Publications by authors named "Daniel L Popkin"

27 Publications

  • Page 1 of 1

A Protease-Activated Fluorescent Probe Allows Rapid Visualization of Keratinocyte Carcinoma during Excision.

Cancer Res 2020 05 4;80(10):2045-2055. Epub 2020 Mar 4.

Department of Dermatology, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio.

Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-3067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7231632PMC
May 2020

The use of 3-dimensionally printed models to optimize patient education and alleviate perioperative anxiety in Mohs micrographic surgery: A randomized controlled trial.

J Am Acad Dermatol 2019 Dec 1;81(6):1339-1345. Epub 2019 Jun 1.

Case Western Reserve University School of Medicine, Cleveland, Ohio; Department of Dermatology, University Hospitals Cleveland Medical Center, Cleveland, Ohio.

Background: Perioperative patient anxiety in Mohs micrographic surgery (MMS) is associated with increased postoperative pain and decreased satisfaction.

Objective: To determine whether a 3-dimensionally printed MMS model with standardized education (SE) improves perioperative patient understanding and anxiety.

Methods: An unblinded, randomized controlled trial was conducted, with patients randomly assigned to receive the MMS model plus SE or SE alone. Baseline and poststage understanding and anxiety were evaluated with the Visual Analog Scale (VAS) and State-Trait Anxiety Inventory (STAI). Additionally, patients completed a 6-item knowledge assessment.

Results: Eighty-two patients were enrolled, 42 in the MMS model and 40 in the SE group, with similar group mean age (67.8 years), sex (59.8% male), and previous MMS experience (47.6%). Both groups experienced significant reductions in VAS anxiety and State-Trait Anxiety Inventory scores and significant increases in VAS understanding. Compared with SE alone, the MMS model group had larger VAS anxiety reduction (change, -1.31; approaching significance) than the SE group (change, -0.52; P = .052) and 5.59 (93.25%) correct responses versus 5.15 (85.83%) correct responses in the SE group (P < .028).

Limitations: Overestimations of baseline patient anxiety in our population and 91.1% recruitment of the intended study population limited study power.

Conclusion: A 3-dimensionally printed MMS model with SE may improve patient understanding of MMS and decrease perioperative anxiety.
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http://dx.doi.org/10.1016/j.jaad.2019.05.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031844PMC
December 2019

Molecular imaging and validation of margins in surgically excised nonmelanoma skin cancer specimens.

J Med Imaging (Bellingham) 2019 Jan 18;6(1):016001. Epub 2019 Mar 18.

Case Western Reserve University, Department of Biomedical Engineering, Cleveland, Ohio, United States.

In an effort to increase the efficiency and cure rate of nonmelanoma skin cancer (NMSC) excisions, we have developed a point-of-care method of imaging and evaluation of skin cancer margins. We evaluate the skin surgical specimens using a smart, near-infrared probe (6qcNIR) that fluoresces in the presence of cathepsin proteases overexpressed in NMSC. Imaging is done with an inverted, flying-spot fluorescence scanner that reduces scatter, giving a 70% improved step response as compared to a conventional imaging system. We develop a scheme for careful comparison of fluorescent signals to histological annotation, which involves image segmentation, fiducial-based registration, and nonrigid free-form deformation on fluorescence images, corresponding color images, "bread-loafed" tissue images, hematoxylin and eosin (H&E)-stained slides, and pathological annotations. From epidermal landmarks, spatial accuracy in the bulk of the sample is , which when extrapolated with a linear stretch model, suggests an error at the margin of , within clinical reporting standards. Cancer annotations on H&E slides are transformed and superimposed on the fluorescence images to generate the final results. Using this methodology, fluorescence cancer signals are generally found to correspond spatially with histological annotations. This method will allow us to accurately analyze molecular probes for imaging skin cancer margins.
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http://dx.doi.org/10.1117/1.JMI.6.1.016001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423324PMC
January 2019

Soluble Fc-Disabled Herpes Virus Entry Mediator Augments Activation and Cytotoxicity of NK Cells by Promoting Cross-Talk between NK Cells and Monocytes.

J Immunol 2019 04 15;202(7):2057-2068. Epub 2019 Feb 15.

Department of Dermatology, Case Western Reserve University School of Medicine, Cleveland, OH 44106;

CD160 is highly expressed by NK cells and is associated with cytolytic effector activity. Herpes virus entry mediator (HVEM) activates NK cells for cytokine production and cytolytic function via CD160. Fc-fusions are a well-established class of therapeutics, where the Fc domain provides additional biological and pharmacological properties to the fusion protein including enhanced serum and interaction with Fc receptor-expressing immune cells. We evaluated the specific function of HVEM in regulating CD160-mediated NK cell effector function by generating a fusion of the HVEM extracellular domain with human IgG1 Fc bearing CD16-binding mutations (Fc*) resulting in HVEM-(Fc*). HVEM-(Fc*) displayed reduced binding to the Fc receptor CD16 (i.e., Fc-disabled HVEM), which limited Fc receptor-induced responses. HVEM-(Fc*) functional activity was compared with HVEM-Fc containing the wild type human IgG1 Fc. HVEM-(Fc*) treatment of NK cells and PBMCs caused greater IFN-γ production, enhanced cytotoxicity, reduced NK fratricide, and no change in CD16 expression on human NK cells compared with HVEM-Fc. HVEM-(Fc*) treatment of monocytes or PBMCs enhanced the expression level of CD80, CD83, and CD40 expression on monocytes. HVEM-(Fc*)-enhanced NK cell activation and cytotoxicity were promoted via cross-talk between NK cells and monocytes that was driven by cell-cell contact. In this study, we have shown that soluble Fc-disabled HVEM-(Fc*) augments NK cell activation, IFN-γ production, and cytotoxicity of NK cells without inducing NK cell fratricide by promoting cross-talk between NK cells and monocytes without Fc receptor-induced effects. Soluble Fc-disabled HVEM-(Fc*) may be considered as a research and potentially therapeutic reagent for modulating immune responses via sole activation of HVEM receptors.
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http://dx.doi.org/10.4049/jimmunol.1801449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424646PMC
April 2019

Evidence That Combination Therapy With CD16-Bearing NK-92 Cells and FDA-Approved Alefacept Can Selectively Target the Latent HIV Reservoir in CD4+ CD2hi Memory T Cells.

Front Immunol 2018 5;9:2552. Epub 2018 Nov 5.

Department of Dermatology, Case Western Reserve University School of Medicine, Cleveland, OH, United States.

Elimination of the latent HIV reservoir remains the biggest hurdle to achieve HIV cure. In order to specifically eliminate HIV infected cells they must be distinguishable from uninfected cells. CD2 was recently identified as a potential marker enriched in the HIV-1 reservoir on CD4+ T cells, the largest, longest-lived and best-characterized constituent of the HIV reservoir. We previously proposed to repurpose FDA-approved alefacept, a humanized α-CD2 fusion protein, to reduce the HIV reservoir in CD2hi CD4+ memory T cells. Here, we show the first evidence that alefacept can specifically target and reduce CD2hi HIV infected cells . We explore a variety of natural killer (NK) cells as mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) including primary NK cells, expanded NK cells as well as the CD16 transduced NK-92 cell line which is currently under study in clinical trials as a treatment for cancer. We demonstrate that CD16.NK-92 has a natural preference to kill CD2hi CD45RA- memory T cells, specifically CD45RA- CD27+ central memory/transitional memory (T) subset in both healthy and HIV patient samples as well as to reduce HIV DNA from HIV samples from donors well controlled on antiretroviral therapy. Lastly, alefacept can combine with CD16.NK-92 to decrease HIV DNA in some patient samples and thus may yield value as part of a strategy toward sustained HIV remission.
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http://dx.doi.org/10.3389/fimmu.2018.02552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230627PMC
September 2019

Soluble Markers of Immune Activation Differentially Normalize and Selectively Associate with Improvement in AST, ALT, Albumin, and Transient Elastography During IFN-Free HCV Therapy.

Pathog Immun 2018 Sep;3(1):149-163

The Louis Stokes VA Medical Center, Cleveland, Ohio.

Background: During chronic hepatitis C virus (HCV) infection, Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) levels mark active liver inflammation and tissue damage, while albumin reflects synthetic liver function and nutritional status. Transient Elastography (TE) is a clinical measure of liver stiffness that facilitates evaluation of liver damage stage. While a portion of the TE score is attributable to liver fibrosis and relatively irreversible damage, another component of the TE score is attributable to liver inflammation or edema. Markers of inflammation during chronic HCV infection include soluble markers of immune activation, which are also associated with morbid outcome (including cardiovascular disease and liver-disease progression). Whether soluble markers of immune activation or changes in their level during HCV therapy relate to normalization of AST, ALT, Albumin, or TE score, is not clear.

Methods: We evaluated soluble markers of immune activation (plasma sCD14, IL-6, sCD163, autotaxin [ATX], and Mac2BP) and TE score, and their relationship in 20 HCV-infected patients before, during, and after HCV-directed IFN-free direct-acting antiviral (DAA) therapy. We evaluated normalization of parameters and the relationship between each over a 6-month window.

Results: Before therapy, serum AST levels positively correlated with plasma levels of sCD14, sCD163, and Mac2BP, while ALT levels positively correlated with Mac2BP. Serum albumin level negatively correlated with plasma IL-6 and ATX levels. IFN-free therapy uniformly resulted in sustained virological response at 12 and 24 weeks after therapy completion. After initiation of therapy AST and ALT normalized, while levels of ATX, Mac2BP, sCD163, and TE score partially normalized over 6 months. Additionally, change in AST level and APRI score correlated with change in sCD163, IL-6, and Mac2BP levels, and change in ALT correlated with change in IL-6 and Mac2BP levels. Improvement in TE score correlated with a decrease in the level of sCD14 at week 4, and almost statistically significant with decrease in sCD14 at weeks 20-24 after initiation of IFN-free HCV therapy.

Conclusions: Soluble markers of immune activation normalize or partially normalize at different rates after initiation of curative HCV DAA therapy, and TE scores improve, with wide variability in the degree of absolute improvement in liver stiffness from patient to patient. Decline magnitude of sCD14 was associated with improvement in TE score, while magnitude of improvement in AST correlated with reduction in sCD163 levels. These data provide support for a model where monocyte/Kupffer cell activation may account for a portion of the liver inflammation and edema, which is at least partially reversible following initiation of HCV DAA therapy.
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http://dx.doi.org/10.20411/pai.v3i1.242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201254PMC
September 2018

A requirement for slc15a4 in imiquimod-induced systemic inflammation and psoriasiform inflammation in mice.

Sci Rep 2018 09 27;8(1):14451. Epub 2018 Sep 27.

Department of Dermatology, Case Western Reserve University Hospitals, Cleveland, OH, 44106, USA.

There is competing evidence that plasmacytoid dendritic cells (pDC), the most potent source of IFN-I, may initiate psoriasis. We targeted pDC function using the slc15a4 loss-of-function mouse whose pDC are unresponsive to TLR agonists. slc15a4 treated with the topical TLR7-agonist imiquimod (IMQ) demonstrated decreased epidermal thickening 24 hours post-treatment which was more pronounced by day 5 as compared to wildtype mice. These findings were specific to the acute IMQ model and not the protracted IL23 model that drives inflammation downstream of TLR activation. Systemically, slc15a4 was required for IMQ-induced weight loss and cutaneous accumulation of CD4+ and Siglec H+, but not CD11b+ cells. Consistent with this phenotype and the function of slc15a4, induction of IFN-I was virtually absent systemically and via cutaneous gene expression. Induction of other inflammatory cytokines (cytokine storm) was modestly blunted in slc15a4 except for inflammasome-associated genes consistent with slc15a4 being required for TLR7-mediated (but not inflammasome-mediated) inflammation downstream of IMQ. Surprisingly, only IFN-I gene expression was suppressed within IMQ-treated skin. Other genes including conserved psoriasiform trademark gene expression were augmented in slc15a4 versus littermate controls. Taken together, we have identified a role for slc15a4 but not canonical psoriasiform genes in the imiquimod model of psoriasiform dermatitis.
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http://dx.doi.org/10.1038/s41598-018-32668-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160456PMC
September 2018

Characterization of the facial microbiome in twins discordant for rosacea.

Exp Dermatol 2018 03;27(3):295-298

Department of Dermatology, Case Western Reserve University, Cleveland, OH, USA.

Previously, we determined that genetic and environmental factors contributed equally towards rosacea in twins. To assess an environmental factor, we characterized the malar cheek bacterial microbiome from twins discordant for rosacea. We found no significant difference in facial microbiome alpha and beta diversity between related twins discordant for rosacea. However, the relative percentage abundance of Gordonia and Geobacillus, low-abundant genera, was positively and negatively associated with rosacea severity, respectively. Our data demonstrate a significant correlation between facial microbiome and severity of rosacea in genetically matched twins and importantly that overall microbiome composition is largely unchanged.
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http://dx.doi.org/10.1111/exd.13491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635131PMC
March 2018

Pre-vaccine plasma levels of soluble inflammatory indices negatively predict responses to HAV, HBV, and tetanus vaccines in HCV and HIV infection.

Vaccine 2018 01 16;36(4):453-460. Epub 2017 Dec 16.

The Louis Stokes VA Medical Center, Cleveland, OH, USA; Department of Medicine, University Hospitals Medical Center, and the Center for AIDS Research, Case Western Reserve University, Cleveland, OH, USA. Electronic address:

Background: Chronic hepatitis C virus (HCV) and HIV infections are associated with impaired responses to neo-antigens contained in hepatitis A virus (HAV)/hepatitis B virus (HBV) vaccines, yet responsible mechanisms are unclear.

