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Item Open Access A novel mutation of the ACADM gene (c.145C>G) associated with the common c.985A>G mutation on the other ACADM allele causes mild MCAD deficiency: a case report.(Orphanet J Rare Dis, 2010-10-05) Dessein, Anne-Frédérique; Fontaine, Monique; Andresen, Brage S; Gregersen, Niels; Brivet, Michèle; Rabier, Daniel; Napuri-Gouel, Silvia; Dobbelaere, Dries; Mention-Mulliez, Karine; Martin-Ponthieu, Annie; Briand, Gilbert; Millington, David S; Vianey-Saban, Christine; Wanders, Ronald JA; Vamecq, JosephA female patient, with normal familial history, developed at the age of 30 months an episode of diarrhoea, vomiting and lethargy which resolved spontaneously. At the age of 3 years, the patient re-iterated vomiting, was sub-febrile and hypoglycemic, fell into coma, developed seizures and sequels involving right hemi-body. Urinary excretion of hexanoylglycine and suberylglycine was low during this metabolic decompensation. A study of pre- and post-prandial blood glucose and ketones over a period of 24 hours showed a normal glycaemic cycle but a failure to form ketones after 12 hours fasting, suggesting a mitochondrial β-oxidation defect. Total blood carnitine was lowered with unesterified carnitine being half of the lowest control value. A diagnosis of mild MCAD deficiency (MCADD) was based on rates of 1-14C-octanoate and 9, 10-3H-myristate oxidation and of octanoyl-CoA dehydrogenase being reduced to 25% of control values. Other mitochondrial fatty acid oxidation proteins were functionally normal. De novo acylcarnitine synthesis in whole blood samples incubated with deuterated palmitate was also typical of MCADD. Genetic studies showed that the patient was compound heterozygous with a sequence variation in both of the two ACADM alleles; one had the common c.985A>G mutation and the other had a novel c.145C>G mutation. This is the first report for the ACADM gene c.145C>G mutation: it is located in exon 3 and causes a replacement of glutamine to glutamate at position 24 of the mature protein (Q24E). Associated with heterozygosity for c.985A>G mutation, this mutation is responsible for a mild MCADD phenotype along with a clinical story corroborating the emerging literature view that patients with genotypes representing mild MCADD (high residual enzyme activity and low urinary levels of glycine conjugates), similar to some of the mild MCADDs detected by MS/MS newborn screening, may be at risk for disease presentation.Item Embargo Aging Clocks: Circadian Factors Control Antiviral Immunity of the Skin(2024) Kirchner, StephenAs human skin ages, its ability to both repair wounds and protect them from infection declines. Several factors play major roles in this, including thinning of epidermis and loss of collagen leading to skin fragility, as well as the decline of innate immune function, though the latter has been less distinctly linked to skin aging. Given the rising aging population globally, understanding how the skin responds to injury across the life spectrum is increasingly important. This work attempts to understand specifically how innate antiviral immunity of the skin is downregulated in age. To do so, we leveraged a discovery where we determined that aging skin has a differential circadian clock, a known immunological regulator, compared to that of younger skin. The circadian rhythm is a biological clock that uses a transcriptional-translational feedback loop to set up patterns of activity throughout the body. This loop uses positive transcription factors BMAL1 and CLOCK, which set up their own repressors, including the PER and CRY family of proteins. This rhythm also influences biological functions throughout the body. In the scope of this work, we became interested in the fact that circadian rhythms were found to influence epithelial repair in injury, antiviral immunity and interferon stimulated genes. To begin our study, we asked whether the known repair and immune dysfunctions of aging skin could be possibly tied to a dysfunctional circadian rhythm. Using qRT-PCR, we found that aging murine skin has a decreased circadian transcription when compared to that of younger skin. Such a phenotype was replicated by human keratinocyte studies using serial passaging as an aging surrogate. Having determined that aging does indeed play a role in regulating cutaneous circadian rhythms, we set out to determine what immune mechanisms of the skin are regulated by this aging-circadian axis. Specifically, we tested circadian regulation of antiviral proteins. Antiviral proteins of distinct families and functions all protect the skin from pathogen invasion. Prior work by our lab had shown that antiviral proteins were induced by skin wounding in a pathway dependent on the cytokine IL-27. Using approaches including qRT-PCR, flow cytometry, and immunofluorescence, we determined that aging skin wounds not only have an attenuated antiviral protein response, but also contained reduced numbers of CD301b+ immune cells that produce IL-27. These distinct immune deficiencies lead to an unprotected skin wound microenvironment in aging skin. However, little is understood about the molecular mechanisms responsible for the antiviral immune deficiencies in the aging skin. To address this, we began by probing publicly available datasets, where we found that the expression of antiviral proteins had 24-hour rhythms of expression in murine skin. Similarly, we found that that rhythmic expression of antiviral proteins occurs in human keratinocytes that were synchronized in a circadian fashion. Additional support for a direct line of circadian regulation of antiviral proteins came from circadian siRNA studies, where we knocked down expression of circadian gene CLOCK and saw an associated downregulation in antiviral proteins within human keratinocytes. Further, we demonstrate via qRT-PCR that murine skin harvested at different time points have different antiviral protein mRNA levels. Subsequent computational analysis showed that Bmal1-/- murine skin is deficient in antiviral protein expression, establishing a direct link between circadian factors and antiviral proteins. In order to better understand the effect of circadian rhythms on wound immune responses, we made use of a number of experimental models, including both Bmal1-/- and ClockΔ19 mutant mice, as well as wild type animals. We wounded wild type animals at distinct time-of-day, and found that the level of antiviral proteins display time-of-day responses, peaking at 8pm. Using circadian mutant mice, we found that these animals have attenuated wound responses with respect to antiviral protein induction; specifically, wounded ClockΔ19 mice do not produce antiviral proteins to the same extent as wild type mice. We were able to tie this directly to IL-27 signaling in two distinct manners. Firstly, using flow cytometry, we found that the CD301b+ cells that produce IL-27 in response to wounding are reduced in number in circadian mutant mouse skin, and moreover, produce less IL-27 as measured by median fluorescence intensity. To determine the role of IL-27 in the time-of-day response of wound-induced antiviral protein expression, we wounded IL-27fl/fl-LysM-Cre mice at two distinct times-of-day and measured antiviral protein production. These mice lack IL-27 production from myeloid cell lineages, including CD301b+ cells. We found that loss of IL-27 diminished time-of-day differential expression of antiviral proteins in wounds, further suggesting that the link between circadian rhythms and antiviral proteins was in fact in part mediated by IL-27. To further our understanding of the cytokine milieu of circadian wounds, we also wounded mice that lacked Type I interferon receptor (IFNAR1) expression and found that loss of Type I interferon signaling also blunted time-of-day antiviral protein responses. These data support a role of both interferons and IL-27 in circadian antiviral protein induction. In order to provide a functional aspect to these findings, we infected wildtype and circadian disrupted keratinocytes and human skin with Herpes Simplex Virus Type I (HSV1). We measured HSV expression in the skin by both immunofluorescence and visual characterization as well by qPCR for viral component UL29. We found that circadian disruption of either BMAL1 or CLOCK sensitizes keratinocytes to HSV1 infection in vitro. On the other side of this spectrum, we questioned whether circadian enhancing drugs, including the compounds SR8278 and nobiletin, can activate circadian rhythms in skin cells and improve skin defense against HSV1 infection. Using a BMAL1:Luciferase reporter, we characterized both drugs as having a circadian augmenting effect in keratinocytes. Upon infection with HSV1, both SR8278 and nobiletin protected human skin from viral spread. Further, we found that SR8278’s antiviral effect was predicated on circadian activity, as BMAL1 and CLOCK siRNA knockdown in keratinocytes lessened the drugs effect. To determine mechanism of circadian drug’s antiviral activity, we tested whether our circadian drugs activate canonical antiviral signaling pathways, such as OAS and IFITM. We found that via qPCR, circadian drugs require the presence of these proteins to fight virus effectively. As a study of clinical relevance, we evaluated the role of acyclovir treatments alongside our circadian drugs. We found that circadian drugs SR8278 and nobiletin did not synergize their effects with acyclovir at a variety of doses tested. We found that acyclovir, as expected, broadly suppressed HSV1 activity at even low doses in keratinocytes, an effect that circadian augmentation was unable to potentiate. This could be due to a number of factors, including dosage optimization and viral susceptibility to drug. However, given the rise of acyclovir resistant HSV, our novel approach may be clinically viable. In particular, we believe this may be a viable treatment platform for aging skin infections; to this end, we tested the ability of SR8278 to suppress HSV1 infection in the skin of mice over a year of age. SR8278 significantly reduced viral spread in this model, suggesting that circadian augmentation may be a useful clinical adjunct in aging skin infections. To determine if these findings were applicable to other non-herpes family viruses that infect the skin, we turned to a model of West Nile Virus infection. West Nile Virus is a mosquito borne illness with increasing range and infection number in the United States. Moreover, it is inoculated through the skin before causing neurological infection, a pathway similar to herpes viruses. Also similar to herpes viruses, West Nile virus is a far more pressing clinical issue in aging populations, who fare worse with this viral infection. Most importantly, there are no currently specific treatments for West Nile Virus infections. Using our HSV infection data as a starting point, we found that circadian drug treatments suppressed West Nile Virus levels in infected keratinocytes. Other work conducted over the course of this PhD encompassed aspects of both circadian and IL-27 signaling in the skin. Using human keratinocytes, we endeavored to understand what environmental factors could drive altered circadian rhythms in the skin, for either elderly or younger tissue. While dogmatically, circadian rhythms are patterned from the brain to the whole body, we built on recent work showing a light dependent murine cutaneous clock by showing that mock sunlight can alter circadian expression in human keratinocytes, without other stimuli present. Further study is needed to understand how our skin’s clock responds to sun mechanistically. Overall, my work over the course of this PhD has established a link between aging, circadian rhythms, and antiviral immunity, and underpinned the important role of the cytokine IL-27 and type I interferon on cutaneous wound responses to a variety of pathogens. This work will provide possible therapeutic avenues, particularly for aging skin, in how to address skin wound care in safe, biologically relevant ways via circadian rhythm exploitation.
Item Open Access Bioengineered Approaches to Prevent Hypertrophic Scar Contraction(2016) Lorden, Elizabeth RBurn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.
In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.
In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.
In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.
In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.
Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.
Item Open Access Bioluminescence imaging of glucose in tissue surrounding polyurethane and glucose sensor implants.(J Diabetes Sci Technol, 2010-09-01) Prichard, Heather L; Schroeder, Thies; Reichert, William M; Klitzman, BruceBACKGROUND: The bioluminescence technique was used to quantify the local glucose concentration in the tissue surrounding subcutaneously implanted polyurethane material and surrounding glucose sensors. In addition, some implants were coated with a single layer of adipose-derived stromal cells (ASCs) because these cells improve the wound-healing response around biomaterials. METHODS: Control and ASC-coated implants were implanted subcutaneously in rats for 1 or 8 weeks (polyurethane) or for 1 week only (glucose sensors). Tissue biopsies adjacent to the implant were immediately frozen at the time of explant. Cryosections were assayed for glucose concentration profile using the bioluminescence technique. RESULTS: For the polyurethane samples, no significant differences in glucose concentration within 100 μm of the implant surface were found between bare and ASC-coated implants at 1 or 8 weeks. A glucose concentration gradient was demonstrated around the glucose sensors. For all sensors, the minimum glucose concentration of approximately 4 mM was found at the implant surface and increased with distance from the sensor surface until the glucose concentration peaked at approximately 7 mM at 100 μm. Then the glucose concentration decreased to 5.5-6.5 mM more than 100 μmm from the surface. CONCLUSIONS: The ASC attachment to polyurethane and to glucose sensors did not change the glucose profiles in the tissue surrounding the implants. Although most glucose sensors incorporate a diffusion barrier to reduce the gradient of glucose and oxygen in the tissue, it is typically assumed that there is no steep glucose gradient around the sensors. However, a glucose gradient was observed around the sensors. A more complete understanding of glucose transport and concentration gradients around sensors is critical.Item Open Access Characterization of the Role of Transferrin receptor 1 (Tfr1) in the Intestinal Epithelium, Pancreas and Skin(2015) Chen, AlanTransferrin receptor 1 (Tfr1) serves as a receptor for transferrin, an iron-binding protein in the blood, in its canonical role of iron assimilation. Tfr1 is expressed ubiquitously in many tissues and is believed to be required for iron uptake by most cells.
The Tfr1 global knockout mouse highlights the requirement for Tfr1 in erythrocyte precursors. The erythron is the tissue with the highest iron requirement, to enable hemoglobin production. Tfr1-null embryos die by embryonic day 12.5 with anemia, which has been assumed to cause lethality of the knockout mice. Due to the embryonic lethality of the mice, the role of Tfr1 has not been well characterized in other tissues in vivo. This thesis examines the role of Tfr1 in other tissues through the generation and characterization of conditional knockout mouse models of Tfr1 deletion in the intestinal epithelium, pancreas, and skin.
Tfr1 is expressed on the basolateral surface of proliferating cells in the intestinal epithelium. Deletion of Tfr1 specifically in the intestinal epithelium resulted in the loss of intestinal epithelial homeostasis, loss of proliferation, lipid accumulation, gene expression indicating epithelial to mesenchymal transition of intestinal epithelial cells, and early neonatal lethality. These phenotypes were mostly alleviated by forced expression of a mutant Tfr1 allele which is unable to bind to iron-loaded transferrin, suggesting that Tfr1 has a novel role independent of its canonical iron-assimilatory ability.
Deletion of Tfr1 in the pancreas resulted in juvenile death due to perturbed homeostasis of both endocrine and exocrine tissues, resulting in symptoms associated with pancreatitis and diabetes. No diabetic phenotype was detected in the conditional knockout mouse model of Tfr1 deletion specifically in β-cells, suggesting that the primary effect of the loss of Tfr1 was limited to the exocrine tissue.
Deletion of Tfr1 in the epidermis of the skin caused neonatal lethality with abnormal hair follicle morphology and a significant reduction in dermal adipocytes.
These results indicate that the loss of Tfr1 has pleiotropic effects, depending on the cell type affected. Furthermore, Tfr1 appears to have non-canonical functions in the intestinal epithelium, a novel discovery.
Item Open Access Cooption of Innate Immune Cells in Promoting and Combating Infections(2018) Arifuzzaman, MohammadThe key components of innate immune defense to pathogens are various migratory as well as tissue resident innate immune cells, however, their interactions with pathogens as well as their immune-orchestrating roles are often poorly understood. While immune cells encounter pathogens at barrier sites and mount the first line of defense, pathogens are well adapted to bypass, inactivate and even exploit the functions of these cells. Better understanding of the interactions between pathogens and innate immune cells can teach us how pathogens avoid or exploit immune cells and how to overcome these mechanisms of pathogenesis by therapeutic interventions. In this work, we examined two scenarios of pathogen invasion and sought to understand the complex ways of external targeting of innate immunocytes that can either benefit the pathogen or the host.
First, we studied the migratory innate immunocytes in draining lymph nodes upon entry of Yersinia pestis via the skin and identified how this plague-causing bacterium coopted host cell death pathways of infiltrated mononuclear phagocytes. By employing time-lapse microscopy and flow cytometry, we demonstrated that within the confines of infected lymph nodes, bacteria-triggered necroptotic cell death resulted in the release of intracellular bacteria into the extracellular environment and attracted neighboring phagocytic cells, promoting their infection by these recently released bacteria. This expansion of bacteria-bearing immune cells which eventually migrate to secondary lymph nodes, enables large numbers of Y. pestis to disseminate from one node to the next via the lymphatic system. We show this mechanism of dissemination being essential for the transition of plague from a bubonic to septicemic stage and demonstrate immunotherapeutic potential of necroptosis inhibitors.
Next, we focused on mast cells, a resident innate immunocyte in the context of skin infection by Staphylococcus aureus. We showed that connective tissue mast cells promoted recruitment of neutrophils at the early stage and CD301b+ dendritic cells at the later stages of infection, which played critical roles in infection control and repair, respectively. We further demonstrated that exogenous activation of skin mast cells via a mast cell-specific G protein-coupled receptor controlled infection as well as enhanced mobilization of dendritic cells to draining lymph nodes in a mast-cell dependent manner and protected mice from re-infection. Therefore, selective activation of mast cells appears to orchestrate immunomodulation integrating both the innate and adaptive immune arms.
These studies reveal the yin and yang of innate immune cells in two very different infectious settings. They emphasize how different strategies to target these cells at the immune checkpoints can be beneficial for host-directed therapy against bacterial infections.
Item Open Access Differential labelling of UDP-N-acetylglucosamine in Huntington's-chorea fibroblasts.(Biochem J, 1981-05-15) Hung, WY; Tourian, AThe hypothesis that there is impaired endogenous synthesis of glucosamine 6-phosphate in Huntington's-chorea fibroblasts was tested by double labelling matched pairs of fibroblasts in culture with carrier-free H3 32PO4 and [U-14C]glucosamine. The [32P]UDP-N-acetyl[14C]glucosamine and [14C]glucosamine 6-[32P]phosphate of the cellular soluble fraction was isolated by charcoal column and paper chromatography. There is no quantitative difference in 32P but a significant difference in 14C in these two sugars in a ratio of approx. 1.5 for Huntington's-chorea fibroblasts compared with normal fibroblasts.Item Open Access Dual infection with Ehrlichia chaffeensis and a spotted fever group rickettsia: a case report.(Emerging infectious diseases, 1998-04) Sexton, DJ; Corey, GR; Carpenter, C; Kong, LQ; Gandhi, T; Breitschwerdt, E; Hegarty, B; Chen, SM; Feng, HM; Yu, XJ; Olano, J; Walker, DH; Dumler, SJWell-documented cases of simultaneous human infection with more than one tick-borne pathogen are rare. To our knowledge only two dual infections have been reported: simultaneous human infection with the agent of human granulocytic ehrlichiosis and Borrelia burgdorferi and simultaneous human infection with B. burgdorferi and Babesia microti (1-2). Rocky Mountain spotted fever has long been known to be endemic in North Carolina; cases of human ehrlichial infection were recognized there soon after Ehrlichia chaffeensis was recognized as an important cause of tick-borne disease in the southeastern United States. Because both Rocky Mountain spotted fever and ehrlichiosis are prevalent in North Carolina, occasional cases of simultaneous human infection by rickettsial and ehrlichial agents would not be surprising; however, no such cases seem to have been reported.Item Open Access Empiric antibiotic treatment of erythema migrans-like skin lesions as a function of geography: a clinical and cost effectiveness modeling study.(Vector Borne Zoonotic Dis, 2013-12) Lantos, Paul M; Brinkerhoff, R Jory; Wormser, Gary P; Clemen, RobertThe skin lesion of early Lyme disease, erythema migrans (EM), is so characteristic that routine practice is to treat all such patients with antibiotics. Because other skin lesions may resemble EM, it is not known whether presumptive treatment of EM is appropriate in regions where Lyme disease is rare. We constructed a decision model to compare the cost and clinical effectiveness of three strategies for the management of EM: Treat All, Observe, and Serology as a function of the probability that an EM-like lesion is Lyme disease. Treat All was found to be the preferred strategy in regions that are endemic for Lyme disease. Where Lyme disease is rare, Observe is the preferred strategy, as presumptive treatment would be expected to produce excessive harm and increased costs. Where Lyme disease is rare, clinicians and public health officials should consider observing patients with EM-like lesions who lack travel to Lyme disease-endemic areas.Item Open Access IL-27 Facilitates Skin Wound Healing through Induction of Epidermal Proliferation and Host Defense.(The Journal of investigative dermatology, 2017-05) Yang, Bin; Suwanpradid, Jutamas; Sanchez-Lagunes, Roberto; Choi, Hae Woong; Hoang, Peter; Wang, Donghai; Abraham, Soman N; MacLeod, Amanda SSkin wound repair requires a coordinated program of epithelial cell proliferation and differentiation as well as resistance to invading microbes. However, the factors that trigger epithelial cell proliferation in this inflammatory process are incompletely understood. In this study, we demonstrate that IL-27 is rapidly and transiently produced by CD301b+ cells in the skin after injury. The functional role of IL-27 and CD301b+ cells is demonstrated by the finding that CD301b-depleted mice exhibit delayed wound closure in vivo, which could be rescued by topical IL-27 treatment. Furthermore, genetic ablation of the IL-27 receptor (Il27Ra-/-) attenuates wound healing, suggesting an essential role for IL-27 signaling in skin regeneration in vivo. Mechanistically, IL-27 feeds back on keratinocytes to stimulate cell proliferation and re-epithelialization in the skin, whereas IL-27 leads to suppression of keratinocyte terminal differentiation. Finally, we identify that IL-27 potently increases expression of the antiviral oligoadenylate synthetase 2, but does not affect expression of antibacterial human beta defensin 2 or regenerating islet-derived protein 3-alpha. Together, our data suggest a previously unrecognized role for IL-27 in regulating epithelial cell proliferation and antiviral host defense during the normal wound healing response.Item Open Access Improving the lens design and performance of a contemporary electromagnetic shock wave lithotripter.