In vivo selection of anti-HIV-1 gene-modified human hematopoietic stem/progenitor cells to enhance engraftment and HIV-1 inhibition.

Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds great promise to eradicate HIV-1 or to provide long-term remission through a continuous supply of anti-HIV-1 gene-modified cells without ongoing antiretroviral therapy. However, achieving sufficient engraftment levels of anti-HIV gene-modified HSPC to provide therapeutic efficacy has been a major limitation. Here, we report an in vivo selection strategy for anti-HIV-1 gene-modified HSPC by introducing 6-thioguanine (6TG) chemoresistance through knocking down hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression using RNA interference (RNAi). We developed a lentiviral vector capable of co-expressing short hairpin RNA (shRNA) against HPRT alongside two anti-HIV-1 genes: shRNA targeting HIV-1 co-receptor CCR5 and a membrane-anchored HIV-1 fusion inhibitor, C46, for efficient in vivo selection of anti-HIV-1 gene-modified human HSPC. 6TG-mediated preconditioning and in vivo selection significantly enhanced engraftment of HPRT-knockdown anti-HIV-1 gene-modified cells (>2-fold, p < 0.0001) in humanized bone marrow/liver/thymus (huBLT) mice. Viral load was significantly reduced (>1 log fold, p < 0.001) in 6TG-treated HIV-1-infected huBLT mice compared to 6TG-untreated mice. We demonstrated that 6TG-mediated preconditioning and in vivo selection considerably improved engraftment of HPRT-knockdown anti-HIV-1 gene-modified HSPC and repopulation of anti-HIV-1 gene-modified hematopoietic cells in huBLT mice, allowing for efficient HIV-1 inhibition.

Production of nascent ribosome precursors within the nucleolar microenvironment of Saccharomyces cerevisiae.

35S rRNA transcripts include a 5'-external transcribed spacer followed by rRNAs of the small and large ribosomal subunits. Their processing yields massive precursors that include dozens of assembly factor proteins. In Saccharomyces cerevisiae, nucleolar assembly factors form 2 coaxial layers/volumes around ribosomal DNA. Most of these factors are cyclically recruited from a latent state to an operative state, and are extensively conserved. The layers match, at least approximately, known subcompartments found in higher eukaryotic cells. ∼80% of assembly factors are essential. The number of copies of these assembly factors is comparable to the number of nascent transcripts. Moreover, they exhibit "isoelectric balance," with RNA-binding candidate "nucleator" assembly factors being notably basic. The physical properties of pre-small subunit and pre-large subunit assembly factors are similar, as are their 19 motif signatures detected by hierarchical clustering, unlike motif signatures of the 5'-external transcribed spacer rRNP. Additionally, many assembly factors lack shared motifs. Taken together with the progression of rRNP composition during subunit maturation, and the realization that the ribosomal DNA cable is initially bathed in a subunit-nonspecific assembly factor reservoir/microenvironment, we propose a "3-step subdomain assembly model": Step (1): predominantly basic assembly factors sequentially nucleate sites along nascent rRNA; Step (2): the resulting rRNPs recruit numerous less basic assembly factors along with notably basic ribosomal proteins; Step (3): rRNPs in nearby subdomains consolidate. Cleavages of rRNA then promote release of rRNPs to the nucleoplasm, likely facilitated by the persistence of assembly factors that were already associated with nucleolar precursors.

Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias.

BACKGROUND: Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment. METHODS: Humanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias. RESULTS: A total of 897 HSPC clones were longitudinally tracked in hu-BLT mice-providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions. CONCLUSIONS: Human repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.

Fixational eye movements in passive versus active sustained fixation tasks.

Human fixational eye movements are so small and precise that high-speed, accurate tools are needed to fully reveal their properties and functional roles. Where the fixated image lands on the retina and how it moves for different levels of visually demanding tasks is the subject of the current study. An Adaptive Optics Scanning Laser Ophthalmoscope (AOSLO) was used to image, track and present a variety of fixation targets (Maltese cross, disk, concentric circles, Vernier and tumbling-E letter) to healthy subjects. During these different passive (static) or active (discriminating) tasks under natural eye motion, the landing position of the target on the retina was tracked in space and time over the retinal image directly with high spatial (<1 arcmin) and temporal (960 Hz) resolution. We computed both the eye motion and the exact trajectory of the fixated target's motion over the retina. We confirmed that compared to passive tasks, active tasks elicited a partial inhibition of microsaccades, leading to longer drift periods compensated by larger corrective saccades. Consequently, the overall fixation stability during active tasks was on average 57% larger than during passive tasks. The preferred retinal locus of fixation was the same for each task and did not coincide with the location of the peak cone density.

