Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments

C-Reactive Protein, Erythrocyte Sedimentation Rate and Orthopedic Implant Infection

BACKGROUND: C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have been shown to be useful for diagnosis of prosthetic hip and knee infection. Little information is available on CRP and ESR in patients undergoing revision or resection of shoulder arthroplasties or spine implants. METHODS/RESULTS: We analyzed preoperative CRP and ESR in 636 subjects who underwent knee (n=297), hip (n=221) or shoulder (n=64) arthroplasty, or spine implant (n=54) removal. A standardized definition of orthopedic implant-associated infection was applied. Receiver operating curve analysis was used to determine ideal cutoff values for differentiating infected from non-infected cases. ESR was significantly different in subjects with aseptic failure infection of knee (median 11 and 53.5 mm/h, respectively, p=<0.0001) and hip (median 11 and 30 mm/h, respectively, p=<0.0001) arthroplasties and spine implants (median 10 and 48.5 mm/h, respectively, p=0.0033), but not shoulder arthroplasties (median 10 and 9 mm/h, respectively, p=0.9883). Optimized ESR cutoffs for knee, hip and shoulder arthroplasties and spine implants were 19, 13, 26, and 45 mm/h, respectively. Using these cutoffs, sensitivity and specificity to detect infection were 89 and 74% for knee, 82 and 60% for hip, and 32 and 93% for shoulder arthroplasties, and 57 and 90% for spine implants. CRP was significantly different in subjects with aseptic failure and infection of knee (median 4 and 51 mg/l, respectively, p<0.0001), hip (median 3 and 18 mg/l, respectively, p<0.0001), and shoulder (median 3 and 10 mg/l, respectively, p=0.01) arthroplasties, and spine implants (median 3 and 20 mg/l, respectively, p=0.0011). Optimized CRP cutoffs for knee, hip, and shoulder arthroplasties, and spine implants were 14.5, 10.3, 7, and 4.6 mg/l, respectively. Using these cutoffs, sensitivity and specificity to detect infection were 79 and 88% for knee, 74 and 79% for hip, and 63 and 73% for shoulder arthroplasties, and 79 and 68% for spine implants. CONCLUSION: CRP and ESR have poor sensitivity for the diagnosis of shoulder implant infection. A CRP of 4.6 mg/l had a sensitivity of 79 and a specificity of 68% to detect infection of spine implants

MiR-155 Induction by F. novicida but Not the Virulent F. tularensis Results in SHIP Down-Regulation and Enhanced Pro-Inflammatory Cytokine Response

The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella

M-CSF Induces Monocyte Survival by Activating NF-κB p65 Phosphorylation at Ser276 via Protein Kinase C

Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-κB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-κB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-κB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCα/β inhibitor Gö-6976, overexpression of dominant negative PKCα constructs and PKCα siRNA reduced NF-κB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-κBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCα as important upstream kinases for M-CSF-induced NF-κB transcriptional activation, NF-κB-regulated gene expression, NF-κB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-κB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-κB activation in human mononuclear phagocytes

Thrombospondin-1 Contributes to Mortality in Murine Sepsis through Effects on Innate Immunity

BACKGROUND:Thrombospondin-1 (TSP-1) is involved in many biological processes, including immune and tissue injury response, but its role in sepsis is unknown. Cell surface expression of TSP-1 on platelets is increased in sepsis and could activate the anti-inflammatory cytokine transforming growth factor beta (TGFβ1) affecting outcome. Because of these observations we sought to determine the importance of TSP-1 in sepsis. METHODOLOGY/PRINCIPAL FINDINGS:We performed studies on TSP-1 null and wild type (WT) C57BL/6J mice to determine the importance of TSP-1 in sepsis. We utilized the cecal ligation puncture (CLP) and intraperitoneal E. coli injection (i.p. E. coli) models of peritoneal sepsis. Additionally, bone-marrow-derived macrophages (BMMs) were used to determine phagocytic activity. TSP-1-/- animals experienced lower mortality than WT mice after CLP. Tissue and peritoneal lavage TGFβ1 levels were unchanged between animals of each genotype. In addition, there is no difference between the levels of major innate cytokines between the two groups of animals. PLF from WT mice contained a greater bacterial load than TSP-1-/- mice after CLP. The survival advantage for TSP-1-/- animals persisted when i.p. E. coli injections were performed. TSP-1-/- BMMs had increased phagocytic capacity compared to WT. CONCLUSIONS:TSP-1 deficiency was protective in two murine models of peritoneal sepsis, independent of TGFβ1 activation. Our studies suggest TSP-1 expression is associated with decreased phagocytosis and possibly bacterial clearance, leading to increased peritoneal inflammation and mortality in WT mice. These data support the contention that TSP-1 should be more fully explored in the human condition

Detection of microRNA Expression in Human Peripheral Blood Microvesicles

MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function.This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA

Understanding the burden of interstitial lung disease post-COVID-19: the UK Interstitial Lung Disease-Long COVID Study (UKILD-Long COVID)

Introduction The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD). Methods and analysis The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment. Ethics and dissemination All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals. Conclusion This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD