Circulating exosomal microRNA expression patterns distinguish cardiac sarcoidosis from myocardial ischemia.

OBJECTIVE: Cardiac sarcoidosis is difficult to diagnose, often requiring expensive and inconvenient advanced imaging techniques. Circulating exosomes contain genetic material, such as microRNA (miRNA), that are derived from diseased tissues and may serve as potential disease-specific biomarkers. We thus sought to determine whether circulating exosome-derived miRNA expression patterns would distinguish cardiac sarcoidosis (CS) from acute myocardial infarction (AMI). METHODS: Plasma and serum samples conforming to CS, AMI or disease-free controls were procured from the Biologic Specimen and Data Repository Information Coordinating Center repository and National Jewish Health. Next generation sequencing (NGS) was performed on exosome-derived total RNA (n = 10 for each group), and miRNA expression levels were compared after normalization using housekeeping miRNA. Quality assurance measures excluded poor quality RNA samples. Differentially expressed (DE) miRNA patterns, based upon >2-fold change (p < 0.01), were established in CS compared to controls, and in CS compared to AMI. Relative expression of several DE-miRNA were validated by qRT-PCR. RESULTS: Despite the advanced age of the stored samples (~5-30 years), the quality of the exosome-derived miRNA was intact in ~88% of samples. Comparing plasma exosomal miRNA in CS versus controls, NGS yielded 18 DE transcripts (12 up-regulated, 6 down-regulated), including miRNA previously implicated in mechanisms of myocardial injury (miR-92, miR-21) and immune responses (miR-618, miR-27a). NGS further yielded 52 DE miRNA in serum exosomes from CS versus AMI: 5 up-regulated in CS; 47 up-regulated in AMI, including transcripts previously detected in AMI patients (miR-1-1, miR-133a, miR-208b, miR-423, miR-499). Five miRNAs with increased DE in CS included two isoforms of miR-624 and miR-144, previously reported as markers of cardiomyopathy. CONCLUSIONS: MiRNA patterns of exosomes derived from CS and AMI patients are distinct, suggesting that circulating exosomal miRNA patterns could serve as disease biomarkers. Further studies are required to establish their specificity relative to other cardiac disorders.

Chronic elevation of plasma vascular endothelial growth factor-A (VEGF-A) is associated with a history of blast exposure.

Mounting evidence points to the significance of neurovascular-related dysfunction in veterans with blast-related mTBI, which is also associated with reduced [18F]-fluorodeoxyglucose (FDG) uptake. The goal of this study was to determine whether plasma VEGF-A is altered in veterans with blast-related mTBI and address whether VEGF-A levels correlate with FDG uptake in the cerebellum, a brain region that is vulnerable to blast-related injury 72 veterans with blast-related mTBI (mTBI) and 24 deployed control (DC) veterans with no lifetime history of TBI were studied. Plasma VEGF-A was significantly elevated in mTBIs compared to DCs. Plasma VEGF-A levels in mTBIs were significantly negatively correlated with FDG uptake in cerebellum. In addition, performance on a Stroop color/word interference task was inversely correlated with plasma VEGF-A levels in blast mTBI veterans. Finally, we observed aberrant perivascular VEGF-A immunoreactivity in postmortem cerebellar tissue and not cortical or hippocampal tissues from blast mTBI veterans. These findings add to the limited number of plasma proteins that are chronically elevated in veterans with a history of blast exposure associated with mTBI. It is likely the elevated VEGF-A levels are from peripheral sources. Nonetheless, increasing plasma VEGF-A concentrations correlated with chronically decreased cerebellar glucose metabolism and poorer performance on tasks involving cognitive inhibition and set shifting. These results strengthen an emerging view that cognitive complaints and functional brain deficits caused by blast exposure are associated with chronic blood-brain barrier injury and prolonged recovery in affected regions.

Circulating MicroRNAs and Extracellular Vesicle-Containing MicroRNAs as Response Biomarkers of Anti-programmed Cell Death Protein 1 or Programmed Death-Ligand 1 Therapy in NSCLC.

INTRODUCTION: Anti-programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) antibody therapy is a standard treatment for advanced NSCLC, and PD-L1 immunohistochemistry is used as a predictive biomarker for therapeutic response. However, because not all patients with NSCLC with high PD-L1 respond, and some patients with low PD-L1 expression exhibit durable benefit, more accurate predictive biomarkers are needed. Circulating microRNA (miRNA) and miRNA packaged in extracellular vesicles (EVs) are believed to play a role in intercellular communication among immune cells and between immune cells and tumor cells and may represent a good source of mechanism-related biomarkers. METHODS: Pretreatment plasma of patients with advanced NSCLC treated with single-agent anti-PD-1 or anti-PD-L1 antibody was used in this study. Plasma EVs were isolated using size-exclusion chromatography. Whole plasma and EV-containing RNAs were extracted. The miRNA profile was analyzed with a next-generation sequencing platform. RESULTS: Samples from 14 responders (patients who exhibited partial response or stable disease ≥6 mo) and 15 nonresponders (patients who exhibited progressive disease as per Response Evaluation Criteria in Solid Tumors) were analyzed. In total, 32 miRNAs (p = 0.0030-0.0495) from whole plasma and seven EV-associated miRNAs (p = 0.041-0.0457) exhibited significant concentration differences between responders and nonresponders. The results of some of these circulating miRNAs were validated in a separate cohort with eight responders and 13 nonresponders. The tumor PD-L1 level was also assessed using immunohistochemistry for patients involved in both cohorts. CONCLUSIONS: Specific circulating miRNAs in whole plasma and plasma EVs are differentially expressed between responders and nonresponders and have potential as predictive biomarkers for anti-PD-1/PD-L1 treatment response.

Alterations in Plasma microRNA and Protein Levels in War Veterans with Chronic Mild Traumatic Brain Injury.

Blast-related mild traumatic brain injury (mTBI) is considered the "signature" injury of the wars in Iraq and Afghanistan. Identifying biomarkers that could aid in diagnosis and assessment of chronic mTBI are urgently needed, as little progress has been made toward identifying blood-based biomarkers of repetitive mTBI in the chronic state. Addressing this knowledge gap is especially important in the population of military veterans who are receiving assessment and care often years after their last exposure. Circulating microRNAs (miRNAs), especially those encapsulated in extracellular vesicles (EVs), have gained interest as a source of biomarkers for neurological conditions. To identify biomarkers for chronic mTBI, we used next generation sequencing (NGS) to analyze miRNAs in plasma and plasma-derived EVs from 27 Iraq and Afghanistan war veterans with blast-related chronic mTBI, 11 deployed veteran non-TBI controls, and 31 civilian controls. We identified 32 miRNAs in plasma and 45 miRNAs in EVs that significantly changed in the chronic mTBI cohort compared with control groups. These miRNAs were predominantly associated with pathways involved in neuronal function, vascular remodeling, blood-brain barrier integrity, and neuroinflammation. In addition, the plasma proteome was analyzed and showed that the concentrations of C-reactive protein (CRP) and membrane metalloendopeptidase (MME) were elevated in chronic mTBI samples. These plasma miRNAs and proteins could potentially be used as biomarkers and provide insights into the molecular processes associated with the long-term health outcomes associated with blast-related chronic mTBI.

Extracellular Vesicle Encapsulated MicroRNAs in Patients with Type 2 Diabetes Are Affected by Metformin Treatment.

Recently, microRNAs (miRNAs) in circulating extracellular vesicles (EVs), have emerged as a source of potential biomarkers for various pathophysiological conditions, including metabolic disorders such as diabetes. Type 2 diabetes mellitus (T2DM), is the most prevalent form of diabetes in the USA, with 30 million diagnosed patients. Identifying miRNA biomarkers that can be used to assess response to glucose lowering treatments would be useful. Using patient plasma samples from a subset of the Danish Metagenomics of the Human Intestinal Tract (MetaHIT) cohort, we characterized miRNAs from whole plasma, plasma-derived EVs, and EV-depleted plasma by small RNA-sequencing to identify T2DM associated miRNAs. We identified several miRNAs that exhibited concentration changes between controls and non-metformin treated T2DM patients and we validated a subset of these by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results showed that the concentrations of many T2DM-affected miRNAs in EV (but not in whole or EV-depleted plasma) decreased to levels close to those of healthy controls following metformin treatment. Among other potential uses of these differentially expressed miRNAs, some might be useful in assessing the response to metformin in T2DM patients.

Circulating RNAs as predictive markers for the progression of type 2 diabetes.

Type 2 Diabetes Mellitus (T2DM) is the most prevalent form of diabetes in the USA, thus, the identification of biomarkers that could be used to predict the progression from prediabetes to T2DM would be greatly beneficial. Recently, circulating RNA including microRNAs (miRNAs) present in various body fluids have emerged as potential biomarkers for various health conditions, including T2DM. Whereas studies that examine the changes of miRNA spectra between healthy controls and T2DM individuals have been reported, the goal of this study is to conduct a baseline comparison of prediabetic individuals who either progress to T2DM, or remain prediabetic. Using an advanced small RNA sequencing library construction method that improves the detection of miRNA species, we identified 57 miRNAs that showed significant concentration differences between progressors (progress from prediabetes to T2DM) and non-progressors. Among them, 26 have been previously reported to be associated with T2DM in either body fluids or tissue samples. Some of the miRNAs identified were also affected by obesity. Furthermore, we identified miRNA panels that are able to discriminate progressors from non-progressors. These results suggest that upon further validation these miRNAs may be useful to predict the risk of conversion to T2DM from prediabetes.

Genome-wide Profiling of Urinary Extracellular Vesicle microRNAs Associated With Diabetic Nephropathy in Type 1 Diabetes.

INTRODUCTION: Diabetic nephropathy (DN) is a form of progressive kidney disease that often leads to end-stage renal disease (ESRD). It is initiated by microvascular complications due to diabetes. Although microalbuminuria (MA) is the earliest clinical indication of DN among patients with type 1 diabetes (T1D), it lacks the sensitivity and specificity to detect the early onset of DN. Recently, microRNAs (miRNAs) have emerged as critical regulators in diabetes as well as various forms of kidney disease, including renal fibrosis, acute kidney injury, and progressive kidney disease. Additionally, circulating extracellular miRNAs, especially miRNAs packaged in extracellular vesicles (EVs), have garnered significant attention as potential noninvasive biomarkers for various diseases and health conditions. METHODS: As part of the University of Pittsburgh Epidemiology of Diabetes Complications (EDC) study, urine was collected from individuals with T1D with various grades of DN or MA (normal, overt, intermittent, and persistent) over a decade at prespecified intervals. We isolated EVs from urine and analyzed the small-RNA using NextGen sequencing. RESULTS: We identified a set of miRNAs that are enriched in urinary EVs compared with EV-depleted samples, and identified a number of miRNAs showing concentration changes associated with DN occurrence, MA status, and other variables, such as hemoglobin A1c levels. CONCLUSION: Many of the miRNAs associated with DN occurrence or MA status directly target pathways associated with renal fibrosis (including transforming growth factor-ß and phosphatase and tensin homolog), which is one of the major contributors to the pathology of DN. These miRNAs are potential biomarkers for DN and MA.

mRNA transcript distribution bias between Borrelia burgdorferi bacteria and their outer membrane vesicles.

Borrelia burgdorferi, a bacterium in the spirochete phylum, is the causative agent of Lyme disease. Borrelia burgdorferi has a linear chromosome with a number of circular and linear plasmids. Bacteria, including B. burgdorferi, release spherical outer membrane vesicles (OMVs) that are known to carry secretory products including metabolites, nucleic acids and proteins. Herein, we provide the first comparative transcriptomic analysis of the vesicles released from B. burgdorferi. We identified a total of ∼1200 unique transcripts with at least one mapped read from the bacterial cell and its OMVs. We compared the spectrum of transcripts between bacterial cell and its OMVs, and found a biased distribution based on the source of transcripts, i.e. plasmid-encoded transcripts are more likely to be enriched in the OMVs. We validated the distribution for some of the transcripts by qPCR. This analysis provides the first evidence that some of the B. burgdorferi transcripts are preferentially packaged in OMV, which further suggest that the bacteria might use its OMVs for bacteria-bacteria or bacteria-host communications. This report also suggests a possible involvement of Borrelia-derived OMVs in the development of Lyme disease in both early and post disease syndromes.

Study protocol: rationale and design of the community-based prospective cohort study of kidney function and diabetes in rural New Mexico, the COMPASS study.

BACKGROUND: Rural areas in the state of New Mexico have been the "ground-zero" for the epidemic of diabetic Chronic Kidney Disease (CKD) in the United States. However, there is limited research about risk factors of diabetic CKD in this area and scarce data regarding the performance of emerging markers of renal filtration and epigenetic biomarkers of renal function and diabetes in this area with its unique ethnic/racial population. We designed the COMPASS study as a community-based program in rural New Mexico aiming to screen for CKD and to discover CKD-related translational biomarkers. METHODS/DESIGN: The study involves a prospective, longitudinal cohort design involving individuals living in rural New Mexico. Participants undergo a screening for kidney disease using markers of abnormal renal filtration (impaired glomerular filtration rate) or damage (albuminuria). Those found to have CKD on the basis of these tests or those at risk for CKD are enrolled in a prospective longitudinal cohort. We measure markers of renal function, insulin resistance and epigenetics (microRNAs) on patients. Individuals are invited to participate in interviews and focus groups in order to characterize their attitudes towards research and barriers or facilitators to participation in future research studies about kidney disease. DISCUSSION: This study will provide important data about the local epidemiology of kidney disease in a high-risk rural setting and the utility of emerging renal filtration markers (Beta 2 Microglobulin and Cystatin C), while generating data and methods for the analyses of microRNA biomarkers. The qualitative research subproject will identify factors associated with increased willingness to participate in future translational research projects. With its geographical focus, this study will address a critical disparity in kidney disease research, while generating novel epigenetic data that are relevant for future studies in the general population.

The C. elegans Spalt-like protein SEM-4 functions through the SoxC transcription factor SEM-2 to promote a proliferative blast cell fate in the postembryonic mesoderm.

Proper development of a multicellular organism relies on well-coordinated regulation of cell fate specification, cell proliferation and cell differentiation. The C. elegans postembryonic mesoderm provides a useful system for uncovering factors involved in these processes and for further dissecting their regulatory relationships. The single Spalt-like zinc finger containing protein SEM-4/SALL is known to be involved in specifying the proliferative sex myoblast (SM) fate. We have found that SEM-4/SALL is sufficient to promote the SM fate and that it does so in a cell autonomous manner. We further showed that SEM-4/SALL acts through the SoxC transcription factor SEM-2 to promote the SM fate. SEM-2 is known to promote the SM fate by inhibiting the expression of two BWM-specifying transcription factors. In light of recent findings in mammals showing that Sall4, one of the mammalian homologs of SEM-4, contributes to pluripotency regulation by inhibiting differentiation, our work suggests that the function of SEM-4/SALL proteins in regulating pluripotency versus differentiation appears to be evolutionarily conserved.

Recent progress toward the use of circulating microRNAs as clinical biomarkers.

MicroRNAs (miRNAs) have been shown to be critical mediators of many cellular and developmental processes and have been implicated in different human diseases. Since the observation of extracellular miRNAs present in various biofluids, much attention and excitement have been garnered toward understanding the functional roles of these circulating extracellular miRNAs and establishing their potential use as noninvasive diagnostic biomarkers. Here, we will review the current state of miRNA biomarkers for many human diseases, including their emerging use in toxicological applications, and discuss the current challenges in the field, with an emphasis on technical issues that often hinder discovery-based miRNA biomarker studies.

