Oral Metformin Inhibits Choroidal Neovascularization by Modulating the Gut-Retina Axis.

Purpose: Emerging data indicate that metformin may prevent the development of age-related macular degeneration (AMD). Whereas the underlying mechanisms of metformin's anti-aging properties remain undetermined, one proposed avenue is the gut microbiome. Using the laser-induced choroidal neovascularization (CNV) model, we investigate the effects of oral metformin on CNV, retinal pigment epithelium (RPE)/choroid transcriptome, and gut microbiota. Methods: Specific pathogen free (SPF) male mice were treated via daily oral gavage of metformin 300 mg/kg or vehicle. Male mice were selected to minimize sex-specific differences to laser induction and response to metformin. Laser-induced CNV size and macrophage/microglial infiltration were assessed by isolectin and Iba1 immunostaining. High-throughput RNA-seq of the RPE/choroid was performed using Illumina. Fecal pellets were analyzed for gut microbiota composition/pathways with 16S rRNA sequencing/shotgun metagenomics, as well as microbial-derived metabolites, including small-chain fatty acids and bile acids. Investigation was repeated in metformin-treated germ-free (GF) mice and antibiotic-treated/GF mice receiving fecal microbiota transplantation (FMT) from metformin-treated SPF mice. Results: Metformin treatment reduced CNV size (P < 0.01) and decreased Iba1+ macrophage/microglial infiltration (P < 0.005). One hundred forty-five differentially expressed genes were identified in the metformin-treated group (P < 0.05) with a downregulation in pro-angiogenic genes Tie1, Pgf, and Gata2. Furthermore, metformin altered the gut microbiome in favor of Bifidobacterium and Akkermansia, with a significant increase in fecal levels of butyrate, succinate, and cholic acid. Metformin did not suppress CNV in GF mice but colonization of microbiome-depleted mice with metformin-derived FMT suppressed CNV. Conclusions: These data suggest that oral metformin suppresses CNV, the hallmark lesion of advanced neovascular AMD, via gut microbiome modulation.

Using Advanced Bioinformatics Tools to Identify Novel Therapeutic Candidates for Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is the dreaded cause of failure following retinal detachment repair; however, no cures or preventative therapies exist to date. The purpose of this study was to use bioinformatics tools to identify drugs or compounds that interact with biomarkers and pathways involved in PVR pathogenesis that could be eligible for further testing for the prevention and treatment of PVR. Methods: We queried PubMed to compile a comprehensive list of genes described in PVR to date from human studies, animal models, and genomic studies found in the National Center for Biotechnology Information database. Gene enrichment analysis was performed using ToppGene on PVR-related genes against drug-gene interaction databases to construct a pharmacome and estimate the statistical significance of overrepresented compounds. Compounds with no clinical indications were filtered out from the resulting drug lists. Results: Our query identified 34 unique genes associated with PVR. Out of 77,146 candidate drugs or compounds in the drug databases, our analysis revealed multiple drugs and compounds that have significant interactions with genes involved in PVR, including antiproliferatives, corticosteroids, cardiovascular agents, antioxidants, statins, and micronutrients. Top compounds, including curcumin, statins, and cardiovascular agents such as carvedilol and enalapril, have well-established safety profiles and potentially could be readily repurposed for PVR. Other significant compounds such as prednisone and methotrexate have shown promising results in ongoing clinical trials for PVR. Conclusions: This bioinformatics approach of studying drug-gene interactions can identify drugs that may affect genes and pathways implicated in PVR. Predicted bioinformatics studies require further validation by preclinical or clinical studies; however, this unbiased approach could identify potential candidates among existing drugs and compounds that could be repurposed for PVR and guide future investigations. Translational Relevance: Novel repurposable drug therapies for PVR can be found using advanced bioinformatics models.

Using Computational Drug-Gene Analysis to Identify Novel Therapeutic Candidates for Retinal Neuroprotection.

Retinal cell death is responsible for irreversible vision loss in many retinal disorders. No commercially approved treatments are currently available to attenuate retinal cell loss and preserve vision. We seek to identify chemicals/drugs with thoroughly-studied biological functions that possess neuroprotective effects in the retina using a computational bioinformatics approach. We queried the National Center for Biotechnology Information (NCBI) to identify genes associated with retinal neuroprotection. Enrichment analysis was performed using ToppGene to identify compounds related to the identified genes. This analysis constructs a Pharmacome from multiple drug-gene interaction databases to predict compounds with statistically significant associations to genes involved in retinal neuroprotection. Compounds with known deleterious effects (e.g., asbestos, ethanol) or with no clinical indications (e.g., paraquat, ozone) were manually filtered. We identified numerous drug/chemical classes associated to multiple genes implicated in retinal neuroprotection using a systematic computational approach. Anti-diabetics, lipid-lowering medicines, and antioxidants are among the treatments anticipated by this analysis, and many of these drugs could be readily repurposed for retinal neuroprotection. Our technique serves as an unbiased tool that can be utilized in the future to lead focused preclinical and clinical investigations for complex processes such as neuroprotection, as well as a wide range of other ocular pathologies.

