Contribution of Extracellular Particles Isolated from Morus sp. (Mulberry) Fruit to Their Reported Protective Health Benefits: An In Vitro Study.

Morus sp. (mulberry) has a long tradition of use as a medicinal treatment, including for cardiovascular disease and type 2 diabetes, being shown to have antioxidant properties and to promote wound healing. Extracellular vesicles (EVs) are sub-micron, membrane-enclosed particles that were first identified in mammalian bodily fluids. EV-like particles have been described in plants (PDVs) and shown to have similar characteristics to mammalian EVs. We hypothesised that some of the health benefits previously attributed to the fruit of Morus sp. could be due to the release of PDVs. We isolated PDVs from Morus nigra and Morus alba via ultracentrifugation and incubated THP-1 monocytes, differentiated THP-1 macrophages, or HMEC-1 endothelial cells with pro-oxidant compounds DMNQ (THP-1) and glucose oxidase (HMEC-1) or lipopolysaccharide (LPS) in the presence of different fractions of mulberry EVs. Mulberry EVs augmented ROS production with DMNQ in THP-1 and caused the downregulation of ROS in HMEC-1. Mulberry EVs increased LPS-induced IL-1ß secretion but reduced CCL2 and TGF-ß secretion in THP-1 macrophages. In scratch wound assays, mulberry EVs inhibited HMEC-1 migration but increased proliferation in both low and high serum conditions, suggesting that they have opposing effects in these two important aspects of wound healing. One of the limitations of plant-derived therapeutics has been overcoming the low bioavailability of isolated compounds. We propose that PDVs could provide the link between physiological dose and therapeutic benefit by protecting plant active compounds in the GIT as well as potentially delivering genetic material or proteins that contribute to previously observed health benefits.

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues.

Extracellular Vesicles: Evolutionarily Conserved Mediators of Intercellular Communication.

Extracellular vesicles (EV) are sub-micron circulating vesicles found in all bodily fluids and in all species so far tested. They have also recently been identified in seawater and it has further been shown that they are released from microorganisms and may participate in interspecies communication in the gut. EV are typically composed of a lipid bilayer formed from the plasma membrane and which encases a cargo that can include genetic material, proteins, and lipids. At least two different processes of formation and release have been described in mammalian cells. The exosome population (50 to 150nm size) are produced via a lyso-endosomal pathway, while microvesicles (100 to 1000nm) are formed by budding of the plasma membrane in a calcium dependent process. Both pathways are highly regulated and appear to be conserved amongst different species. EV release has been shown to be upregulated in a number of human chronic diseases including cardiovascular disease, metabolic disorders, obesity, and cancer; evaluation of their presence in veterinary samples may aid diagnosis in the future. This review will provide insight into the formation of EV and their detection in bodily fluids from different veterinary species and how they may provide a novel addition to the veterinary toolkit of the future.

"Patients who can't get an appointment go to the ER": access to specialty care for publicly insured children.

STUDY OBJECTIVE: Emergency departments (EDs) frequently refer patients for needed outpatient specialty care, but little is known about the dynamics of these referrals when patients are publicly insured. Hence, we explored factors, including the role of ED referrals, associated with specialists' willingness to accept patients covered by Medicaid and the Children's Health Insurance Program (CHIP). METHODS: We conducted semistructured qualitative interviews with a purposive sample of 26 specialists and 14 primary care physicians in Cook County, Illinois, from April to September 2009, until theme saturation was reached. Transcripts and notes were entered into ATLAS.ti and analyzed using an iterative coding process to identify patterns of responses, ensure reliability, examine discrepancies, and achieve consensus through content analysis. RESULTS: Themes that emerged indicate that primary care physicians face considerable barriers getting publicly insured patients into outpatient specialty care and use the ED to facilitate this process. Specialty physicians reported that decisions to refuse or limit the number of patients with Medicaid/CHIP are due to economic strain or direct pressure from their institutions. Factors associated with specialist acceptance of patients with Medicaid/CHIP included high acuity or complexity, personal request from or an informal economic relationship with the primary care physician, geography, and patient hardship. Referral through the ED was a common and expected mechanism for publicly insured patients to access specialty care. CONCLUSION: These exploratory findings suggest that specialists are willing to see children with Medicaid/CHIP if they are referred from an ED. As health systems restructure, EDs have the potential to play a role in improving care coordination and access to outpatient specialty care.

Health Benefits of Montmorency Tart Cherry Juice Supplementation in Adults with Mild to Moderate Ulcerative Colitis: A Protocol for a Placebo Randomized Controlled Trial.

Ulcerative colitis, characterized by its relapsing and remissive nature, negatively affects perception, body image, and overall quality of life. The associated financial burden underscores the need for alternative treatment approaches with fewer side effects, alongside pharmaceutical interventions. Montmorency tart cherries, rich in anthocyanins, have emerged as a potential natural anti-inflammatory agent for ulcerative colitis. This manuscript outlines the study protocol for a randomized placebo-controlled trial investigating the effects of Montmorency tart cherry in individuals with ulcerative colitis. The trial aims to recruit 40 participants with mild to moderate disease activity randomly assign them to either a Montmorency tart cherry or placebo group. The intervention will span 6 weeks, with baseline and 6-week assessments. The primary outcome measure is the Inflammatory Bowel Disease Quality of Life Questionnaire. Secondary outcomes include other health-related questionnaires and biological indices. Statistical analysis will adhere to an intention-to-treat approach using linear mixed effect models. Ethical approval has been obtained from the University of Hertfordshire (cLMS/SF/UH/05240), and the trial has been registered as a clinical trial (NCT05486507). The trial findings will be disseminated through a peer-reviewed publication in a scientific journal.

Exploration of Mediators Associated with Myocardial Remodelling in Feline Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) affects both humans and cats and exhibits considerable interspecies similarities that are exemplified by underlying pathological processes and clinical presentation to the extent that developments in the human field may have direct relevance to the feline disease. Characteristic changes on histological examination include cardiomyocyte hypertrophy and interstitial and replacement fibrosis. Clinically, HCM is characterised by significant diastolic dysfunction due to a reduction in ventricular compliance and relaxation associated with extracellular matrix (ECM) remodelling and the development of ventricular hypertrophy. Studies in rodent models and human HCM patients have identified key protein mediators implicated in these pathological changes, including lumican, lysyl oxidase and TGF-ß isoforms. We therefore sought to quantify and describe the cellular location of these mediators in the left ventricular myocardium of cats with HCM and investigate their relationship with the quantity and structural composition of the ECM. We identified increased myocardial content of lumican, LOX and TGF-ß2 mainly attributed to their increased expression within cardiomyocytes in HCM cats compared to control cats. Furthermore, we found strong correlations between the expressions of these mediators that is compatible with their role as important components of cellular pathways promoting remodelling of the left ventricular myocardium. Fibrosis and hypertrophy are important pathological changes in feline HCM, and a greater understanding of the mechanisms driving this pathology may facilitate the identification of potential therapies.

MIBlood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.

Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor-dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre-analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood-EV), a tool to record and report information about pre-analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre-analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence-based optimization of pre-analytical protocols through meta-analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter-laboratory comparative EV studies, and (iv) improve the peer review process.

Inhibition of macrophage inflammatory cytokine secretion by chylomicron remnants is dependent on their uptake by the low density lipoprotein receptor.

Secretion of pro-inflammatory chemokines and cytokines by macrophages is a contributory factor in the pathogenesis of atherosclerosis. In this study, the effects of chylomicron remnants (CMR), the lipoproteins which transport dietary fat in the blood, on the production of pro-inflammatory chemokine and cytokine secretion by macrophages was investigated using CMR-like particles (CRLPs) together with THP-1 macrophages or primary human macrophages (HMDM). Incubation of CRLPs or oxidized CRLPs (oxCRLPs) with HMDM or THP-1 macrophages for up to 24h led to a marked decrease in the secretion of the pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1) and the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß (-50-90%), but these effects were reduced or abolished when CRLPs protected from oxidation by incorporation of the antioxidant drug, probucol, (pCRLPs) were used. In macrophages transfected with siRNA targeted to the low density lipoprotein receptor (LDLr), neither CRLPs nor pCRLPs had any significant effect on chemokine/cytokine secretion, but in cells transfected with siRNA targeted to the LDLr-related protein 1 (LRP1) both types of particles inhibited secretion to a similar extent to that observed with CRLPs in mock transfected cells. These findings demonstrate that macrophage pro-inflammatory chemokine/cytokine secretion is down-regulated by CMR, and that these effects are positively related to the lipoprotein oxidative state. Furthermore, uptake via the LDLr is required for the down-regulation, while uptake via LRP1 does not bring about this effect. Thus, the receptor-mediated route of uptake of CMR plays a crucial role in modulating their effects on inflammatory processes in macrophages.

