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A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Northwestern University

MicroRNAs (miRNAs) are small, non-coding RNAs of ~18-25 nucleotides that have gained extensive attention as critical regulators in complex gene networks including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and function. Many viruses encode miRNAs that directly downregulate the expression of factors of the innate immune system, which includes proteins involved in promoting apoptosis and recruitment. In this study, we examined the expression profiles of three previously identified viral miRNAs (v-miRs) from the human herpesvirus (HHV) family, HSV-1 (miR-H1), KSHV (miR-K12-3-3p), and HCMV (miR-US4) in healthy and diseased periodontal tissues and observed increased levels of v-miRs in diseased tissues. To understand the significance of this increase, we overexpressed v-miRs in human oral keratinocytes (HOK), a common target for various HHV, and analyzed the impact of miR-H1 and miR-K12-3-3p on the host transcriptome. More than 1300 genes were altered in HOK overexpressing miR-H1 and miR-K12-3-3p. Global pathway analysis of deregulated genes identified several key cellular pathways that may favor viral persistence. Using bioinformatic analysis, we predicted hundreds of potential v-miR binding sites on genes downregulated by miR-H1 and miR-K12-3-3p and validated three novel target v-miR sites suggesting widespread direct and indirect modulation of numerous host genes/pathways by a single v-miR. Finally, in vitro HSV-1 infection assays showed that miR-H1 can regulate viral entry and infection in human oral keratinocytes (HOK). Overall, our results demonstrate clinical and functional relevance of pathogenic viral molecules viz., v-miRs that regulate both host and viral functions and may contribute to the pathogenesis of inflammatory oral diseases.

Herpesvirus-encoded microRNAs detected in human gingiva alter host cell transcriptome and regulate viral infection.

Current drug-delivery systems are designed primarily for parenteral applications and are either lipid or polymer drug conjugates. In our quest to inhibit herpes simplex virus infection via the compounds found in commonly used cosmetic products, we found that activated carbon particles inhibit infection and, in addition, substantially improve topical delivery and, hence, the efficacy of a common antiviral drug, acyclovir (ACV). Our in vitro studies demonstrate that highly porous carbon structures trapped virions, blocked infection and substantially improved efficacy when ACV was loaded onto them. Also, using murine models of corneal and genital herpes infections, we show that the topical use of drug-encapsulated carbon (DECON) reduced dosing frequency, shortened treatment duration, and exhibited higher therapeutic efficacy than currently approved topical or systemic antivirals alone. DECON is a nontoxic, cost-effective and nonimmunogenic alternative to current topical drug-delivery systems that is uniquely triggered for drug release by virus trapping.

Drug-encapsulated carbon (DECON): A novel platform for enhanced drug delivery.

Vitiligo is a T-cell mediated skin disorder characterized by progressive loss of skin color. In individuals genetically predisposed to the disease, various triggers contribute to the initiation of vitiligo. Precipitating factors can stress the skin, leading to T-cell activation and recruitment. Though hereditary factors are implicated in the pathogenesis of vitiligo, it is unknown whether precipitating, stressful events play a role in vitiligo. To understand this, we utilized a validated perceived stress scale (PSS) to measure this parameter in vitiligo patients compared to persons without vitiligo. Additionally, we probed a clinical database, using a knowledge linking software called ROCKET, to gauge stress-related conditions in the vitiligo patient population. From a pool of patients in an existing database, a hundred individuals with vitiligo and twenty-five age- and sex-matched comparison group of individuals without vitiligo completed an online survey to quantify their levels of perceived stress. In parallel, patients described specifics of their disease condition, including the affected body sites, the extent, duration and activity of their vitiligo. Perceived stress was significantly higher among vitiligo individuals compared to those without vitiligo. ROCKET analyses suggested signs of metabolic-related disease (i.e., 'stress') preceding vitiligo development. No correlation was found between perceived stress and the stage or the extent of disease, suggesting that elevated stress may not be a consequence of pigment loss alone. The data provide further support for stress as a precipitating factor in vitiligo development.

The relationship between stress and vitiligo: Evaluating perceived stress and electronic medical record data.

Patients with lymphangioleiomyomatosis (LAM) develop pulmonary cysts associated with neoplastic, smooth muscle-like cells that feature neuroendocrine cell markers. The disease preferentially affects pre-menopausal women. Existing therapeutics do not cure LAM. As gp100 is a diagnostic marker expressed by LAM lesions, we proposed to target this immunogenic glycoprotein using TCR transgenic T cells. To reproduce the genetic mutations underlying LAM, we cultured Tsc2-/- kidney tumor cells from aged Tsc2 heterozygous mice and generated a stable gp100-expressing cell line by lentiviral transduction. T cells were isolated from MHC-matched TCR transgenic pmel-1 mice to measure cytotoxicity in vitro, and 80% cytotoxicity was observed within 48 hrs. Antigen-specific cytotoxicity was likewise observed using pmel-1 TCR transduced mouse T cells, suggesting that transgenic T cells may likewise be useful to treat LAM in vivo. Upon IV injection, slow-growing gp100+ LAM-like cells formed lung nodules that were readily detectable in SCID/beige mice. Adoptive transfer of gp100-reactive but not wildtype T cells into mice significantly shrunk established lung tumors, even in the absence of anti-PD-1 therapy. These results demonstrate the treatment potential of adoptively transferred T cells to eliminate pulmonary lesions in LAM.

Dinesh Jaishankar

Robert Lurie Comprehensive Cancer Center and Department of Dermatology,
Feinberg School of Medicine,
Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine

Northwestern University

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Dinesh Jaishankar

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Northwestern University

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Robert Lurie Comprehensive Cancer Center and Department of Dermatology,
Feinberg School of Medicine,
Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine