Microparticle Counts Research Articles

Platelet-, leucocyte- and red cell-derived microparticles in stored whole blood, with and without leucofiltration, with and without ionising radiation.

Storage lesion, including microparticle formation, has been partially characterised in whole blood, but not in all combinations of pre-storage leucofiltration and/or irradiation. Single-donor whole blood products were processed into four subunits: with and without leucofiltration, with and without X-irradiation (25 Gy). Platelet-, leucocyte-, and erythrocyte-derived microparticles and free haemoglobin were measured periodically throughout 42 days of storage. Pre-storage leucofiltration substantially reduced platelet- and leucocyte-derived microparticle counts throughout storage. Irradiation, in contrast, had no significant effect on microparticle counts. A gate for all microparticles showed a substantial time-dependent increase in unfiltered whole blood. A time-dependent increase in free haemoglobin was greatest in unfiltered, irradiated whole blood. This study indicates that leucofiltration can prevent the formation of leucocyte- and platelet-derived microparticles, and might reduce haemolysis in irradiated whole blood, either by removing factors that provoke haemolysis, or by selective retention of senescent or effete red cells most prone to haemolysis.

37 1,25(OH)2D3 suppresses oxidative stress – Induced microparticle release by placental trophoblasts

Introduction Emerging evidence showed that microparticles (MPs) derived from placental trophoblasts (TC) contribute to maternal vascular dysfunction and promote endothelial inflammatory response in preeclampsia (PE). Vitamin D insufficiency has been linked to placental dysfunction in pregnancy disorders such as PE and IUGR. Objective To determine: (1) if TC from PE placentas release more MPs than TC from normal placentas; (2) if vitamin D could suppress oxidative stress-induced MP release by placental TC; and (3) potential mechanism of vitamin D protective effects on placental TCs. Methods TC were isolated from normal and PE placentas by trypsin digestion and purified with Percoll gradient centrifugation. Freshly isolated TC (5×06cells/well) were seeded into 6 well plates and cultured with DMEM containing 5% FBS and antibiotics for 72h. For the oxidative stress experiment, cobalt chloride (CoCl 2 , a hypoxic mimicking agent) was used to induce TC oxidative stress and 1,25(OH)2D3 was used as bioactive vitamin D. MPs were isolated from culture supernatants by a two-step centrifugation procedure (420g for 20min and then 20,000g for 60min) and quantified by annexin V positive staining measured by flow cytometry with TruCount beads to determine absolute counts of MP for each sample. Data are expressed as annexin V positive MP/μg cell protein/well. Total cellular protein and MP protein were also collected and protein expression for caveolin-1, eNOS, pro-caspase-3, cleaved caspase-3, and ROCK1 were determined by Western blot. Data were analyzed by paired or unpaired t-test or ANOVA with Newman-Keuls test as a post hoc test. A p level Results TC isolated from PE placentas ( n =6) released significantly more MP than cells isolated from normal placentas ( n =6), 295.20±47.67 vs. 117.20±18.02/μg protein/well, p 2 compared to control TC, p 2 was significantly reduced in TC treated with 1,25(OH)2D3+CoCl 2 , p 2 . This phenomenon could be reversed when cells were treated with 1,25(OH)2D3. Moreover, reduced pro-caspase-3 and ROCK1 expression in TC induced by CoCl 2 could also be attenuated in cells treated with1,25(OH)2D3. Conclusions MP release was significantly increased in TC from PE placenta. 1,25(OH)2D3 suppresses oxidative stress-induced MP release by placental TC. Caveolin-1 is an essential structural component of caveolae and eNOS is associated with plasma membrane caveolae. Therefore, reduced caveolin-1 and increased eNOS expression together with reduced MP release in TC treated with 1,25(OH)2D3 demonstrate that vitamin D could protect placental TC from oxidative stress-induced injury during pregnancy. Since caspase-3 cleavage and ROCK1 activation are key steps of MP formation and release, suppression of caspase-3 cleavage and ROCK1 activation by vitamin D could be a plausible mechanism of vitamin D inhibition of increased MP release induced by oxidative stress.

