Faculty Bibliography

Developmental eye defects in X-linked Ocular Albinism type I (OA1) are caused by G-Protein Coupled Receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most GPCRs, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for OA1. Many GPCRs require association with other proteins to function. These GPCR-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 co-immunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when co-expressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using Fluorescence Resonance Energy Transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis.

Atopic dermatitis is one of the most common complaints presenting to dermatologists, and patients typically inquire as to appropriate bathing recommendations. Although many dermatologists, allergists, and primary-care practitioners provide explicit bathing instructions, recommendations regarding frequency of bathing, duration of bathing, and timing related to emollient and medication application relative to bathing vary widely. Conflicting and vague guidelines stem from knowledge related to the disparate effects of water on skin, as well as a dearth of studies, especially randomized controlled trials, evaluating the effects of water and bathing on the skin of patients with atopic dermatitis. We critically review the literature related to bathing and associated atopic dermatitis treatments, such as wet wraps, bleach baths, bath additives, and balneotherapy. We aim to provide readers with a comprehensive understanding of the impact of water and related therapies on atopic dermatitis as well as recommendations based upon the published data.

Increasing percentages of children are being born to older fathers. This has resulted in concerns about the potential adverse effects of advanced paternal age. To help clinicians counsel couples, a systemic review was performed to attempt to address questions that these couples may ask: Should routine sperm testing be performed in older males? Should preimplantation genetic diagnosis (PGD) be performed? How do providers counsel patients about risk? Should young males freeze sperm if they plan to delay paternity? Using the terms "advanced paternal age", "semen testing", "preimplantation genetic diagnosis/screening", and "cryopreservation", a comprehensive search was performed in PubMed and the Cochrane Library, and numerous international societal guidelines were reviewed. In total, 42 articles or guidelines were reviewed. There were no limits placed on the timing of the articles. Thirty articles were found to be relevant and beneficial to answering the above questions. Each question was answered separately by the supporting literature. While primary research exists to support the role of semen testing, PGD/preimplantation genetic screening, and sperm banking in males who may be affected by advancing age, comprehensive studies on the possible clinical benefit of these interventions have yet to be performed. As a result, societal guidelines have yet to incorporate distinct best-practice guidelines on advanced paternal age.

Selective serotonin re-uptake inhibitors (SSRIs) are one of the most commonly prescribed antidepressants worldwide. Recent studies have linked chronic SSRI use to osteoporosis and an increased fracture risk. To date there are no studies investigating the effect of SSRIs on fracture healing. Here, we examined the effects of SSRIs on osteoprogenitor cells (OPCs) in vitro, followed by a comprehensive analysis in two murine fracture models. Bone marrow-derived OPCs were treated with varying doses of fluoxetine. Cell proliferation and differentiation were assessed with standard tests including BrdU, PCNA, qPCR for collagen type 1, runx2, osteocalcin, osteopontin, osterix, alkaline phosphatase and alizarin red staining. In vivo fracture healing was studied using a monocortical drill hole and a fracture model in adult C57/BL6 mice after a 3-week course of oral fluoxetine. Mice were euthanized at 7, 14 and 28 days post injury. In vitro treatment of primary OPCs with fluoxetine resulted in a significant reduction of proliferation compared to the control (p<=0.05). Next, we treated cells with osteogenic differentiation media +/- fluoxetine for 7 days and then performed an mineralization assay (Fig. 1A). After 7 days, we found a significant reduction in Alizarin Red staining after fluoxetine treatment (p=0.001). qPCR revealed that osteoblastic markers, such as runx2, collagen type 1, osterix, osteocalcin and ALP were downregulated in the fluoxetine treated cells (p<0.002)(Fig. 1B). Injured tibiae and femurs from control and SSRI-treated mice were analyzed by histomorphometry, microCT and molecular and cellular analyses. At all time points, the callus volume was significantly smaller in mice treated with fluoxetine, confirming the in vitro findings in these two vivo model (Fig. 1C,D). These experiments demonstrate that SSRIs inhibit osteoprogenitor cell proliferation and impedes osteogenic differentiation both in vitro and in vivo. Animal research and human clinical data have unmistakably shown that chronic SSRI use leads to osteoporosis, thus putting patients at risk for fragility fractures. If in fact SSRIs have a negative effect on bone regeneration after fracture, then this patient cohort will be prone for delayed unions and non-unions

ATG16L1T300A, a major risk polymorphism in Crohn's disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1alpha. IRE1alpha accumulates in Paneth cells of Atg16l1DeltaIEC mice, and humans homozygous for ATG16L1T300A exhibit a corresponding increase of IRE1alpha in intestinal epithelial crypts. In contrast to a protective role of the IRE1beta isoform, hyperactivated IRE1alpha also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1DeltaIEC mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1alpha, and optineurin deficiency amplifies IRE1alpha levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1DeltaIEC mice as predicted from impaired Paneth cell antimicrobial function, such structural alteration of the microbiota does not trigger ileitis but, rather, aggravates dextran sodium sulfate-induced colitis. Hence, we conclude that defective autophagy in IECs may predispose to CD ileitis via impaired clearance of IRE1alpha aggregates during ER stress at this site.

Stem cells are not resistant to the aging process. Osteoprogenitor cells (OPCs) from healthy people over age 65 make less bone compared to OPCs from people under age 35, irrespective of their sex [1]. We hypothesize that age-related chronic inflammation contributes to a decline in the osteogenic capacity of OPCs. In this study, we compared young, (1) 12 week-old mice with aged, (2) 52 week-old mice and (3) aged mice that were treated with an anti-inflammatory drug for 3 months with the goal to inhibit age-related inflammation. First, we assessed the systemic inflammatory state of these three groups and showed that a 3 months course of NSAID treatment reversed the inflammatory cytokine profile of aged mice to that of young mice (Fig.1A). Next, we quantified age-dependent alterations in proliferation and osteogenic differentiation of a pure population of OPCs from these three different groups. We used the Leptin-receptor as a marker for OPCs [2] and demonstrated an age-related decline in OPC number (Fig.1B). However, this decline was less significant in animals treated with NSAIDs. In addition, when we employed a battery of in vitro analyses to test the proliferative and osteogenic differentiation capacity of these three cell populations we learned that NSAID treatment of aged mice resulted in a reversal of the age-related decline in proliferation and osteogenic differentiation. In detail, the expression of osteocalcin, osterix and alkaline phosphatase returned to levels similar to those observed in young animals (Fig.1C). Mineralization assays showed a complete reversal of the osteogenic differentiation capacity after NSAID treatment (Fig.1D). These experiments demonstrate for the first time that age-related chronic inflammation is responsible for the decreased proliferative and osteogenic potential of aged OPCs and that this process is reversible by anti-inflammatory treatment. The findings from this study may have a profound translational impact: If we could restore the regenerative potential of the aged skeleton by treating age-related inflammation, then theoretically, we may have a tool at hand to improve the healing process of osteoporotic fracture patients

BACKGROUND:Surgical manipulation of skin may result in undesired puckering of excess tissue, which is generally assumed to settle over time. In this article, the authors address the novel question of how this excess tissue remodels. METHODS:Purse-string sutures (6-0 nylon) were placed at the midline dorsum of 22 wild-type BALB/c mice in a circular pattern marked with tattoo ink. Sutures were cinched and tied under tension in the treatment group, creating an excess tissue deformity, whereas control group sutures were tied without tension. After 2 or 4 weeks, sutures were removed. The area of tattooed skin was measured up to 56 days after suture removal. Histologic analysis was performed on samples harvested 14 days after suture removal. RESULTS:The majority of excess tissue deformities flattened within 2 days after suture removal. However, the sutured skin in the treatment group decreased in area by an average of 18 percent from baseline (n = 9), compared to a 1 percent increase in the control group (n = 10) at 14 days after suture removal (p < 0.05). This was similarly observed at 28 days (treatment, -11.7 percent; control, 4.5 percent; n = 5; p = 0.0243). Despite flattening, deformation with purse-string suture correlated with increased collagen content of skin, in addition to increased numbers of myofibroblasts. Change in area did not correlate with duration of suture placement. CONCLUSIONS:Excess dermal tissue deformities demonstrate the ability to remodel with gross flattening of the skin, increased collagen deposition, and incomplete reexpansion to baseline area. Further studies will reveal whether our findings in this mouse model translate to humans.

There are ~350 million chronic carriers of hepatitis B (HBV). While a prophylactic vaccine and drug regimens to suppress viremia are available, chronic HBV infection is rarely cured. HBV's limited host tropism leads to a scarcity of susceptible small animal models and is a hurdle to developing curative therapies. Mice that support engraftment with human hepatoctyes have traditionally been generated through crosses of murine liver injury models to immunodeficient backgrounds. Here, we describe the disruption of fumarylacetoacetate hydrolase directly in the NOD Rag1-/- IL2RgammaNULL (NRG) background using zinc finger nucleases. The resultant human liver chimeric mice sustain persistent HBV viremia for >90 days. When treated with standard of care therapy, HBV DNA levels decrease below detection but rebound when drug suppression is released, mimicking treatment response observed in patients. Our study highlights the utility of directed gene targeting approaches in zygotes to create new humanized mouse models for human diseases.

Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-beta (TGF-beta). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-beta signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.