Absence of Gut Microbiota Is Associated with RPE/Choroid Transcriptomic Changes Related to Age-Related Macular Degeneration Pathobiology and Decreased Choroidal Neovascularization.

Studies have begun to reveal significant connections between the gut microbiome and various retinal diseases, including age-related macular degeneration (AMD). As critical supporting tissues of the retina, the retinal pigment epithelium (RPE) and underlying choroid play a critical role in retinal homeostasis and degeneration. However, the relationship between the microbiome and RPE/choroid remains poorly understood, particularly in animal models of AMD. In order to better elucidate this role, we performed high-throughput RNA sequencing of RPE/choroid tissue in germ-free (GF) and specific pathogen-free (SPF) mice. Furthermore, utilizing a specialized laser-induced choroidal neovascularization (CNV) model that we developed, we compared CNV size and inflammatory response between GF and SPF mice. After correction of raw data, 660 differentially expressed genes (DEGs) were identified, including those involved in angiogenesis regulation, scavenger and cytokine receptor activity, and inflammatory response-all of which have been implicated in AMD pathogenesis. Among lasered mice, the GF group showed significantly decreased CNV lesion size and microglial infiltration around CNV compared to the SPF group. Together, these findings provide evidence for a potential gut-RPE/choroidal axis as well as a correlation with neovascular features of AMD.

Using Advanced Bioinformatics Tools to Identify Novel Therapeutic Candidates for Age-Related Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is the most common cause of aging-related blindness in the developing world. Although medications can slow progressive wet AMD, currently, no drugs to treat dry-AMD are available. We use a systems or in silico biology analysis to identify chemicals and drugs approved by the Food and Drug Administration for other indications that can be used to treat and prevent AMD. Methods: We queried National Center for Biotechnology Information to identify genes associated with AMD, wet AMD, dry AMD, intermediate AMD, and geographic atrophy to date. We combined genes from various AMD subtypes to reflect distinct stages of disease. Enrichment analysis using the ToppGene platform predicted molecules that can influence AMD genes. Compounds without clinical indications or with deleterious effects were manually filtered. Results: We identified several drug/chemical classes that can affect multiple genes involved in AMD. The drugs predicted from this analysis include antidiabetics, lipid-lowering agents, and antioxidants, which could theoretically be repurposed for AMD. Metformin was identified as the drug with the strongest association with wet AMD genes and is among the top candidates in all dry AMD subtypes. Curcumin, statins, and antioxidants are also among the top drugs correlating with AMD-risk genes. Conclusions: We use a systematic computational process to discover potential therapeutic targets for AMD. Our systematic and unbiased approach can be used to guide targeted preclinical/clinical studies for AMD and other ocular diseases. Translational Relevance: Advanced bioinformatics models identify novel chemicals and approved drug candidates that can be efficacious for different subtypes of AMD.

Gut microbiome and retinal diseases: an updated review.

PURPOSE OF REVIEW: The gut microbiome, trillions of microorganisms residing in our digestive tract, is now believed to play a significant role in retinal diseases. Breakthroughs in computational biology and specialized animal models have allowed researchers not only to characterize microbes associated with retinal diseases, but also to provide early insights into the function of the microbiome in relation to biological processes in the retinal microenvironment. This review aims to provide an update on recent advances in the current knowledge on the relationship between the gut microbiome and retinal disorders. RECENT FINDINGS: Recent work demonstrates distinct gut microbial compositions associated with retinal diseases such as agerelated macular degeneration and retinopathy of prematurity. Currently, it is believed that gut dysbiosis leads to increased gut permeability, elevated circulation of bacterial products, microbial metabolites and inflammatory mediators that result in immune dysregulation at distant anatomic sites including the retina. SUMMARY: Emerging evidence for the gut-retina axis can elucidate previously unknown pathways involved in retinal diseases and also presents an exciting potential therapeutic avenue. Further preclinical and clinical studies are necessary to establish causation and delineate the precise relationship of the gut microbiome with retinal disorders.

Refractory Ventricular Tachycardia Originating From an Intracardiac Metastasis Treated With Catheter Ablation.

A 45-year-old man with stage IV melanoma presented with incessant nonsustained wide complex tachycardia. He was found to have a right ventricular intracardiac metastasis that created a nidus for ventricular tachycardia refractory to multiple therapeutic interventions. The patient underwent catheter ablation for this rare indication, with successful arrhythmia control by direct ablation over the tumor surface. (Level of Difficulty: Advanced.).

Gnotobiotic Operations and Assembly for Development of Germ-Free Animal Model of Laser-Induced Choroidal Neovascularization.

Purpose: Compelling new evidence reveals a close link between the gut microbiome and the pathogenesis of neovascular age-related macular degeneration (nAMD). Germ-free (GF) animal models are the current gold standard for studying host the microbe interactions in vivo; yet, no GF animal models of nAMD are available today. This protocol describes gnotobiotic operations and assembly for a laser-induced choroidal neovascularization (CNV) model in GF mice to study the gut microbiome in neovascular AMD. Methods: We developed a step-wise approach to performing retinal laser photocoagulation in GF C57BL/6J mice that were bred and maintained at the gnotobiotic facility. Following a strict sterility protocol, we administered laser photocoagulation via an Argon 532-nm laser attached to a customized slit-lamp delivery system. Sterility was confirmed by weekly fecal cultures and reverse transcriptase-polymerase chain reaction. Results: The experiment was repeated twice at different time points using seven mice (14 eyes). Stool cultures and RT-PCR remained negative for 14 days post-procedure in all mice. Lectin immunostaining performed on choroidal flatmounts confirmed the presence of CNV lesions 2 weeks after laser treatment. Conclusions: We established a GF mouse model of nAMD with detailed guidelines to deliver retinal laser in GF mice maintaining sterility after the laser procedure. Translational Relevance: To our knowledge, this is the first protocol that describes a GF murine model of laser-induced CNV. In addition to nAMD, this animal model can be used to investigate host-microbial interactions in other eye diseases with laser-induced mouse models such as glaucoma and retinal vein occlusion.

High-Fat Diet Alters the Retinal Transcriptome in the Absence of Gut Microbiota.

The relationship between retinal disease, diet, and the gut microbiome has shown increasing importance over recent years. In particular, high-fat diets (HFDs) are associated with development and progression of several retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy. However, the complex, overlapping interactions between diet, gut microbiome, and retinal homeostasis are poorly understood. Using high-throughput RNA-sequencing (RNA-seq) of whole retinas, we compare the retinal transcriptome from germ-free (GF) mice on a regular diet (ND) and HFD to investigate transcriptomic changes without influence of gut microbiome. After correction of raw data, 53 differentially expressed genes (DEGs) were identified, of which 19 were upregulated and 34 were downregulated in GF-HFD mice. Key genes involved in retinal inflammation, angiogenesis, and RPE function were identified. Enrichment analysis revealed that the top 3 biological processes affected were regulation of blood vessel diameter, inflammatory response, and negative regulation of endopeptidase. Molecular functions altered include endopeptidase inhibitor activity, protease binding, and cysteine-type endopeptidase inhibitor activity. Human and mouse pathway analysis revealed that the complement and coagulation cascades are significantly affected by HFD. This study demonstrates novel data that diet can directly modulate the retinal transcriptome independently of the gut microbiome.

A Review of the Role of the Intestinal Microbiota in Age-Related Macular Degeneration.

Blindness from age-related macular degeneration (AMD) is an escalating problem, yet AMD pathogenesis is incompletely understood and treatments are limited. The intestinal microbiota is highly influential in ocular and extraocular diseases with inflammatory components, such as AMD. This article reviews data supporting the role of the intestinal microbiota in AMD pathogenesis. Multiple groups have found an intestinal dysbiosis in advanced AMD. There is growing evidence that environmental factors associated with AMD progression potentially work through the intestinal microbiota. A high-fat diet in apo-E-/- mice exacerbated wet and dry AMD features, presumably through changes in the intestinal microbiome, though other independent mechanisms related to lipid metabolism are also likely at play. AREDS supplementation reversed some adverse intestinal microbial changes in AMD patients. Part of the mechanism of intestinal microbial effects on retinal disease progression is via microbiota-induced microglial activation. The microbiota are at the intersection of genetics and AMD. Higher genetic risk was associated with lower intestinal bacterial diversity in AMD. Microbiota-induced metabolite production and gene expression occur in pathways important in AMD pathogenesis. These studies suggest a crucial link between the intestinal microbiota and AMD pathogenesis, thus providing a novel potential therapeutic target. Thus, the need for large longitudinal studies in patients and germ-free or gnotobiotic animal models has never been more pressing.