Evaluation of concurrent perineal hernia in adult male dogs presenting with nontraumatic, acquired inguinal hernias.

A possible association between the development of nontraumatic, acquired inguinal hernias (NAIH) and perineal hernias (PH) has been postulated in adult dogs. The objective of this study was to evaluate the frequency of concurrent diagnosis of PH in dogs presented with NAIH and determine potential risk factors for concurrent PH and NAIH. Medical records of adult male dogs presented for NAIH to 4 hospitals between 2007 and 2017 were retrospectively reviewed. Twenty-one dogs with NAIH were included, 8 of which had concurrent PH. There were no significant differences between dogs with and without PH; however, among dogs with both conditions, intact dogs (8.1 ± 1.4 years) were younger than neutered dogs (11.7 ± 1.0 years; P = 0.007). Thirty-eight percent of male dogs presenting for NAIH had concurrent PH, indicating that these conditions commonly occur together. Dogs presenting for NAIH should be carefully evaluated for concurrent PH before surgical intervention.

Évaluation d'hernie périnéale concomitante chez des chiens mâles adultes présentant des hernies inguinales acquises non traumatiques. Une association possible entre le développement d'hernies inguinales acquises non traumatiques (NAIH) et les hernies périnéales (PH) a été postulée chez les chiens adultes. L'objectif de cette étude était d'évaluer la fréquence des diagnostics simultanés d'HP chez les chiens présentés avec NAIH et de déterminer les facteurs de risque potentiels de PH et NAIH concomitantes. Les dossiers médicaux de chiens mâles adultes présentés pour NAIH à quatre hôpitaux entre 2007 et 2017 ont été revus rétrospectivement. Vingt et un chiens atteints de NAIH ont été inclus, dont huit avaient une PH concomitante. Il n'y avait aucune différence significative entre les chiens avec et sans PH; cependant, parmi les chiens atteints des deux conditions, les chiens intacts (8,1 ± 1,4 ans) étaient plus jeunes que les chiens castrés (11,7 ± 1,0 ans; P = 0,007). Trente-huit pour cent des chiens mâles se présentant pour NAIH avaient une PH concomitante, ce qui indique que ces conditions se produisent généralement ensemble. Les chiens présentant un NAIH doivent être soigneusement évalués pour une PH concomitante avant une intervention chirurgicale.(Traduit par Dr Serge Messier).

Investigation of the use of microwave ablation with and without cooling urethral perfusion for thermal ablation of the prostate gland in canine cadavers.

OBJECTIVE: To investigate the use of microwave ablation (MWA) with cooling urethral perfusion and with no perfusion (MWA-UP and MWA-NP, respectively) for prostate gland ablation in canine cadavers. ANIMALS: Cadavers of 18 sexually intact male dogs. PROCEDURES: After technique refinement in 2 cadavers, laparotomy with ultrasound-guided MWA-UP (n = 8) or MWA-NP (8) of the prostate gland was performed in 16 cadavers. Normograde cystourethroscopy was performed before and after treatment; recorded images were reviewed in a blinded manner for scoring of urethral mucosal discoloration and loss of integrity. Difficulty with cystoscope insertion was recorded if present. Excised prostate glands were fixed for serial sectioning, gross measurements, and calculation of percentage ablation. Percentages of prostate tissue necrosis from MWA, denuded urethral mucosa, and depth of epithelial surface loss in an adjacent section of the colon were estimated histologically. Variables of interest were statistically analyzed. RESULTS: Difficulty with cystoscope insertion after treatment was significantly more common and scores for urethral mucosal discoloration and loss of integrity were significantly higher (indicating more severe lesions) for the MWA-NP group than for the MWA-UP group. The histologically assessed percentage of denuded urethral mucosa was also greater for the MWA-NP group. Overall median percentage prostate gland ablation was 73%; this result was not associated with prostate gland volume or chronological order of treatment. CONCLUSIONS AND CLINICAL RELEVANCE: MWA-UP induced subtotal thermal necrosis of prostate glands in canine cadavers while limiting urethral mucosal injury. Further study is required to optimize the technique and evaluate its safety and efficacy in vivo as a future curative-intent treatment for prostatic tumors in dogs.

The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.

