14 somatic point mutations were CC>TT ultraviolet "signature" mutations, never seen as a TSC germline mutation. These occurred exclusively in facial angiofibroma tumors from sun-exposed sites. These results implicate UV-induced DNA damage as a cause of second-hit mutations and development of TSC facial angiofibromas, and suggest that measures to limit UV exposure in TSC children and adults should reduce the frequency and severity of these lesions.
    Human Molecular Genetics 11/2013; · 7.69 Impact Factor
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    ABSTRACT: Lymphangioleiomyomatosis (LAM), sporadic or in women with tuberous sclerosis complex (TSC), is characterized by cystic lung destruction, lymphatic involvement (e.g., chylous pleural effusions, lymphangioleiomyomas) and renal angiomyolipomas (AMLs). The multisystem manifestations of LAM appear to result from metastatic dissemination of LAM cells bearing inactivating mutations or having loss of heterozygosity (LOH) in the tumor suppressor genes, TSC1 or TSC2, which leads to hyper-activation of the mammalian target of rapamycin (mTOR). Sirolimus slows the decline of lung function, reduces chylous effusions and shrinks the size of AMLs. The purpose of this study was to determine the effect of sirolimus on circulating LAM cells. Cells from blood were isolated by a density-gradient fractionation system and from urine and chylous effusions by centrifugation. Cells from blood were incubated with anti-CD45-fluorescein isothiocyanate (FITC) and anti-CD235a-R-Phycoerythrin (PE) antibodies, and urine and chylous effusion cells with anti-CD44v6-FITC and anti-CD9-R-PE antibodies. Cells were sorted and analyzed for TSC2 LOH. LAM cells with TSC2 LOH were identified, in this cohort, in 100% of blood specimens and 75% of urine samples from patients before therapy; over a mean duration of 2.2 ± 0.4 years of sirolimus therapy, detection rates of LAM cells were significantly decreased to 25% in blood (P < 0.001) and 8% in urine (P = 0.003). Following therapy, greater loss of circulating LAM cells was seen in post-menopausal patients (p = 0.025). Patients receiving sirolimus had a progressive loss of circulating LAM cells, which was dependent on time of treatment and menopausal status.
    Chest 09/2013; · 7.13 Impact Factor
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    ABSTRACT: Sex prevalence in lung disease suggests that sex-specific hormones may contribute to the pathogenesis and/or progression of at least some lung diseases, such as lung adenocarcinoma, lymphangioleiomyomatosis (LAM) and benign metastasising leiomyoma (BML). Oestrogen is an important hormone in normal lung development and in the pathogenesis of female predominant pulmonary diseases. In vivo and in vitro studies have facilitated our understanding of disease pathogenesis and discovery of potential therapeutic targets. Oestrogen promoted disease progression in cell and animal models of lung adenocarcinoma, LAM and BML. Specifically, oestrogen enhanced tumour growth and metastasis in animal models of these diseases. Furthermore, 17β-estradiol (E2), the most abundant form of oestrogen in humans, increased the size and proliferation of cultured cells of lung adenocarcinoma and LAM. Coupled with the known mechanisms of oestrogen metabolism and signalling, these model systems may provide insights into the diverse effects of oestrogen and other hormones on lung diseases. Anti-oestrogen treatments that target key events of oestrogen synthesis or signalling, such as aromatase activity, oestrogen receptors and signalling pathways, may offer additional opportunities for clinical trials.
    European Respiratory Review 09/2013; 22(129):302-11.
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    Journal of Investigative Dermatology 08/2013; · 6.19 Impact Factor
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    Journal of Investigative Dermatology 07/2013; · 6.19 Impact Factor
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    Diane C Seibert, Thomas N Darling
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    ABSTRACT: Purpose: Some forms of genetic skin disease are highly prevalent and others are exceedingly rare, but collectively, genetic skin disorders (or genodermatoses) are often poorly understood. The purpose of this article, therefore, is to increase nurses' awareness and understanding of some of the physical, psychological, social, and ethical issues facing patients with inherited skin disorders. Organizing Construct: This article offers an overview of genetic skin diseases; highlights the complexity and prevalence of the genodermatoses; describes inheritance patterns, genetics, and treatment for six genodermatoses; and reviews some of the ethical, privacy, technological, and resource issues nurses should consider when caring for patients with genetic skin disorders. Conclusions: Because genodermatoses are found in all age groups, across all populations, and within all healthcare settings, nurses are uniquely positioned to address the educational and healthcare needs of patients and families with inherited skin disorders. Clinical Relevance: Over the past two decades, genetics has evolved from a niche specialty into general practice. To ensure that patients and their families receive appropriate services and resources, nurses must have a working knowledge of genetic concepts. This article reinforces key genetic concepts while discussing many of the issues and concerns important to caring for patients with genetic skin disease.
    Journal of Nursing Scholarship 01/2013; · 1.61 Impact Factor
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    ABSTRACT: The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.
    Nature Genetics 06/2012; 44(8):928-33. · 35.21 Impact Factor