Steroid-refractory dermatologic and pulmonary toxicity in a patient on rituximab treated with pembrolizumab for progressive urothelial carcinoma: a case report.

BACKGROUND: Increasingly widespread use of programmed cell death protein 1 (PD-1) immune checkpoint inhibitors (ICIs) for treatment of a variety of progressive malignancies continues to reveal a range of immune-related adverse events (irAEs), necessitating immunosuppressive therapy for management. While a single course of systemic corticosteroids may be sufficient for many irAEs, no clear standard-of-care guidelines exist for steroid-refractory cases. We present an unusual case of a patient who developed several steroid-refractory novel irAEs on pembrolizumab despite ongoing B cell-directed immunosuppressive therapy with rituximab, who ultimately noted resolution of symptoms with tacrolimus, a T-cell-directed immunosuppressant. CASE PRESENTATION: This 72-year-old Caucasian man with Waldenstrom's macroglobulinemia and myelin-associated glycoprotein (MAG) immunoglobulin M (IgM) antibody-associated neuropathy was being treated with maintenance rituximab and intravenous immunoglobulin when he was started on pembrolizumab (2.26 mg/kg) for metastatic urothelial cancer 31 months after surgery and adjuvant chemotherapy. After his third dose of pembrolizumab, he developed a painful blistering papular rash of the distal extremities. He received two more doses of pembrolizumab before he also developed diarrhea, and it was held; he was initiated on 1 mg/kg prednisone for presumed ICI-induced dermatitis and colitis. Skin biopsy 10 weeks after cessation of pembrolizumab and taper of steroids to 20  mg daily revealed a unique bullous erythema multiforme. He was then admitted with dyspnea and imaging concerning for necrotizing pneumonia, but did not respond to antibiotic therapy. Bronchoscopy and biopsy revealed acute fibrinous organizing pneumonia. His symptoms failed to fully respond to multiple courses of high-dose systemic corticosteroids and a trial of azathioprine, but pneumonia, diarrhea, and skin rash all improved markedly with tacrolimus. The patient has since completed his therapy for tacrolimus, continues off of ICI, and has not experienced a recurrence of any irAEs, though has more recently experienced progression of his cancer. CONCLUSION: Despite immunosuppression with rituximab and intravenous immunoglobulin, two immunomodulators targeting B cells, ICI cessation, and systemic corticosteroid therapy, our patient developed two high-grade unusual irAEs, bullous erythema multiforme and acute fibrinous organizing pneumonia. Our patient's improvement with tacrolimus can offer critical insight into the pathophysiology of steroid-refractory irAEs.

Chloroquine- and Hydroxychloroquine-Induced Cardiomyopathy: A Case Report and Brief Literature Review.

OBJECTIVES: To present an index case and review the histologic and electron microscopic findings in chloroquine (CQ) and hydroxychloroquine (HCQ) myopathy, focusing primarily on cardiomyopathy. CQ and HCQ are antimalarial drugs with disease-modifying activity in rheumatic diseases (DMARD) and now are among the most widely used DMARDs. Although they are rare, severe adverse effects caused mainly by deposition of intracellular metabolites in both cardiac and skeletal muscle have been described. Currently, both CQ and HCQ have been proposed to have efficacy for patients with coronavirus disease 2019, and several large centers in the United States and other countries have started clinical trials. METHODS: A case of HCQ cardiotoxicity diagnosed on an endomyocardial biopsy is presented. A review of the pathology archives was performed to identify additional cases of CQ or HCQ myopathy, and histologic changes were recorded. A brief literature review with an emphasis on pathologic findings in myopathies was performed. RESULTS: Including the index case, 4 cases of CQ or HCQ myopathy were identified. Light microscopic findings included vacuolated myopathy, and electron microscopic findings included myeloid bodies and curvilinear inclusion bodies. CONCLUSION: CQ and HCQ myopathy can present following long-term administration of the drug. The pathologic findings are nonspecific and overlap with other vacuolated myopathies, necessitating careful correlation of the histologic changes with the patient's medical history.

Lymph Liquid Biopsy for Detection of Cancer Stem Cells.

Collection of a blood sample defined by the term "blood liquid biopsy" is commonly used to detect diagnostic, prognostic, and therapeutic decision-making markers of metastatic tumors including circulating tumor cells (CTCs). Many tumors also release CTCs and other markers into lymph fluid, but the utility of lymphatic markers largely remains unexplored. Here, we introduce lymph liquid biopsy through collection of peripheral (afferent) and central (thoracic duct [TD]) lymph samples and demonstrates its feasibility for detection of stem-like CTCs potentially responsible for metastasis development and tumor relapse. Stemness of lymphatic CTCs (L-CTCs) was determined by spheroid-forming assay in vitro. Simultaneously, we tested blood CTCs by conventional blood liquid biopsy, and monitored the primary tumor size, early metastasis in a sentinel lymph node (SLN) and distant metastasis in lungs. Using a mouse model at early melanoma stage with no distant metastasis, we identified stem-like L-CTCs in lymph samples from afferent lymphatic vessels. Since these vessels transport cells from the primary tumor to SLN, our finding emphasizes the significance of the lymphatic pathway in development of SLN metastasis. Surprisingly, in pre-metastatic disease, stem-like L-CTCs were detected in lymph samples from the TD, which directly empties lymph into blood circulation. This suggests a new contribution of the lymphatic system to initiation of distant metastasis. Integration of lymph and blood liquid biopsies demonstrated that all mice with early melanoma had stem-like CTCs in at least one of three samples (afferent lymph, TD lymph, and blood). At the stage of distant metastasis, spheroid-forming L-CTCs were detected in TD lymph, but not in afferent lymph. Altogether, our results demonstrated that lymph liquid biopsy and testing L-CTCs holds promise for diagnosis and prognosis of early metastasis. © 2020 International Society for Advancement of Cytometry.

In Vivo Lymphatic Circulating Tumor Cells and Progression of Metastatic Disease.

The dissemination of circulating tumor cells (CTCs) by lymph fluid is one of the key events in the development of tumor metastasis. However, little progress has been made in studying lymphatic CTCs (L-CTCs). Here, we demonstrate the detection of L-CTCs in preclinical mouse models of melanoma and breast cancer using in vivo high-sensitivity photoacoustic and fluorescent flow cytometry. We discovered that L-CTCs are be detected in pre-metastatic disease stage. The smallest primary tumor that shed L-CTCs was measured as 0.094mm×0.094mm, its volume was calculated as 0.0004 mm3; and its productivity was estimated as 1 L-CTC per 30 minutes. As the disease progressed, primary tumors continued releasing L-CTCs with certain individual dynamics. The integrated assessment of lymph and blood underlined the parallel dissemination of CTCs at all disease stages. However, the analysis of links between L-CTC counts, blood CTC (B-CTC) counts, primary tumor size and metastasis did not reveal statistically significant correlations, likely due to L-CTC heterogeneity. Altogether, our results showed the feasibility of our diagnostic platform using photoacoustic flow cytometry for preclinical L-CTC research with translational potential. Our findings also demonstrated new insights into lymphatic system involvement in CTC dissemination. They help to lay the scientific foundation for the consideration of L-CTCs as prognostic markers of metastasis and to emphasize the integrative assessment of lymph and blood.

Educational Case: Asthma: Clinical Features and Morphologic Findings.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.1.

Massive Myocardial Calcium Deposition: Hardened Heart.

A 25-year-old African-American woman with end-stage renal disease presented with new-onset heart failure. Transthoracic echocardiography indicated a significantly hyperechoic myocardium, and computed tomography noted a circumferential hyperattenuated myocardium. Endomyocardial biopsy revealed focal interstitial and intramyocyte calcium deposition in the heart, confirming a rare diagnosis of massive myocardial calcium deposition. (Level of Difficulty: Beginner.).

Educational Case: Myocardial Infarction: Histopathology and Timing of Changes.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.1.

Current understanding of treatment and management protocol for adult diabetic in-patients at a tertiary care hospital.

OBJECTIVE: To assess the current understanding of treatment and management protocols for adult diabetic inpatients at a tertiary care hospital. METHODS: This cross-sectional study, conducted at the Civil Hospital Karachi from July to September 2009, involved 450 participants, who were interviewed through a well-structured questionnaire regarding the patient's demography, clinical features, past medical history, type of diabetes mellitus, duration, associated complications, and also involved patient notes for laboratory tests and management. SPSSv15.0 was used for descriptive analysis. RESULTS: The study population of 450 diabetics had 144 (32%) males and 306 (68%) females. Of the total, 435 (96.7%) patients had type 2 diabetes. There were 231 (51%) patients using insulin, 168 (37.3%) oral hypoglycaemic drugs, and 51 (11.3%) using both. Among patients using insulin, regular insulin usage stood at 30% followed by a combination of regular insulin and NPH (26.7%) and NPH alone at 6%. The most popular drug used was metformin (27.3%) and the least used drug was glitazones (4%). In the study population, 73.3% patients controlled their diabetes with diet, and 24.7% with regular exercise. CONCLUSION: Majority of the study population had type 2 diabetes with a female preponderance. Insulin was prescribed for half the patients. Metformin was the most frequently used oral hypoglycaemic drug.