(Proc Natl Acad Sci U S A, 2014-04-01) Neisius, Andreas; Smith, Nathan B; Sankin, Georgy; Kuntz, Nicholas John; Madden, John Francis; Fovargue, Daniel E; Mitran, Sorin; Lipkin, Michael Eric; Simmons, Walter Neal; Preminger, Glenn M; Zhong, PeiThe efficiency of shock wave lithotripsy (SWL), a noninvasive first-line therapy for millions of nephrolithiasis patients, has not improved substantially in the past two decades, especially in regard to stone clearance. Here, we report a new acoustic lens design for a contemporary electromagnetic (EM) shock wave lithotripter, based on recently acquired knowledge of the key lithotripter field characteristics that correlate with efficient and safe SWL. The new lens design addresses concomitantly three fundamental drawbacks in EM lithotripters, namely, narrow focal width, nonidealized pulse profile, and significant misalignment in acoustic focus and cavitation activities with the target stone at high output settings. Key design features and performance of the new lens were evaluated using model calculations and experimental measurements against the original lens under comparable acoustic pulse energy (E+) of 40 mJ. The -6-dB focal width of the new lens was enhanced from 7.4 to 11 mm at this energy level, and peak pressure (41 MPa) and maximum cavitation activity were both realigned to be within 5 mm of the lithotripter focus. Stone comminution produced by the new lens was either statistically improved or similar to that of the original lens under various in vitro test conditions and was significantly improved in vivo in a swine model (89% vs. 54%, P = 0.01), and tissue injury was minimal using a clinical treatment protocol. The general principle and associated techniques described in this work can be applied to design improvement of all EM lithotripters.Item Open Access Needle-Free Injection of Exosomes Derived from Human Dermal Fibroblast Spheroids Ameliorates Skin Photoaging.(ACS nano, 2019-10) Hu, Shiqi; Li, Zhenhua; Cores, Jhon; Huang, Ke; Su, Teng; Dinh, Phuong-Uyen; Cheng, KeHuman dermal fibroblasts (HDFs), the main cell population of the dermis, gradually lose their ability to produce collagen and renew intercellular matrix with aging. One clinical application for the autologous trans-dermis injection of HDFs that has been approved by the Food and Drug Administration aims to refine facial contours and slow down skin aging. However, the autologous HDFs used vary in quality according to the state of patients and due to many passages they undergo during expansion. In this study, factors and exosomes derived from three-dimensional spheroids (3D HDF-XOs) and the monolayer culture of HDFs (2D HDF-XOs) were collected and compared. 3D HDF-XOs expressed a significantly higher level of tissue inhibitor of metalloproteinases-1 (TIMP-1) and differentially expressed miRNA cargos compared with 2D HDF-XOs. Next, the efficacy of 3D HDF-XOs in inducing collagen synthesis and antiaging was demonstrated in vitro and in a nude mouse photoaging model. A needle-free injector was used to administer exosome treatments. 3D HDF-XOs caused increased procollagen type I expression and a significant decrease in MMP-1 expression, mainly through the downregulation of tumor necrosis factor-alpha (TNF-α) and the upregulation of transforming growth factor beta (TGF-β). In addition, the 3D-HDF-XOs group showed a higher level of dermal collagen deposition than bone marrow mesenchymal stem cell-derived exosomes. These results indicate that exosomes from 3D cultured HDF spheroids have anti-skin-aging properties and the potential to prevent and treat cutaneous aging.Item Open Access Quantifiable biomarkers of normal aging in the Japanese medaka fish (Oryzias latipes).(PLoS One, 2010-10-11) Hinton, DEBACKGROUND: Small laboratory fish share many anatomical and histological characteristics with other vertebrates, yet can be maintained in large numbers at low cost for lifetime studies. Here we characterize biomarkers associated with normal aging in the Japanese medaka (Oryzias latipes), a species that has been widely used in toxicology studies and has potential utility as a model organism for experimental aging research. PRINCIPAL FINDINGS: The median lifespan of medaka was approximately 22 months under laboratory conditions. We performed quantitative histological analysis of tissues from age-grouped individuals representing young adults (6 months old), mature adults (16 months old), and adults that had survived beyond the median lifespan (24 months). Livers of 24-month old individuals showed extensive morphologic changes, including spongiosis hepatis, steatosis, ballooning degeneration, inflammation, and nuclear pyknosis. There were also phagolysosomes, vacuoles, and residual bodies in parenchymal cells and congestion of sinusoidal vessels. Livers of aged individuals were characterized by increases in lipofuscin deposits and in the number of TUNEL-positive apoptotic cells. Some of these degenerative characteristics were seen, to a lesser extent, in the livers of 16-month old individuals, but not in 6-month old individuals. The basal layer of the dermis showed an age-dependent decline in the number of dividing cells and an increase in senescence-associated β-galactosidase. The hearts of aged individuals were characterized by fibrosis and lipofuscin deposition. There was also a loss of pigmented cells from the retinal epithelium. By contrast, age-associated changes were not apparent in skeletal muscle, the ocular lens, or the brain. SIGNIFICANCE: The results provide a set of markers that can be used to trace the process of normal tissue aging in medaka and to evaluate the effect of environmental stressors.Item Open Access Regulated spindle orientation buffers tissue growth in the epidermis.(eLife, 2019-10) Morrow, Angel; Underwood, Julie; Seldin, Lindsey; Hinnant, Taylor; Lechler, TerryTissue homeostasis requires a balance between progenitor cell proliferation and loss. Mechanisms that maintain this robust balance are needed to avoid tissue loss or overgrowth. Here we demonstrate that regulation of spindle orientation/asymmetric cell divisions is one mechanism that is used to buffer changes in proliferation and tissue turnover in mammalian skin. Genetic and pharmacologic experiments demonstrate that asymmetric cell divisions were increased in hyperproliferative conditions and decreased under hypoproliferative conditions. Further, active K-Ras also increased the frequency of asymmetric cell divisions. Disruption of spindle orientation in combination with constitutively active K-Ras resulted in massive tissue overgrowth. Together, these data highlight the essential roles of spindle orientation in buffering tissue homeostasis in response to perturbations.Item Open Access Rickettsia rickettsii transmission by a lone star tick, North Carolina.(Emerg Infect Dis, 2011-05) Breitschwerdt, Edward B; Hegarty, Barbara C; Maggi, Ricardo G; Lantos, Paul M; Aslett, Denise M; Bradley, Julie MOnly indirect or circumstantial evidence has been published to support transmission of Rickettsia rickettsii by Amblyomma americanum (lone star) ticks in North America. This study provides molecular evidence that A. americanum ticks can function, although most likely infrequently, as vectors of Rocky Mountain spotted fever for humans.Item Open Access Straight Hair, Brown Skin, and a Killa' Shape: Beauty Standards for Young Black Girls(2011-05-04) Johnson, NaomiMy goal in conducting this research is to include black adolescent women in the discourse on body image, respond to the shortcomings of the literature and research attempting to speak on behalf of black adolescent women, and empower black adolescent women to address and complicate these prevailing misconceptions by encouraging them to speak on their own behalf. Based on the qualitative research that I conducted with the 7th and 8th grade young black girls participating in “Girl Talk”, I found that there is a recognizable standard of beauty that young black girls desire to live up to. The girls identified straight, long, flowy hair, medium skin, and a curvy but skinny shape as an approved template for attaining beauty. On the other hand, the girls felt insecure when they did not have their hair done, were poorly dressed, or felt that their skin was too dark. As the girls actively negotiated black womanhood, they remained both connected and disconnected to a legacy that preceded them. The issues that the girls were facing such as a desire for agency, self-definition, inclusion and mobility were very similar to the desires of black women in the past. As the girls navigated beauty ideals, their awareness of the battle they were simultaneously fighting against racism (and the extent to which their social norms have been intoxicated with white ideals) remained vastly underdeveloped.Item Open Access The secreted metalloprotease ADAMTS20 is required for melanoblast survival.(PLoS Genet, 2008-02-29) Silver, Debra L; Hou, Ling; Somerville, Robert; Young, Mary E; Apte, Suneel S; Pavan, William JADAMTS20 (Adisintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kit(tm1Alf)/+ and bt/bt;Kitl(Sl)/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases.Item Open Access UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.(Proc Natl Acad Sci U S A, 2013-08-20) Moore, Carlene; Cevikbas, Ferda; Pasolli, H Amalia; Chen, Yong; Kong, Wei; Kempkes, Cordula; Parekh, Puja; Lee, Suk Hee; Kontchou, Nelly-Ange; Yeh, Iwei; Jokerst, Nan Marie; Fuchs, Elaine; Steinhoff, Martin; Liedtke, Wolfgang BAt our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca(2+) response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.