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    ABSTRACT: Cystic fibrosis (CF) is the most common autosomal recessive disease in the USA and Europe, whose life-limiting phenotype is manifest on epithelial cells throughout the body. The principal cause of morbidity and mortality is a massively proinflammatory condition in the lung. The mutation responsible for most cases of CF is [ΔF508]CFTR. However, the penetrance of the disease is quite variable, and adverse events leading to hospitalization cannot be easily predicted. Thus, there is a strong need for prognostic endpoints that might serve to identify impending clinical problems long before they happen. Our approach has been to search for proteomic signatures in easily accessed biological fluids that might identify the molecular basis for adverse events. We describe here a workflow that begins with patient-derived bronchial brush biopsies and progresses to analysis of serum and plasma from patients on antibody microarrays.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 823:179-200. · 1.29 Impact Factor
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    ABSTRACT: Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox) mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.
    PLoS ONE 01/2012; 7(11):e47842. · 3.53 Impact Factor
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    ABSTRACT: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).
    New England Journal of Medicine 08/2011; 365(7):611-9. · 54.42 Impact Factor
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    ABSTRACT: Tuberous sclerosis complex (TSC) is associated with tumor development in the brain, retina, kidney, skin, heart, and lung. Seizures, intellectual disability, and characteristic skin lesions commonly manifest in early childhood, but some findings, notably renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM), emerge later, placing adults with undiagnosed TSC at increased risk for morbidity and mortality. To describe the clinical presentation and severity of TSC in adult women. Retrospective cohort study. National Institutes of Health Clinical Center, Bethesda, Maryland, 1995 to 2010. 79 women aged 18 years or older who were enrolled in an observational cohort study of TSC to evaluate disease manifestations. History, physical examination, pulmonary function testing, chest radiography, abdominal computed tomography, high-resolution chest computed tomography, and brain magnetic resonance imaging were used to evaluate patients. Among the 45 patients who received a diagnosis of TSC in adulthood, 21 presented with symptoms due to LAM, 19 with renal angiomyolipomas, and 10 with seizures. Of the 45 patients, 30 met clinical criteria for TSC in childhood that remained undiagnosed for a median of 21.5 years and 15 were older than 18 years before meeting the clinical criteria for TSC. Patients diagnosed in adulthood and those diagnosed in childhood had similar occurrences of pneumothorax, shortness of breath, hemoptysis, nephrectomy, and death. No men were included in the study, and selection was biased toward patients having pulmonary LAM. Women who received a TSC diagnosis in adulthood had minimal morbidity during childhood but were still at risk for life-threatening pulmonary and renal manifestations. Intramural Research Program, National Institutes of Health, National Heart, Lung, and Blood Institute.
    Annals of internal medicine 06/2011; 154(12):806-13, W-294. · 13.98 Impact Factor
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    ABSTRACT: Hamartomas are composed of cells native to an organ but abnormal in number, arrangement or maturity. In the tuberous sclerosis complex (TSC), hamartomas develop in multiple organs because of mutations in TSC1 or TSC2. Here we show that TSC2-null fibroblast-like cells grown from human TSC skin hamartomas induced normal human keratinocytes to form hair follicles and stimulated hamartomatous changes. Follicles were complete with sebaceous glands, hair shafts and inner and outer root sheaths. TSC2-null cells surrounding the hair bulb expressed markers of the dermal sheath and dermal papilla. Tumour xenografts recapitulated characteristics of TSC skin hamartomas with increased mammalian target of the rapamycin complex 1 (mTORC1) activity, angiogenesis, mononuclear phagocytes and epidermal proliferation. Treatment with an mTORC1 inhibitor normalized these parameters and reduced the number of tumour cells. These studies indicate that TSC2-null cells are the inciting cells for TSC skin hamartomas, and suggest that studies on hamartomas will provide insights into tissue morphogenesis and regeneration.
    Nature Communications 03/2011; 2:235. · 10.74 Impact Factor
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    Journal of the American Academy of Dermatology 02/2011; 64(2):442-4. · 4.91 Impact Factor

Publication Stats

1k Citations
454.07 Total Impact Points

Institutions

  • 2004–2014
    • Uniformed Services University of the Health Sciences
      • Department of Dermatology
      Maryland, United States
  • 2013
    • Harvard Medical School
      • Department of Medicine
      Boston, Massachusetts, United States
  • 2011–2013
    • National Human Genome Research Institute
      Maryland, United States
  • 2005–2013
    • National Heart, Lung, and Blood Institute
      • Translational Medicine Branch
      Maryland, United States
  • 2007–2011
    • Walter Reed National Military Medical Center
      • Department of Dermatology
      Washington, Washington, D.C., United States
  • 2010
    • U.S. Department of Veterans Affairs
      Washington, Washington, D.C., United States
  • 1996–2010
    • National Institutes of Health
      • Branch of Dermatology
      Maryland, United States
  • 1997–1998
    • National Cancer Institute (USA)
      • Dermatology Branch
      Maryland, United States