While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

Association between biliary tree manipulation and outcome in dogs undergoing cholecystectomy for gallbladder mucocele: A multi-institutional retrospective study.

OBJECTIVE: To determine whether catheterization of the common bile duct (CBD) is associated with outcome in dogs undergoing cholecystectomy for gallbladder mucocele and to determine whether this association is modified by the catheterization method. STUDY DESIGN: Multi-institutional retrospective cohort study. ANIMALS: Dogs (n = 252) that underwent cholecystectomy for gallbladder mucocele. METHODS: Dogs were identified via electronic medical record review at four veterinary teaching hospitals. Baseline dog characteristics, surgical findings, and methods including normograde vs retrograde CBD catheterization, intraoperative outcomes, and postoperative outcomes and complications were recorded. Variables were compared between dogs with and without catheterization. RESULTS: Catheterized dogs had higher American Society of Anesthesiologists scores (P = .04), higher total bilirubin (P = .01), and were more likely to have dilated CBD at the time of surgery (P < .01). Incidence of major and minor intraoperative complications was similar between the two groups. Surgical time was longer for the catheterized group (P = .01). The overall incidence of postoperative complications was similar between the groups; however, postoperative pancreatitis was associated with performing CBD catheterization (P = .01). This association was retained as an independent association in a multivariable model that addressed baseline group differences (P = .04). Likelihood of developing postoperative pancreatitis was not different between normograde and retrograde catheterization (P = .57). CONCLUSION: Catheterization of the CBD was associated with development of postoperative pancreatitis. This was not influenced by the method of catheterization. CLINICAL SIGNIFICANCE: The requirement for catheterization of the CBD during open cholecystectomy in dogs should be carefully considered, particularly in dogs without evidence of biliary obstruction because the procedure may induce postoperative pancreatitis.

Mechanisms of virus dissemination in bone marrow of HIV-1-infected humanized BLT mice.

Immune progenitor cells differentiate in bone marrow (BM) and then migrate to tissues. HIV-1 infects multiple BM cell types, but virus dissemination within BM has been poorly understood. We used light microscopy and electron tomography to elucidate mechanisms of HIV-1 dissemination within BM of HIV-1-infected BM/liver/thymus (BLT) mice. Tissue clearing combined with confocal and light sheet fluorescence microscopy revealed distinct populations of HIV-1 p24-producing cells in BM early after infection, and quantification of these populations identified macrophages as the principal subset of virus-producing cells in BM over time. Electron tomography demonstrated three modes of HIV-1 dissemination in BM: (i) semi-synchronous budding from T-cell and macrophage membranes, (ii) mature virus association with virus-producing T-cell uropods contacting putative target cells, and (iii) macrophages engulfing HIV-1-producing T-cells and producing virus within enclosed intracellular compartments that fused to invaginations with access to the extracellular space. These results illustrate mechanisms by which the specialized environment of the BM can promote virus spread locally and to distant lymphoid tissues.

Long-term outcome and risk factors associated with death or the need for revision surgery in dogs with permanent tracheostomies.

OBJECTIVE: To evaluate long-term outcomes and identify factors associated with death or the need for revision surgery in dogs with permanent tracheostomies (PTs). DESIGN: Retrospective cohort study. ANIMALS: 69 client-owned dogs that received a PT between January 2002 and June 2016 at 1 of 4 veterinary teaching hospitals. PROCEDURES: Medical records were reviewed, and data extracted included signalment, history, clinical signs, radiographic and laryngeal examination findings, presence of esophageal abnormalities, date and reason for receiving a PT, postoperative complications, cause of death, and survival time. Dogs surviving < 2 weeks after receiving a PT were excluded. RESULTS: Major complications occurred in 42 of 69 (61%) dogs, with aspiration pneumonia (13 [19%]), skinfold occlusion (13 [19%]), and stoma stenosis (12 [17%]) being most common. Revision surgery was performed in 24 of 69 (35%) dogs, most commonly because of stoma stenosis or skinfold occlusion (9/24 [38%] each). Brachycephalic dogs were more likely (OR, 3.5; 95% confidence interval, 1.2 to 10.2) to require revision surgery than were nonbrachycephalic dogs. The overall median survival time was 1,825 days, and dogs that received corticosteroids before receiving a PT, had tracheal collapse, or were older had shorter survival times. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study indicated that creation of a PT was a viable treatment option for obstructive upper airway diseases in dogs and that long-term survival after receiving a PT was possible; however, a PT may not reduce the risk of aspiration pneumonia in dogs.

Challenges in managing patients referred for eosinophilic esophagitis: A telephone survey and retrospective review.

Background: Eosinophilic esophagitis (EoE) in patients referred to allergists can be difficult to manage. This is due to multiple factors, including overlaps in presentation with gastroesophageal reflux disease and proton-pump inhibitor responsive eosinophilia, remaining uncertainties regarding the role of different forms of allergy testing, and a variety of patient adherence issues. Objective: To highlight, in an objectively studied fashion, complications that can be faced when managing patients referred for EoE. Methods: We conducted a telephone survey of 78 patients (pediatric and adult) who were referred to our academic allergy clinic for EoE. The survey focused on patients' perspectives regarding their symptoms and treatments. We then conducted a chart review to determine if there had been a proton-pump inhibitor (PPI) trial before diagnosis, and we compared patient responses with documented allergy test results, treatment plans, and biopsy results. Results: Only 22 of 78 patients (28%) had a ≥8-week PPI trial before diagnosis and/or referral. There was considerable variability in the type of allergy testing done for patients, and how the results were used to guide therapy. More than one-third of the patients reported being on a different treatment regimen (PPI, swallowed steroid, and/or diet) than planned, and the majority of patients on dietary therapy reported being on a different diet than planned. Also, nearly half of the adult patients did not have follow-up biopsies done despite recommendations for this. Conclusion: We identified several challenges in EoE management, including potential misdiagnosis or overtreatment, lack of standardization in testing and dietary recommendations, and patient adherence issues. We hope this information will prompt increased vigilance for these issues and promote solutions when needed.

Owner Perception of Outcome Following Permanent Tracheostomy in Dogs.

The purpose of this study was to evaluate owner perception of outcome following permanent tracheostomy (PT) in dogs. Medical records of dogs who received PT from 2002 to 2016 were reviewed. A questionnaire was given to owners verbally or by e-mail to ascertain their perception of their dog's outcome after PT. Median time to questionnaire administration from PT surgery was 608 days (64-3,708). Owner satisfaction after PT was high (89.7%), with the majority stating they would have the procedure performed again (79.5%). Owners reported an improvement in their dog's personality (30.8%) and increased activity (41%). Median survival time was 1,825 days (64-2,663), with 6 of 39 dogs (15.4%) alive at study end. Of the 33 dogs who died, 11 (33.3%) died from underlying respiratory conditions suspected to be related to the PT. The overall complication rate was 82.1%, with mucus secretion being the most common. Revision surgery was required in 30.8% of dogs (most commonly due to skin occlusion), and aspiration pneumonia occurred in 17.9% of dogs. Overall, owner satisfaction after PT in dogs is high despite intensive postoperative management, and long survival times can be achieved.

Characterization of Vancomycin Reactions and Linezolid Utilization in the Pediatric Population.

BACKGROUND: Red man syndrome (RMS) occurs because of non-IgE-mediated histamine release. Unlike vancomycin allergy, which necessitates the use of an alternative drug (often linezolid), RMS does not typically preclude further vancomycin use. Care should be taken to differentiate these reaction types from one another to prevent unnecessary vancomycin avoidance. OBJECTIVE: To characterize vancomycin reaction types in our population, and to determine whether having a reaction consistent with RMS is associated with otherwise unexplained vancomycin avoidance and linezolid use. METHODS: We retrospectively reviewed charts for children with documented vancomycin reactions. We classified the in-hospital reactions via an objective analysis and estimated the prevalence of different reaction types. We then identified children who received linezolid over 3 years, and investigated reasons for linezolid use instead of vancomycin. RESULTS: Of the 78 in-hospital reactions we characterized, 72 (92%) were objectively consistent with RMS, 5 we could not objectively classify (2 most likely RMS, 3 more suspicious for possible IgE-mediated allergy), and 1 was a non-RMS/non-IgE reaction. Of 60 children who received linezolid, 19 had previous reactions consistent with RMS, which should not preclude further vancomycin. Nevertheless, only 7 of 19 (37%) had a clear explanation for receiving linezolid instead of vancomycin compared with 32 of 39 (82%) children without previous vancomycin reactions (P < .001). CONCLUSIONS: The vast majority of patients had vancomycin reactions consistent with RMS. These patients are at risk for unnecessary vancomycin avoidance and linezolid utilization. We propose that this may be related to how reactions appear in the electronic medical record.

Pigment epithelium-derived factor restoration increases bone mass and improves bone plasticity in a model of osteogenesis imperfecta type VI via Wnt3a blockade.

Null mutations in for pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, are the cause of osteogenesis imperfecta (OI) type VI. The PEDF-knockout (KO) mouse captures crucial elements of the human disease, including diminished bone mineralization and propensity to fracture. Our group and others have demonstrated that PEDF directs human mesenchymal stem cell (hMSC) commitment to the osteoblast lineage and modulates Wnt/ß-catenin signaling, a major regulator of bone development; however, the ability of PEDF to restore bone mass in a mouse model of OI type VI has not been determined. In this study, PEDF delivery increased trabecular bone volume/total volume by 52% in 6-mo-old PEDF-KO mice but not in wild-type mice. In young (19-d-old) PEDF-KO mice, PEDF restoration increased bone volume fraction by 35% and enhanced biomechanical parameters of bone plasticity. A Wnt-green fluorescent protein reporter demonstrated dynamic changes in Wnt/ß-catenin signaling characterized by early activation and marked suppression during terminal differentiation of hMSCs. Continuous Wnt3a exposure impeded mineralization of hMSCs, whereas the combination of Wnt3a and PEDF potentiated mineralization. Interrogation of the PEDF sequence identified a conserved motif found in other Wnt modulators, such as the dickkopf proteins. Mutation of a single amino acid on a 34-mer PEDF peptide increased mineralization of hMSC cultures compared with the native peptide sequence. These results indicate that PEDF counters Wnt signaling to allow for osteoblast differentiation and provides a mechanistic insight into how the PEDF null state results in OI type VI.-Belinsky, G. S., Sreekumar, B., Andrejecsk, J. W., Saltzman, W. M., Gong, J., Herzog, R. I., Lin, S., Horsley, V., Carpenter, T. O., Chung, C. Pigment epithelium-derived factor restoration increases bone mass and improves bone plasticity in a model of osteogenesis imperfecta type VI via Wnt3a blockade.

Novel glycylated sugar alcohols protect ESC-specific microRNAs from degradation in iPS cells.

Excessive accumulation of embryonic stem cell (ESC)-specific microRNAs occurs in both ESCs and induced pluripotent stem cells (iPSC); yet, the mechanism involved is unknown. In iPSCs, we for the first time found that novel glycylated sugar alcohols, particularly glycylglycerins, are tightly bound with ESC-specific microRNA precursors (pre-miRNA), such as pre-miR-302. Among these isolated glycylglycerins, we further identified that 1,3-diglycylglycerin and 1,2,3-triglycylglycerin are two major compounds bonded with negatively charged nucleic acids via electro-affinity and subsequently forming sugar-like coats in the hairpin-like double helix structures of pre-miRNAs. As a result, such glycylglycerin-formed coating serves as a protection layer against miRNA degradation. Moreover, we found that the pH value of iPSC cytosol determines the charges of these glycylglycerins. During iPSC differentiation, the cytosol pH is increased and hence neutralizes the charges of glycylglycerins, consequently leading to fast miRNA degradation. Therefore, the current findings not only explain how ESC-specific miRNAs are preserved and accumulated in iPSCs and ESCs but also demonstrate an important function of glycylglycerins in protecting the structural integrity of highly degradable miRNAs, providing a useful means for maintaining miRNA/siRNA function as well as developing the related RNA interference (RNAi) applications.

Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis.

Immunosuppressive therapies using calcineurin inhibitors, such as cyclosporine A, are associated with a higher incidence of squamous cell carcinoma formation in mice and humans. Calcineurin is believed to suppress tumorigenesis in part through Nfatc1, a transcription factor expressed primarily in hair follicle bulge stem cells in mice. However, mice overexpressing a constitutively active Nfatc1 isoform in the skin epithelium developed increased spontaneous skin squamous cell carcinomas. Because follicular stem cells can contribute to skin tumorigenesis, whether the endogenous expression of Nfatc1 inhibits or enhances skin tumorigenesis is unclear. Here we show that loss of the endogenous expression of Nfatc1 suppresses the rate of DMBA/TPA-induced skin tumorigenesis. Inducible deletion of Nfatc1 in follicular stem cells before tumor initiation significantly reduces the rate of tumorigenesis and the contribution of follicular stem cells to skin tumors. We find that skin tumors from mice lacking Nfatc1 display reduced Hras codon 61 mutations. Furthermore, Nfatc1 enhances the expression of genes involved in DMBA metabolism and increases DMBA-induced DNA damage in keratinocytes. Together these data implicate Nfatc1 in the regulation of skin stem cell-initiated tumorigenesis via the regulation of DMBA metabolism.

Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

A review of the evidence linking eosinophilic esophagitis and food allergy.

Eosinophilic esophagitis (EoE) is a chronic inflammation of the esophagus that has been considered an allergic phenomenon based on its similarities to other allergic conditions. More specifically, EoE has been considered a form of food allergy because of patient sensitizations to foods and improvements in symptoms and inflammation after food eliminations. This article presents the currently available evidence regarding the classification of EoE as an allergic condition, the involvement of foods in disease pathogenesis, and the value of different types of allergy testing and elimination diets in management of EoE. Using the search engines PubMed and Ovid, English literature in the past 10 years was reviewed with the use of the following key words: eosinophilic esophagitis, EoE epidemiology, EoE pathophysiology, food allergy, eosinophils, skin-prick testing, atopy patch testing, elemental diet, test directed elimination diet, six food elimination diet. Studies of EoE epidemiology and pathophysiology support the link between EoE and allergy in general, and studies of food allergy testing and elimination diets have supported a link between EoE and food allergy. Although food elimination diets cause resolution of symptoms and pathology in pediatric EoE, the results of testing and diet elimination studies are not as clear in adults, and aeroallergen sensitizations may play a larger role in adult EoE pathophysiology. Although several studies in children and adults support considering EoE a form of food allergy, the usefulness of skin-prick testing and atopy patch testing for food allergies and the optimal elimination diet for disease management are still uncertain.

Alteration of the EphA2/Ephrin-A signaling axis in psoriatic epidermis.

EphA2 is a receptor tyrosine kinase (RTK) that triggers keratinocyte differentiation upon activation and subsequent downregulation by ephrin-A1 ligand. The objective of this study was to determine whether the EphA2/ephrin-A1 signaling axis was altered in psoriasis, an inflammatory skin condition in which keratinocyte differentiation is abnormal. Microarray analysis of skin biopsies from psoriasis patients revealed increased mRNA transcripts for several members of this RTK family in plaques, including the EphA1, EphA2, and EphA4 subtypes prominently expressed by keratinocytes. Of these, EphA2 showed the greatest upregulation, a finding that was confirmed by quantitative reverse-transcriptase-PCR, immunohistochemistry (IHC), and ELISA. In contrast, psoriatic lesions exhibited reduced ephrin-A ligand immunoreactivity. Exposure of primary keratinocytes induced to differentiate in high calcium or a three-dimensional (3D) raft culture of human epidermis to a combination of growth factors and cytokines elevated in psoriasis increased EphA2 mRNA and protein expression while inducing S100A7 and disrupting differentiation. Pharmacological delivery of a soluble ephrin-A1 peptidomimetic ligand led to a reduction in EphA2 expression and ameliorated proliferation and differentiation in raft cultures exposed to EGF and IL-1α. These findings suggest that ephrin-A1-mediated downregulation of EphA2 supports keratinocyte differentiation in the context of cytokine perturbation.

Eph/ephrin signaling in epidermal differentiation and disease.

Eph receptor tyrosine kinases mediate cell-cell communication by interacting with ephrin ligands residing on adjacent cell surfaces. In doing so, these juxtamembrane signaling complexes provide important contextual information about the cellular microenvironment that helps orchestrate tissue morphogenesis and maintain homeostasis. Eph/ephrin signaling has been implicated in various aspects of mammalian skin physiology, with several members of this large family of receptor tyrosine kinases and their ligands present in the epidermis, hair follicles, sebaceous glands, and underlying dermis. This review focuses on the emerging role of Eph receptors and ephrins in epidermal keratinocytes where they can modulate proliferation, migration, differentiation, and death. The activation of Eph receptors by ephrins at sites of cell-cell contact also appears to play a key role in the maturation of intercellular junctional complexes as keratinocytes move out of the basal layer and differentiate in the suprabasal layers of this stratified, squamous epithelium. Furthermore, alterations in the epidermal Eph/ephrin axis have been associated with cutaneous malignancy, wound healing defects and inflammatory skin conditions. These collective observations suggest that the Eph/ephrin cell-cell communication pathway may be amenable to therapeutic intervention for the purpose of restoring epidermal tissue homeostasis and integrity in dermatological disorders.