Microparticle formation after co-culture of human whole blood and umbilical artery in a novel in vitro model of flow.

Cardiovascular disease (CVD) is now the largest killer in western society, and the importance of interactions between vascular endothelium and circulating blood components in disease pathogenesis is well established. Microparticles are a heterogeneous population of <1 µm blood borne particles that arise from blebbing or shedding of cell membranes. The microparticle population includes several classes of apoptotic bodies; however, increased numbers of procoagulant microparticles have been described in plasma from people with CVD. We have previously demonstrated that interactions of monocytes and platelets with isolated inflamed endothelial cells lead to production of pro-coagulant tissue factor bearing microparticles under laminar flow conditions. Here we have investigated microparticle production after perfusion of human whole blood through intact inflamed human umbilical artery. When blood was perfused through umbilical arteries which had been pre-stimulated with tumour necrosis factor (TNFα) for 18 h under flow conditions, there was significantly increased production of microparticles from both platelet and non-platelet sources, in particular from erythrocytes. To determine whether microparticles generated during interactions with inflamed endothelium could induce a pro-inflammatory response in trans, we isolated microparticles by centrifugation after co-culture and incubated with isolated quiescent endothelial cells followed by measurement of reactive oxygen species formation. Microparticles derived from co-culture with inflamed endothelium induced significantly enhanced levels of reactive oxygen species (ROS). These data suggest that presence of an inflamed endothelium causes release of pro-inflammatory microparticles from circulating blood cells, which could contribute to prolonged endothelial activation and subsequent atherosclerotic changes in blood vessels subjected to inflammatory insult.

Sensitivity of the Natriuretic Peptide/cGMP System to Hyperammonaemia in Rat C6 Glioma Cells and GPNT Brain Endothelial Cells.

C-type natriuretic peptide (CNP) is the major natriuretic peptide of the central nervous system and acts via its selective guanylyl cyclase-B (GC-B) receptor to regulate cGMP production in neurons, astrocytes and endothelial cells. CNP is implicated in the regulation of neurogenesis, axonal bifurcation, as well as learning and memory. Several neurological disorders result in toxic concentrations of ammonia (hyperammonaemia), which can adversely affect astrocyte function. However, the relationship between CNP and hyperammonaemia is poorly understood. Here, we examine the molecular and pharmacological control of CNP in rat C6 glioma cells and rat GPNT brain endothelial cells, under conditions of hyperammonaemia. Concentration-dependent inhibition of C6 glioma cell proliferation by hyperammonaemia was unaffected by CNP co-treatment. Furthermore, hyperammonaemia pre-treatment (for 1 h and 24 h) caused a significant inhibition in subsequent CNP-stimulated cGMP accumulation in both C6 and GPNT cells, whereas nitric-oxide-dependent cGMP accumulation was not affected. CNP-stimulated cGMP efflux from C6 glioma cells was significantly reduced under conditions of hyperammonaemia, potentially via a mechanism involving changed in phosphodiesterase expression. Hyperammonaemia-stimulated ROS production was unaffected by CNP but enhanced by a nitric oxide donor in C6 cells. Extracellular vesicle production from C6 cells was enhanced by hyperammonaemia, and these vesicles caused impaired CNP-stimulated cGMP signalling in GPNT cells. Collectively, these data demonstrate functional interaction between CNP signalling and hyperammonaemia in C6 glioma and GPNT cells, but the exact mechanisms remain to be established.

Microparticle formation after exposure of blood to activated endothelium under flow.

Increased numbers of circulating microparticles (MPs) are indicative of poor clinical outcome in a number of inflammatory disorders, including atherosclerosis. Platelets and megakaryocytes are a major source of MP and are identified by presence of CD42b on the MP surface. MP shed from activated platelets can be identified by presence of P-selectin (CD62P). Tissue factor (TF) is the principal initiator of blood coagulation and its activity has been identified in MPs derived from patient plasma, which may contribute to thrombosis. Here, we have investigated by flow cytometry the expression of TF and CD62P on MP after exposure of diluted whole blood to TNF-activated endothelial cells (EC) both under static conditions and in our newly established model of flow. MPs were significantly increased in blood subjected to flow and this was further enhanced after exposure of blood to TNF-activated EC. MP surface expression of CD62P or TF was upregulated following exposure to TNF-activated EC under flow compared with flow with nonactivated EC or after static coculture with and without prior EC activation. These data strongly suggest that interactions of blood with inflamed EC can modulate production of CD62P and TF bearing MP under flow conditions, and thus may contribute to a prothrombotic environment.

ICAM-1 signaling in endothelial cells.

Intercellular adhesion molecule-1 (ICAM-1; CD54) is a 90 kDa member of the immunoglobulin (Ig) superfamily and is critical for the firm arrest and transmigration of leukocytes out of blood vessels and into tissues. ICAM-1 is constitutively present on endothelial cells, but its expression is increased by proinflammatory cytokines. The endothelial expression of ICAM-1 is increased in atherosclerotic and transplant-associated atherosclerotic tissue and in animal models of atherosclerosis. Additionally, ICAM-1 has been implicated in the progression of autoimmune diseases. We and others have shown that the ligation of ICAM-1 on the surface of endothelial or smooth muscle cells with monoclonal antibodies, via its main leukocyte ligand, lymphocyte function associated molecule (LFA)-1, or with antibodies derived from patient serum, leads to the activation of several proinflammatory signaling cascades, and to the rearrangement of the actin cytoskeleton. A circulating or soluble form of ICAM-1 (sICAM-1) has been measured in various body fluids, with elevated levels being observed in patients with atherosclerosis, heart failure, coronary artery disease and transplant vasculopathy. sICAM-1 has signaling properties in several cell types, including EC, and invokes a range of proinflammatory responses. Thus, we propose that in addition to acting as a leukocyte adhesion molecule, ICAM-1 directly contributes to inflammatory responses within the blood vessel wall by increasing endothelial cell activation and augmenting atherosclerotic plaque formation.

Use of Technology to Identify and Cover Underweighted Topics in Resident Conference Curriculum.

Emergency medicine training programs face many challenges in creating and maintaining high quality didactic and asynchronous learning experiences. To address these challenges, our team created two tools. First, we designed the Emergency Medicine Curriculum Assessment Tool (EMCAT) to help program leaders compare their didactic program to the Model of Clinical Practice established by the American Board of Emergency Medicine (ABEM). Second, we created a catalog of free, open-access medical education (FOAMed) resources based on the ABEM Model subcategory. Residency leaders can use EMCAT to identify the underweighted topics in their conference program and then access the resource catalog to find educational content matched to their areas of increased need. To date, five programs have implemented EMCAT and users from over 72 countries have accessed nearly 1,000 resources. Both EMCAT and the resource catalog are available free online.

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids ("liquid biopsies"). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.

Expression of endothelial intercellular adhesion molecule-1 is determined by genotype: effects on efficiency of leukocyte adhesion to human endothelial cells.

Two biallelic polymorphisms, previously described in the human intercellular adhesion molecule (ICAM)-1 gene at codon 241 (glycine [G] to arginine [R] substitution) and codon 469 (glutamic acid [E] to lysine [K] substitution) have been associated with a number of diseases including myocardial infarction, transplant rejection, and diabetes. However, the functional significance of these polymorphisms has not been determined. ICAM-1 cell surface expression and ICAM-1-mediated leukocyte adhesion were investigated using Cos7 transfected with ICAM-1 polymorphic variants or human umbilical vein endothelial cells (HUVEC) of different ICAM-1 genotypes. There was significantly higher expression of surface ICAM-1 on Cos7 transfected with a plasmid encoding the GE (G241/E469) ICAM-1 variant or untreated HUVEC of GEGE (G241/E469 homozygous genotype). ICAM-1-mediated adhesion of peripheral blood mononuclear cells (PBMC) to GE-Cos7 cells or TNF-treated GEGE HUVEC was significantly increased. However, there was no significant difference in adhesion of PBMC to recombinant ICAM-1 of each polymorphic variant plated onto plastic wells. We conclude that the GE genotype of ICAM-1 is associated with greater cell surface expression of ICAM-1, which in turn leads to greater adhesion of leukocytes. This may explain the previously described associations of ICAM-1 polymorphisms with chronic inflammatory disease.

C-Type Natriuretic Peptide (CNP) Inhibition of Interferon-γ-Mediated Gene Expression in Human Endothelial Cells In Vitro.

Cardiovascular diseases, including atherosclerosis, now account for more deaths in the Western world than from any other cause. Atherosclerosis has a chronic inflammatory component involving Th1 pro-inflammatory cytokines such as IFN-γ, which is known to induce endothelial cell inflammatory responses. On the other hand CNP, which acts via its receptors to elevate intracellular cGMP, is produced by endothelium and endocardium and is upregulated in atherosclerosis. It is believed to be protective, however its role in vascular inflammation is not well understood. The aim of this study was to investigate the effects of CNP on human endothelial cell inflammatory responses following IFN-γ stimulation. Human umbilical vein endothelial cells were treated with either IFN-γ (10 ng/mL) or CNP (100 nm), or both in combination, followed by analysis by flow cytometry for expression of MHC class I and ICAM-1. IFN-γ significantly increased expression of both molecules, which was significantly inhibited by CNP or the cGMP donor 8-Bromoguanosine 3',5'-cyclic monophosphate (1 µm). CNP also reduced IFN-γ mediated kynurenine generation by the IFN-γ regulated enzyme indoleamine-2,3-deoxygenase (IDO). We conclude that CNP downmodulates IFN-γ induced pro-inflammatory gene expression in human endothelial cells via a cGMP-mediated pathway. Thus, CNP may have a protective role in vascular inflammation and novel therapeutic strategies for CVD based on upregulation of endothelial CNP expression could reduce chronic EC inflammation.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

Leukocyte Adhesion Under Hemodynamic Flow Conditions.

Vascular endothelial cells (ECs) line the luminal side of all blood vessels and act as a selective barrier between blood and tissue. ECs are constantly exposed to biochemical and biomechanical stimuli from the blood and underlying tissue. Fluid shear stress acts in parallel to the vessel wall, resulting from friction of blood against EC. Despite the importance of flow on normal EC function, much of the information regarding EC function and dysfunction has been derived from cells harvested, grown, and studied in static culture.In order to study the effects of shear stress on EC function a number of in vitro models have been developed. This manuscript provides methodology for use of a system which enables recirculation of leukocytes and cell culture medium over the endothelium for a period of several minutes to days and enables investigation of the effects of prolonged leukocyte coculture on both the endothelial and leukocyte populations.

Brugia malayi microfilariae adhere to human vascular endothelial cells in a C3-dependent manner.

Brugia malayi causes the human tropical disease, lymphatic filariasis. Microfilariae (Mf) of this nematode live in the bloodstream and are ingested by a feeding mosquito vector. Interestingly, in a remarkable co-evolutionary adaptation, Mf appearance in the peripheral blood follows a circadian periodicity and reaches a peak when the mosquito is most likely to feed. For the remaining hours, the majority of Mf sequester in the lung capillaries. This circadian phenomenon has been widely reported and is likely to maximise parasite fitness and optimise transmission potential. However, the mechanism of Mf sequestration in the lungs remains largely unresolved. In this study, we demonstrate that B. malayi Mf can, directly adhere to vascular endothelial cells under static conditions and under flow conditions, they can bind at high (but not low) flow rates. High flow rates are more likely to be experienced diurnally. Furthermore, a non-periodic nematode adheres less efficiently to endothelial cells. Strikingly C3, the central component of complement, plays a crucial role in the adherence interaction. These novel results show that microfilariae have the ability to bind to endothelial cells, which may explain their sequestration in the lungs, and this binding is increased in the presence of inflammatory mediators.