Critical involvement of macrophage infiltration in the development of Sjögren's syndrome-associated dry eye.

Lymphocytic infiltration of the lacrimal gland and ocular surface in autoimmune diseases such as Sjögren's syndrome (SS) causes an aqueous-deficient dry eye that is associated with significant morbidity. Previous studies from our laboratory and others have established autoimmune regulator (Aire)-deficient mice as a useful model to examine exocrinopathy and ocular surface disease associated with SS. Consistent with human SS, autoreactive CD4(+) T cells play an indispensible role in the development of exocrine and ocular surface disease in Aire knockout mice. We report that in addition to CD4(+) T cells, a large number of macrophages infiltrate the corneal stroma, limbus, and lacrimal glands of diseased mice. Adoptive transfer of autoreactive CD4(+) T cells from Aire knockout mice led to local infiltration of macrophages and ocular surface damage in immunodeficient recipients. Depletion of local macrophages, through subconjunctival injection of clodronate liposome, attenuated lissamine green staining and improved ocular phenotype. Alternatively, systemic depletion of macrophages had no effect on ocular phenotype but led to significant improvements in lacrimal gland exocrinopathy and tear secretion. Our results suggested that autoreactive CD4(+) T cells provoked macrophage infiltration to the eye and lacrimal gland, where they played a functional role in directing the development of autoimmune dry eye.

Interleukin-1 receptor mediates the interplay between CD4+ T cells and ocular resident cells to promote keratinizing squamous metaplasia in Sjögren's syndrome.

Keratinizing squamous metaplasia (SQM) of the ocular mucosal epithelium is a blinding corneal disease characterized by the loss of conjunctival goblet cells (GCs), pathological ocular surface keratinization and tissue recruitment of immune cells. Using the autoimmune regulator (Aire)-deficient mouse as a model for Sjögren's syndrome (SS)-associated SQM, we identified CD4(+) T lymphocytes as the main immune effectors driving SQM and uncovered a pathogenic role for interleukin-1 (IL-1). IL-1, a pleiotropic cytokine family enriched in ocular epithelia, governs tissue homeostasis and mucosal immunity. Here, we used adoptive transfer of autoreactive CD4(+) T cells to dissect the mechanism whereby IL-1 promotes SQM. CD4(+) T cells adoptively transferred from both Aire knockout (KO) and Aire/IL-1 receptor type 1 (IL-1R1) double KO donors conferred SQM to severe-combined immunodeficiency (scid) recipients with functional IL-1R1, but not scid recipients lacking IL-1R1. In the lacrimal gland, IL-1R1 was primarily immunolocalized to ductal epithelium surrounded by CD4(+) T cells. In the eye, IL-1R1 was expressed on local mucosal epithelial and stromal cells, but not on resident antigen-presenting cells or infiltrating immune cells. In both tissues, autoreactive CD4(+) T-cell infiltration was only observed in the presence of IL-1R1-postive resident cells. Moreover, persistent activation of IL-1R1 signaling led to chronic immune-mediated inflammation by retaining CD4(+) T cells in the local microenvironment. Following IL-1R1-dependent infiltration of CD4(+) T cells, we observed SQM hallmarks in local tissues-corneal keratinization, conjunctival GC mucin acidification and epithelial cell hyperplasia throughout the ocular surface mucosa. Proinflammatory IL-1 expression in ocular epithelial cells significantly correlated with reduced tear secretion, while CD4(+) T-cell infiltration of the lacrimal gland predicted the development of ocular SQM. Collectively, data in this study indicated a central role for IL-1 in orchestrating a functional interplay between immune cells and resident cells of SS-targeted tissues in the pathogenesis of SQM.

Identification of occult metastatic disease via lymphoscintigraphy-guided neck dissection in N0 oral squamous cell carcinoma.

BACKGROUND: The purpose of this study is to assess the efficacy of lymphoscintigraphy-guided neck dissection (LSG-ND) in the treatment of N0 oral squamous cell carcinoma. METHODS: A retrospective cohort study of patients with N0 oral squamous cell carcinoma (SCC) who had either LSG-ND or neck dissection (ND) at our institution between 2008 and 2020. RESULTS: Eighty-seven patients met criteria with N0 oral squamous cell carcinoma with no previous treatment or neck surgery (27 LSG-ND, 60 ND). Sentinel lymph nodes were identified on the contralateral side in 14.8% of patients with unilateral tumors in the LSG-ND group, with 22.2% of cases with unexpected lymphatic drainage outside ipsilateral levels I-III. No recurrences to date have occurred in the LSG-ND cohort, while 13.3% of patients with ND had regional recurrence (p = 0.04). CONCLUSIONS: LSG-ND provides a greater ability to identify occult metastatic disease with a significant reduction in regional recurrence in N0 oral SCC.

Two isoleucyl tRNAs that decode synonymous codons divergently regulate breast cancer metastatic growth by controlling translation of proliferation-regulating genes.

The human genome contains 61 codons encoding 20 amino acids. Synonymous codons representing a given amino acid are decoded by a set of transfer RNAs (tRNAs) called isoacceptors. We report the surprising observation that two isoacceptor tRNAs that decode synonymous codons become modulated in opposing directions during breast cancer progression. Specifically, tRNAIleUAU became upregulated, whereas tRNAIleGAU became repressed as breast cancer cells attained enhanced metastatic capacity. Functionally, tRNAIleUAU promoted and tRNAIleGAU suppressed metastatic colonization in mouse xenograft models. These tRNAs mediated opposing effects on codon-dependent translation of growth-promoting genes, consistent with genomic enrichment or depletion of their cognate codons in mitotic genes. Our findings uncover a specific isoacceptor tRNA pair that act in opposition, divergently impacting growth-regulating genes and a disease phenotype. Degeneracy of the genetic code can thus be biologically exploited by human cancer cells via tRNA isoacceptor shifts that causally facilitate the transition toward a growth-promoting state.

Interleukin-1 as a phenotypic immunomodulator in keratinizing squamous metaplasia of the ocular surface in Sjögren's syndrome.

Chronic inflammation of the ocular surface in Sjögren's syndrome (SS) is associated with a vision-threatening, phenotypic change of the ocular surface, which converts from a nonkeratinized, stratified squamous epithelium to a nonsecretory, keratinized epithelium. This pathological process is known as squamous metaplasia. Based on a significant correlation between ocular surface interleukin (IL)-1beta expression and squamous metaplasia in patients with SS, we investigated the role of IL-1 in the pathogenesis of squamous metaplasia in an animal model that mimics the clinical characteristics of SS. Using autoimmune-regulator (aire)-deficient mice, we assessed lacrimal gland and ocular surface immunopathology by quantifying the infiltration of major histocompatibility complex class II(+) (I-A(d+)) dendritic cells and CD4(+) T cells. We examined squamous metaplasia using a biomarker of keratinization, small proline-rich protein 1B. We used lissamine green staining as a readout for ocular surface epitheliopathy and Alcian blue/periodic acid-Schiff histochemical analysis to characterize goblet cell muco-glycoconjugates. Within 8 weeks, the eyes of aire-deficient mice were pathologically keratinized with significant epithelial damage and altered mucin glycosylation. Although knockdown of IL-1 receptor 1 did not attenuate lymphocytic infiltration of the lacrimal gland or eye, it significantly reduced ocular surface keratinization, epitheliopathy, and muco-glycoconjugate acidification. These data demonstrate a phenotypic modulation role for IL-1 in the pathogenesis of squamous metaplasia and suggest that IL-1 receptor 1-targeted therapies may be beneficial for treating ocular surface disease associated with SS.

Identification of molecular determinants of primary and metastatic tumour re-initiation in breast cancer.

Through in vivo selection of multiple ER-negative human breast cancer populations for enhanced tumour-forming capacity, we have derived subpopulations that generate tumours more efficiently than their parental populations at low cell numbers. Tumorigenic-enriched subpopulations exhibited increased expression of LAMA4, FOXQ1 and NAP1L3­genes that are also expressed at greater levels by independently derived metastatic subpopulations. These genes promote metastatic efficiency. FOXQ1 promotes LAMA4 expression, and LAMA4 enhances clonal expansion following substratum detachment in vitro, tumour re-initiation in multiple organs, and disseminated metastatic cell proliferation and colonization. The promotion of cancer cell proliferation and tumour re-initiation by LAMA4 requires ß1-integrin. Increased LAMA4 expression marks the transition of human pre-malignant breast lesions to malignant carcinomas, and tumoral LAMA4 overexpression predicts reduced relapse-free survival in ER-negative patients. Our findings reveal common features that govern primary and metastatic tumour re-initiation and identify a key molecular determinant of these processes.

Acute drug treatment in the early C. elegans embryo.

Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.