Methods: ACTG 5232 and CFAR0910 were clinical trials where pre-vaccine levels of plasma IP10, IL-6, sCD163 and sCD14 were measured in viremic HCV- (n = 15) or HIV-infected participants (n = 24) and uninfected controls (n = 10). Accelerated dosing HAV/HBV vaccine and tetanus booster were administered and antibody response was measured at 0, 1, 3, 8, and 24 weeks.

Results: Pre-vaccine plasma IP10, IL-6, and sCD14 levels were elevated in both HCV and HIV-infected participants, while sCD163 was also elevated in HCV-infected participants. Pre-immunization tetanus antibody levels were lower in HIV-infected than in uninfected participants, while vaccine induced antibody responses were intact in HCV and HIV-infected participants. After HAV/HBV vaccination, HCV and HIV-infected participants had lower and less durable HAV and HBV antibody responses than uninfected controls. Among HCV-infected participants, pre-vaccine plasma IP10, IL-6, sCD14, and sCD163 levels inversely correlated with HAV, HBV and tetanus antibody responses after vaccine. Low HAV/HBV vaccine responses in HIV-infected participants prohibited assessment of immune correlates.

Conclusions: During HCV and HIV infection markers of systemic inflammation reflect immune dysfunction as demonstrated by poor response to HAV/HBV neo-antigen vaccine.
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http://dx.doi.org/10.1016/j.vaccine.2017.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767517PMC
January 2018

Cetylpyridinium Chloride (CPC) Exhibits Potent, Rapid Activity Against Influenza Viruses and .

Pathog Immun 2017 26;2(2):252-269. Epub 2017 Jun 26.

Division of Pediatric Infectious Diseases, University Hospitals Cleveland Medical Center and Case Western Reserve University, Cleveland, Ohio.

Background: There is a continued need for strategies to prevent influenza. While cetylpyridinium chloride (CPC), a broad-spectrum antimicrobial agent, has an extensive antimicrobial spectrum, its ability to affect respiratory viruses has not been studied in detail.

Objectives: Here, we evaluate the ability of CPC to disrupt influenza viruses and .

Methods: The virucidal activity of CPC was evaluated against susceptible and oseltamivir-resistant strains of influenza viruses. The effective virucidal concentration (EC) of CPC was determined using a hemagglutination assay and tissue culture infective dose assay. The effect of CPC on viral envelope morphology and ultrastructure was evaluated using transmission electron microscopy (TEM). The ability of influenza virus to develop resistance was evaluated after multiple passaging in sub-inhibitory concentrations of CPC. Finally, the efficacy of CPC in formulation to prevent and treat influenza infection was evaluated using the PR8 murine influenza model.

Results: The virucidal effect of CPC occurred within 10 minutes, with mean EC and EC ranging between 5 to 20 μg/mL, for most strains of influenza tested regardless of type and resistance to oseltamivir. Examinations using TEM showed that CPC disrupted the integrity of the viral envelope and its morphology. Influenza viruses demonstrated no resistance to CPC despite prolonged exposure. Treated mice exhibited significantly increased survival and maintained body weight compared to untreated mice.

Conclusions: The antimicrobial agent CPC possesses virucidal activity against susceptible and resistant strains of influenza virus by targeting and disrupting the viral envelope. Substantial virucidal activity is seen even at very low concentrations of CPC without development of resistance. Moreover, CPC in formulation reduces influenza-associated mortality and morbidity .
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http://dx.doi.org/10.20411/pai.v2i2.200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605151PMC
June 2017

Overexpression of AQP3 and AQP10 in the skin exacerbates psoriasiform acanthosis.

Exp Dermatol 2017 10 21;26(10):949-951. Epub 2017 Apr 21.

Department of Dermatology, Case Western Reserve University, Cleveland, OH, USA.

We previously observed that aquaporin-3 and aquaporin-10 are upregulated in the epidermis of hand dermatitis patients (Med. Hypotheses, 84, 2015, 498). To address the functional relevance of this upregulation, we overexpressed AQP3/AQP10 in mice using the human K1 promoter. Combining imiquimod with detergent-containing water challenge, a common trigger in hand and other dermatitis, resulted in an increase in acanthosis in mice overexpressing AQP3 or AQP3 and AQP10. Aquaporin overexpression also drove a trend towards greater weight loss in these animals. These data support a role for cutaneous aquaporins in the pathogenesis of dermatitis and as a potential target in their treatment.
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http://dx.doi.org/10.1111/exd.13307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577374PMC
October 2017

During Hepatitis C Virus (HCV) Infection and HCV-HIV Coinfection, an Elevated Plasma Level of Autotaxin Is Associated With Lysophosphatidic Acid and Markers of Immune Activation That Normalize During Interferon-Free HCV Therapy.

J Infect Dis 2016 Nov 17;214(9):1438-1448. Epub 2016 Aug 17.

Department of Medicine.

Background:  Immune activation predicts morbidity during hepatitis C virus (HCV) infection and human immunodeficiency virus (HIV) infection, although mechanisms underlying immune activation are unclear. Plasma levels of autotaxin and its enzymatic product, lysophosphatidic acid (LPA), are elevated during HCV infection, and LPA activates immunocytes, but whether this contributes to immune activation is unknown.

Methods:  We evaluated plasma levels of autotaxin, interleukin 6 (IL-6), soluble CD14 (sCD14), soluble CD163 (sCD163), and Mac2 binding protein (Mac2BP) during HCV infection, HIV infection, and HCV-HIV coinfection, as well as in uninfected controls, before and after HIV antiretroviral therapy (ART) initiation and during interferon-free HCV therapy.

Results:  We observed greater plasma autotaxin levels in HCV-infected and HCV-HIV-coinfected participants, compared with uninfected participants, primarily those with a higher ratio of aspartate aminotransferase level to platelet count. Autotaxin levels correlated with IL-6, sCD14, sCD163, Mac2BP, and LPA levels in HCV-infected participants and with Mac2BP levels in HCV-HIV-coinfected participants, while in HIV-infected individuals, sCD14 levels correlated with Mac2BP levels. Autotaxin, LPA, and sCD14 levels normalized, while sCD163 and Mac2BP levels partially normalized within 6 months of starting interferon-free HCV therapy. sCD163 and IL-6 levels normalized within 6 months of starting ART for HIV infection. In vitro, LPA activated monocytes.

Conclusions:  These data indicate that elevated levels of autotaxin and soluble markers of immune activation during HCV infection are partially reversible within 6 months of initiating interferon-free HCV treatment and that autotaxin may be causally linked to immune activation during HCV infection and HCV-HIV coinfection.
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http://dx.doi.org/10.1093/infdis/jiw372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281372PMC
November 2016

Tropism of CPMV to Professional Antigen Presenting Cells Enables a Platform to Eliminate Chronic Infections.

ACS Biomater Sci Eng 2015 Nov 20;1(11):1050-1054. Epub 2015 Oct 20.

Department of Dermatology, Case Western Reserve University Hospitals, Cleveland, Ohio 44106, United States; Department of Pathology, Case Western Reserve University Hospitals, Cleveland, Ohio 44106, United States; Department of Molecular Biology and Microbiology, Case Western Reserve University Hospitals, Cleveland, Ohio 44106, United States.

Chronic viral infections (e.g., HIV, HBV, HCV) represent a significant source of morbidity and mortality with over 500 million people infected worldwide. Dendritic cells (DCs) and macrophages are key cell types for productive viral replication and persistent systemic infection. We demonstrate that the plant virus cowpea mosaic virus (CPMV) displays tropism for such antigen presenting cells in both mice and humans, thus making it an ideal candidate for targeted drug delivery toward viral infections. Furthermore, we show inhibition of a key host protein for viral infection, site-1 protease (S1P), using the small molecule PF-429242 in the model pathogen arenavirus lymphocytic choriomeningitis virus (LCMV) limits viral growth. By packaging PF-429242 in CPMV, we are able to control drug release and efficiently deliver the drug. This sets the groundwork for utilizing the natural tropism of CPMV for a therapeutic approach that specifically targets cell types most commonly subverted by chronic viruses.
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http://dx.doi.org/10.1021/acsbiomaterials.5b00344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894745PMC
November 2015

Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection.

Proc Natl Acad Sci U S A 2015 Oct 5;112(42):E5706-14. Epub 2015 Oct 5.

Department of Genetics, The Scripps Research Institute, La Jolla, CA 92037; Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390

Endoplasmic reticulum (ER)-resident proteins are continually retrieved from the Golgi and returned to the ER by Lys-Asp-Glu-Leu (KDEL) receptors, which bind to an eponymous tetrapeptide motif at their substrate's C terminus. Mice and humans possess three paralogous KDEL receptors, but little is known about their functional redundancy, or if their mutation can be physiologically tolerated. Here, we present a recessive mouse missense allele of the prototypical mammalian KDEL receptor, KDEL ER protein retention receptor 1 (KDELR1). Kdelr1 homozygous mutants were mildly lymphopenic, as were mice with a CRISPR/Cas9-engineered frameshift allele. Lymphopenia was cell intrinsic and, in the case of T cells, was associated with reduced expression of the T-cell receptor (TCR) and increased expression of CD44, and could be partially corrected by an MHC class I-restricted TCR transgene. Antiviral immunity was also compromised, with Kdelr1 mutant mice unable to clear an otherwise self-limiting viral infection. These data reveal a nonredundant cellular function for KDELR1, upon which lymphocytes distinctly depend.
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http://dx.doi.org/10.1073/pnas.1515619112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620900PMC
October 2015

Genetic vs Environmental Factors That Correlate With Rosacea: A Cohort-Based Survey of Twins.