Current approaches to epigenetic therapy for the treatment of mantle cell lymphoma.

Epigenetics is the study of heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence. Such changes can include DNA methylation or histone modifications which both serve to silence gene expression. This review describes a new development in pharmacology, epigenetic therapy, which attempts to correct these epigenetic changes for the treatment of mantle cell lymphoma (MCL) and other B cell malignancies for which no consensus on standard therapy exists. One class of drugs utilized are the histone deacetylase inhibitors, (HDACi) which result in the accumulation of acetylated histones. Hyperacetylation of histones and nonhistone proteins are postulated to mediate the anticancer effects of these drugs. Another class of epigenetic agents are hypomethylating agents, that can cause both DNA and histone hypomethylation. Epigenetic drugs may be useful in the treatment of cancer where hypermethylation of tumor suppressor genes is known to lead to silencing of these genes. The purine analog cladribine has been shown to have hypomethylating properties and has activity as a single agent or in combination with other therapies for mantle cell lymphoma. Epigenetic therapy with the DNA hypomethylating agent 5-aza-2-deoxycytidine can also cause restoration of cell surface expression of the CD20 protein and increase rituximab sensitivity in vitro. Combinations of epigenetic agents may act synergistically to further potentiate the efficacy of monoclonal antibodies like rituximab and ofatumumab and improve the treatment outcome in MCL.

Transcriptional regulation of HLH-6-independent and subtype-specific genes expressed in the Caenorhabditis elegans pharyngeal glands.

The Caenorhabditis elegans pharyngeal glands represent one of five cell types in the pharynx. We have previously shown that the bHLH transcription factor, HLH-6, is required for gland development and for expression of many, but not all, gland genes (Smit et al., 2008). Here, we have identified additional gland-expressed genes and find that transcriptional regulatory inputs other than HLH-6 are necessary for their regulation. We demonstrate that at least two hlh-6 independent gland genes, nas-12 and Y8A9A.2, require a cis-acting motif (HRL3- Hlh-6 Regulatory eLement 3), previously described based on its requirement for hlh-6 expression (Ghai and Gaudet, 2008). We also show that expression of the gland-expressed genes, ZK596.1, scl-3, wrt-3, and Y76B12C.3, rely on cis-elements and trans-acting factor(s) other than HLH-6 and HRL3. In addition, we show that negative regulatory mechanisms are employed to refine the spatial expression of some genes, resulting in expression in only a subset of the five gland cells. We show that one of these genes, Y8A9A.2, is negatively regulated by the NHR transcription factor encoded by nhr-48, which represses Y8A9A.2 expression in the g1A cells. We also show that another gene expressed in the reciprocal subset of gland cells, phat-5, is negatively regulated in the g1P and g2 cells by an unknown factor acting through a conserved cis-element in the phat-5 promoter. Overall, this work reveals levels of regulation of gene expression in a single cell type beyond that previously known, and suggests mechanisms by which the different gland sub-types are distinguished.

Cell architecture: surrounding muscle cells shape gland cell morphology in the Caenorhabditis elegans pharynx.

The acquisition and maintenance of shape is critical for the normal function of most cells. Here we investigate the morphology of the pharyngeal glands of Caenorhabditis elegans. These unicellular glands have long cellular processes that extend discrete lengths through the pharyngeal musculature and terminate at ducts connected to the pharyngeal lumen. From a genetic screen we identified several mutants that affect pharyngeal gland morphology. The most severe such mutant is an allele of sma-1, which encodes a ß-spectrin required for embryonic elongation, including elongation of the pharynx. In sma-1 mutants, gland projections form normally but become increasingly abnormal over time, acquiring additional branches, outgrowths, and swelling, suggestive of hypertrophy. Rather than acting in pharyngeal glands, sma-1 functions in the surrounding musculature, suggesting that pharyngeal muscles play a critical role in maintenance of gland morphology by restricting their growth, and analysis of other mutants known to affect pharyngeal muscles supports this hypothesis. We suggest that gland morphology is maintained by a balance of forces from the muscles and the glands.

The CSL transcription factor LAG-1 directly represses hlh-6 expression in C. elegans.

The Caenorhabditis elegans gene hlh-6 is expressed specifically in pharyngeal glands, one of five distinct pharyngeal cell types. Expression of hlh-6 is controlled by a discrete set of cis-regulatory elements, including a negative element called HRL1. Here we demonstrate that HRL1 is a functional binding site for LAG-1, the CSL transcriptional effector of Notch in C. elegans, and that regulation of hlh-6 by LAG-1 is direct. Regulation of hlh-6 by LAG-1 is strictly negative: removal of HRL1 or LAG-1 regulation results in ectopic expression of hlh-6, but does not affect expression in pharyngeal glands. Furthermore, direct regulation of hlh-6 expression does not appear to involve Notch signaling, contrary to the canonical mechanism by which CSL factors regulate target genes. We also identify an additional cis-regulatory element in the hlh-6 promoter that, together with previously identified elements, is sufficient to overcome repression by LAG-1 and activate hlh-6 expression in pharyngeal glands.

Fulminant postcesarean Clostridium difficile pseudomembranous colitis.

BACKGROUND: Pseudomembranous colitis due to Clostridium difficile infection is rarely reported in the obstetric literature. This disease process is associated with prior antibiotic exposure. CASE: A term primigravida was delivered by primary cesarean for failed vacuum extraction. She received Intravenous cefazolin after cord clamping, which was continued for 36 hours for a presumptive diagnosis of endometritis. On day 3, oral amoxicillin and clavulanate was started for suspected cellulitis of the incision. She was readmitted 1 day after her discharge with severe diffuse abdominal pain and distention. Proctoscopy showed pseudomembranous colitis. Colectomy with temporary ileostomy was performed for worsening symptoms and imminent perforation. CONCLUSION: The diagnosis of pseudomembranous colitis should be considered in postpartum women who have low-grade fever, abdominal and gastrointestinal symptoms, and recent antibiotic exposure.

Activity of continuous infusion plus pulse interleukin-2 with famotidine in patients with metastatic kidney cancer or melanoma previously treated with interleukin-2.

Lymphokine-activated killer (LAK) cells generated by high-dose continuous infusion interleukin-2 (IL-2) are able to nonspecifically lyse melanoma and kidney cancer cells. In vitro famotidine enhances cytotoxicity of LAK against tumor cells, possibly by increasing IL-2 uptake at the IL-2 receptor on lymphocytes. Outpatient IL-2 regimens typically have response rates of 15% or less, with most patients eventually experiencing progressive disease. Second-line therapy is, therefore, needed. We treated 11 patients (6 with metastatic melanoma; 5 having metastatic kidney cancer) who had previously experienced progressive disease on prior IL-2 regimens, with a combination of famotidine 20 mg intravenously (i.v.) twice per day and continuous-infusion IL-2 18 MIU/M2/24 hours x 72 hours, followed 24 hours later by a pulse IL-2 dose (18 MIU/M2 over 15 minutes). Cycles were repeated every 3 weeks. Patient characteristics were: 9 males, median age 63 years (range, 57-75), median Eastern Cooperative Oncology Group (ECOG) performance status: 1; most common metastatic sites: lungs, lymph nodes, and soft tissue/subcutaneous (s.c.); median number of cycles received: 4; most common toxicities were fever, nausea/emesis, hypophosphatemia, and hypomagnesemia. Five (5) patients (3 with melanoma, 2 with kidney cancer) have had partial responses. Two (2) patients with kidney cancer have been converted to complete responders with resection of residual disease, remaining without relapse at 5+ and 20+ months. Responding sites are lungs, lymph nodes, abdominal mass, and s.c. Median duration of response was 9.5 months. Median survival was 12 months. This combination has activity in patients with metastatic kidney cancer or melanoma who have received prior IL-2.