JAMA Dermatol 2015 Nov;151(11):1213-9

Department of Dermatology, University Hospitals Case Medical Center, Cleveland, Ohio3Department of Pathology, Case Western Reserve University, Cleveland, Ohio4Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Oh.

Importance: To our knowledge, this is the first study on rosacea to formally define genetic and environmental contributions.

Objectives: To study a cohort of identical and fraternal twins to determine whether genetic factors contribute to rosacea development and, if genetic factors are present, quantitatively estimate the genetic contribution, as well as to identify environmental factors that correlate with rosacea by controlling for genetic susceptibility.

Design, Setting, And Participants: Identical and fraternal twins were surveyed regarding risk factors implicated in rosacea. Faculty dermatologists determined a rosacea score for each twin participant according to the National Rosacea Society (NRS) grading system. Data were collected at the annual Twins Days Festival in Twinsburg, Ohio, on August 4-5, 2012, and August 2-3, 2013. Analysis was conducted for several months after each meeting. A cohort of 550 twin individuals, with most from Ohio, Pennsylvania, and the northeastern United States, participated.

Main Outcomes And Measures: The NRS score and rosacea subtype were assessed using the NRS grading system and physical examination by board-certified dermatologists.

Results: Among the 275 twin pairs (550 individuals), there were 233 identical twin pairs with a mean rosacea score of 2.46 and 42 fraternal twin pairs with a mean rosacea score of 0.75. We observed a higher association of NRS scores between identical vs fraternal twins (r = 0.69 vs r = 0.46; P = .04), demonstrating a genetic contribution. Using the ACE model (proportion of variance in a trait heritable secondary to additive genetics [A] vs the proportions due to a common environment [C] and unique environment [E]), we calculated this genetic contribution to be 46%. A higher NRS score was also significantly associated with the following factors: age (r = 0.38; P < .001) and lifetime UV radiation exposure (r = 0.26; P < .001). These associations remained after use of propensity score matching to adjust for multicollinearity. Other correlated variables included body mass index (r = 0.21; P < .001), smoking (r = 0.10; P < .02), alcohol consumption (r = 0.11; P = .01), cardiovascular comorbidity (r = 0.17; P < .001), and skin cancer comorbidity (r = 0.19; P < .001).

Conclusions And Relevance: The study of twins allows us to separate genetic susceptibility and the influence of environmental factors affecting rosacea. We found that approximately half of the contribution to the NRS score could be accounted for by genetics and the other half by environment. We identified correlations between rosacea and UV radiation exposure, alcohol, smoking, skin cancer history, cardiac comorbidity, and age. These findings may help improve current management and expectations of individuals affected by rosacea.
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http://dx.doi.org/10.1001/jamadermatol.2015.2230DOI Listing
November 2015

Langerhans Cell Hyperplasia From Molluscum Contagiosum.

Am J Dermatopathol 2015 Aug;37(8):e93-5

Department of Dermatology, Case Western Reserve University, Cleveland, OH.

Langerhans cell histiocytosis (LCH) carries a prognosis, which ranges from benign to potentially fatal. There is currently little framework to decipher metrics, which predict the benign versus aggressive nature of LCH. We wanted to determine whether molluscum contagiosum virus (MCV) DNA could be isolated from a cutaneous lesion, demonstrating Langerhans cell hyperplasia resembling LCH in a patient with both. Polymerase chain reaction on biopsy-proven MCV and the hyperplastic lesion has been performed. Two specific regions within the MCV genome were detected from both biopsies. The authors report our findings and suggest that some MCV can produce histological lesions resembling LCH, similar to the literature on scabies mimicking LCH. Efforts to find a reactive "driver" in LCH may significantly inform the clinical scenario.
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http://dx.doi.org/10.1097/DAD.0000000000000201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334759PMC
August 2015

Role of lymphocytic choriomeningitis virus (LCMV) in understanding viral immunology: past, present and future.

Viruses 2012 Oct 29;4(11):2650-69. Epub 2012 Oct 29.

Department of Dermatology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.

Lymphocytic choriomeningitis virus (LCMV) is a common infection of rodents first identified over eighty years ago in St. Louis, MO, U.S.A. It is best known for its application in immunological studies. The history of LCMV closely correlates with the development of modern immunology. With the use of LCMV as a model pathogen several key concepts have emerged: Major Histocompatibility Complex (MHC) restriction, T cell memory, persistent infections, T cell exhaustion and the key role of immune pathology in disease. Given the phenomenal infrastructure within this field (e.g., defined immunodominant and subdominant epitopes to all T cell receptor specificities as well as the cognate tetramers for enumeration in vivo) the study of LCMV remains an active and productive platform for biological research across the globe to this day. Here we present a historical primer that highlights several breakthroughs since the discovery of LCMV. Next, we highlight current research in the field and conclude with our predictions for future directions in the remarkable field of LCMV research.
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http://dx.doi.org/10.3390/v4112650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509666PMC
October 2012

ENU-induced phenovariance in mice: inferences from 587 mutations.

BMC Res Notes 2012 Oct 24;5:577. Epub 2012 Oct 24.

Department of Genetics, The Scripps Research Institute, La Jolla, CA 92037, USA.

Background: We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype.

Findings: In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied.

Conclusions: Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.
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http://dx.doi.org/10.1186/1756-0500-5-577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3532239PMC
October 2012

Slc15a4, a gene required for pDC sensing of TLR ligands, is required to control persistent viral infection.

PLoS Pathog 2012 Sep 13;8(9):e1002915. Epub 2012 Sep 13.

Department of Genetics, The Scripps Research Institute, La Jolla, California, USA.

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.
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http://dx.doi.org/10.1371/journal.ppat.1002915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441671PMC
September 2012

Hypopigmentation and maternal-zygotic embryonic lethality caused by a hypomorphic mbtps1 mutation in mice.

G3 (Bethesda) 2012 Apr 1;2(4):499-504. Epub 2012 Apr 1.

The site 1 protease, encoded by Mbtps1, mediates the initial cleavage of site 2 protease substrates, including sterol regulatory element binding proteins and CREB/ATF transcription factors. We demonstrate that a hypomorphic mutation of Mbtps1 called woodrat (wrt) caused hypocholesterolemia, as well as progressive hypopigmentation of the coat, that appears to be mechanistically unrelated. Hypopigmentation was rescued by transgenic expression of wild-type Mbtps1, and reciprocal grafting studies showed that normal pigmentation depended upon both cell-intrinsic or paracrine factors, as well as factors that act systemically, both of which are lacking in wrt homozygotes. Mbtps1 exhibited a maternal-zygotic effect characterized by fully penetrant embryonic lethality of maternal-zygotic wrt mutant offspring and partial embryonic lethality (~40%) of zygotic wrt mutant offspring. Mbtps1 is one of two maternal-zygotic effect genes identified in mammals to date. It functions nonredundantly in pigmentation and embryogenesis.
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http://dx.doi.org/10.1534/g3.112.002196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337478PMC
April 2012

Specific mutation of a gammaherpesvirus-expressed antigen in response to CD8 T cell selection in vivo.

J Virol 2012 Mar 14;86(5):2887-93. Epub 2011 Dec 14.

Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo.
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http://dx.doi.org/10.1128/JVI.06101-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302239PMC
March 2012

Expanded potential for recombinant trisegmented lymphocytic choriomeningitis viruses: protein production, antibody production, and in vivo assessment of biological function of genes of interest.

J Virol 2011 Aug 25;85(15):7928-32. Epub 2011 May 25.

The Scripps Research Institute, Department of Immunology and Microbial Sciences (IMM-6), 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

The recombinant engineering of trisegmented lymphocytic choriomeningitis virus (LCMV) to express two genes of interest was recently reported. We used this technology to efficiently express green fluorescent protein (GFP) and the immunoregulatory gene product interleukin-10 (IL-10) in vitro, assess IL-10 function in vivo during viral meningitis, and generate specific, robust monoclonal antibody responses to IL-10. Tripartite viruses were attenuated in wild-type and TLR7(-/-) mice. However, IFNAR1(-/-) mice sustained systemic viral replication when 2 nucleotide substitutions from a persistent LCMV variant were present. These findings demonstrate the utility of tripartite LCMV in vitro and in vivo to study genes in the context of a well-defined model system.
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http://dx.doi.org/10.1128/JVI.00486-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147893PMC
August 2011

Hypomorphic mutation in the site-1 protease Mbtps1 endows resistance to persistent viral infection in a cell-specific manner.

Cell Host Microbe 2011 Mar;9(3):212-222

Department of Immunology and Microbial Sciences, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), which naturally persists in rodents, represents a model for HIV, HBV, and HCV. Cleavage of the viral glycoprotein precursor by membrane-bound transcription factor peptidase, site 1 (Mbtps1 or site-1 protease), is crucial for the life cycle of arenaviruses and therefore represents a potential target for therapy. Recently, we reported a viable hypomorphic allele of Mbtps1 (woodrat) encoding a protease with diminished enzymatic activity. Using the woodrat allele, we examine the role of Mbtps1 during persistent LCMV infection. Surprisingly, Mbtps1 inhibition limits persistent but not acute viral infection and is associated with an organ/cell type-specific decrease in viral titers. Analysis of bone marrow-derived dendritic cells from woodrat mice supports their specific role in resolving persistent viral infection. These results support in vivo targeting of Mbtps1 in the treatment of arenavirus infections and demonstrate a critical role for dendritic cells in persistent viral infections.
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http://dx.doi.org/10.1016/j.chom.2011.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058147PMC
March 2011

Gamma interferon blocks gammaherpesvirus reactivation from latency.

J Virol 2006 Jan;80(1):192-200

Washington University School of Medicine, Department of Pathology and Immunology, 660 South Euclid Ave., St. Louis, MO 63110, USA.

Establishment of latent infection and reactivation from latency are critical aspects of herpesvirus infection and pathogenesis. Interfering with either of these steps in the herpesvirus life cycle may offer a novel strategy for controlling herpesvirus infection and associated disease pathogenesis. Prior studies show that mice deficient in gamma interferon (IFN-gamma) or the IFN-gamma receptor have elevated numbers of cells reactivating from murine gammaherpesvirus 68 (gammaHV68) latency, produce infectious virus after the establishment of latency, and develop large-vessel vasculitis. Here, we demonstrate that IFN-gamma is a powerful inhibitor of reactivation of gammaHV68 from latency in tissue culture. In vivo, IFN-gamma controls viral gene expression during latency. Importantly, depletion of IFN-gamma in latently infected mice results in an increased frequency of cells reactivating virus. This demonstrates that IFN-gamma is important for immune surveillance that limits reactivation of gammaHV68 from latency.
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http://dx.doi.org/10.1128/JVI.80.1.192-200.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1317536PMC
January 2006

Widespread annular eruption in a Black man--quiz case.

Arch Dermatol 2005 Jan;141(1):93-8

Washington University School of Medicine, St Louis, MO, USA.

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http://dx.doi.org/10.1001/archderm.141.1.93-aDOI Listing
January 2005

Murine cytomegalovirus paralyzes macrophages by blocking IFN gamma-induced promoter assembly.

Proc Natl Acad Sci U S A 2003 Nov 12;100(24):14309-14. Epub 2003 Nov 12.

Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Macrophages (M phi) are activated by IFN gamma and are important cellular targets for infection by human and murine cytomegalovirus (MCMV), making it advantageous for CMVs to block IFN gamma-induced M phi differentiation. We found that MCMV infection inhibited IFN gamma regulation of many genes in M phi. MCMV infection blocked IFN gamma responses at the level of transcription without blocking Janus kinase/signal transducer and activator of transcription pathway activation and targeted IFN response factor 1- and class II transactivator-dependent and independent promoters. MCMV did not alter basal transcription from IFN gamma-responsive promoters and left the majority of cellular transcripts unchanged even after 48 h of infection. The effects of MCMV infection were specific to chromosomal rather than transiently transfected promoters. Characterization of the IFN gamma-responsive chromosomal class II transactivator promoter revealed that MCMV infection blocked IFN gamma-induced promoter assembly, allowing the virus to transcriptionally paralyze infected M phi responses while allowing basal transcription to proceed.
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http://dx.doi.org/10.1073/pnas.1835673100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC283588PMC
November 2003

Murine cytomegalovirus infection inhibits tumor necrosis factor alpha responses in primary macrophages.

J Virol 2003 Sep;77(18):10125-30

Department of Pathology, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110, USA.

Despite robust host immune responses the betaherpesvirus murine cytomegalovirus (MCMV) is able to establish lifelong infection. This capacity is due at least in part to the virus utilizing multiple immune evasion mechanisms to blunt host responses. Macrophages are an important cell for MCMV infection, dissemination, and latency despite expression in the host of multiple cytokines, including tumor necrosis factor alpha (TNF-alpha), that can induce an antiviral state in macrophages or other cells. In this study, we found that MCMV infection of bone marrow-derived macrophages inhibited TNF-alpha-induced ICAM-1 surface expression and mRNA expression in infected cells via expression of immediate early and/or early viral genes. MCMV infection blocked TNF-alpha-induced nuclear translocation of NF-kappaB. This inhibition of TNF-alpha signaling was explained by a decrease in TNF receptor 1 (TNFR1) and TNFR2 that was due to decreased mRNA for the latter. These findings provide a mechanism by which MCMV can evade the effects of an important host cytokine in macrophages.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC224571PMC
http://dx.doi.org/10.1128/jvi.77.18.10125-10130.2003DOI Listing
September 2003