Increased circulating cell-derived microparticle count is associated with recurrent implantation failure after IVF and embryo transfer

Cell-derived microparticles (cMPs) are small membrane vesicles that are released from many different cell types in response to cellular activation or apoptosis. Elevated cMP counts have been found in almost all thrombotic diseases and pregnancy wastage, such as recurrent spontaneous abortion and in a number of conditions associated with inflammation, cellular activation and angiogenesis. cMP count was investigated in patients experiencing unexplained recurrent implantation failure (RIF). The study group was composed of 30 women diagnosed with RIF (RIF group). The first control group (IVF group) (n = 30) comprised patients undergoing a first successful IVF cycle. The second control group (FER group) included 30 healthy women who had at least one child born at term and no history of infertility or obstetric complications. cMP count was significantly higher in the RIF group compared with the IVF and FER groups (P < 0.05 and P < 0.01, respectively) (RIF group: 15.8 ± 6.2 nM phosphatidylserine equivalent [PS eq]; IVF group: 10.9 ± 5.3 nM PS eq; FER group: 9.6 ± 4.0 nM PS eq). No statistical difference was found in cMP count between the IVF and FER groups. Increased cMP count is, therefore, associated with RIF after IVF and embryo transfer.

Endothelial, platelet, and macrophage microparticle levels do not change acutely following transcatheter aortic valve replacement.

BackgroundPatients with severe aortic stenosis have increased levels of prothrombotic and proinflammatory microparticles (MP), and MPs actively regulate pathological processes that lead to atherothrombotic cardiovascular events. Shear stress is a validated stimulus of MP production, and abnormal shear stress in aortic stenosis increases MP release in ex-vivo studies. We hypothesized that in patients with severe aortic stenosis, percutaneous replacement of the aortic valve (TAVR) would reduce abnormal shear stress and would decrease levels of circulating MPs.FindingsThe experimental protocol utilized flow cytometry (FC) and nanoparticle tracking analysis (NTA) to quantify circulating plasma MP levels in aortic stenosis patients at baseline and 5 days after TAVR. The baseline and 5 day MP counts measured by FC were 6.10⋅105 ± 1.21⋅105 MP/μL and 5.74⋅105 ± 9.54⋅104 MP/μL, respectively (p = 0.91). The baseline and 5 day MP counts measured by NTA were 9.29⋅1013 ± 1.66⋅1013 MP/μL and 3.95⋅1014 ± 3.11⋅1014 MP/μL, respectively (p = 0.91). When MPs were stratified by cell source, there was no difference in pre/post TAVR endothelial, platelet, or leukocyte MP levels.ConclusionLevels of circulating MPs do not change acutely following TAVR therapy for aortic stenosis.Trial registered at clinicaltrials.gov NCT02193035 on July 11, 2014.Electronic supplementary materialThe online version of this article (doi:10.1186/s12952-016-0051-2) contains supplementary material, which is available to authorized users.

Microparticles as biomarkers of lung disease: enumeration in biological fluids using lipid bilayer microspheres.

Extracellular vesicles, specifically microparticles (MPs), are rapidly gaining attention for their capacity to act as biomarkers for diagnosis, prognosis, or responsiveness to therapy in lung disease, in keeping with the concept of precision medicine. However, MP analysis by high-sensitivity flow cytometry (FCM) is complicated by a lack of accurate means for MP enumeration. To address this gap, we report here an enhanced FCM MP gating and enumeration technique based on the use of novel engineered lipid bilayer microspheres (LBMs). By comparison of LBM-based MP enumeration with conventional bead- or fluorescent-based FCM enumeration techniques and a gravimetric consumption gold standard, we found LBMs to be superior to commercial bead preparations, showing the smallest fixed bias and limits of agreement in Bland Altman analyses. LBMs had simultaneous capacity to aid FCM enumeration of MPs in plasma, BAL, and cell culture supernatants. LBM enumeration detected differences in MP counts in mice exposed to intraperitoneal lipopolysaccharide or saline. LBMs provided for 1) higher sensitivity for gating MPs populations, 2) reduced background within MP gates, 3) more appropriate size, and 4) an inexpensive alternative amenable to different fluorescent tags. LBM-based MP enumeration was useful for a series of different FCM systems assessed, whereas LBM gating benefited high- but not low-sensitivity FCM systems compared with fluorescence gating. By offering exclusive advantages over current means of gating and enumerating MPs, LBMs are uniquely suited to realizing the potential of MPs as biomarkers in biological lung fluids and facilitating precision medicine in lung disease.

The interface of inflammation and subclinical atherosclerosis in granulomatosis with polyangiitis (Wegener's): a preliminary study

The objective of this study is to assess the relationship between inflammatory disease in granulomatosis with polyangiitis (GPA, Wegener's) and the development of subclinical atherosclerosis. A total of 46 adult patients with GPA were enrolled. Disease status was measured by Birmingham vasculitis assessment scores as modified for GPA, vasculitis damage index, disease duration, and number of relapses. Classic atherosclerotic risk factors, platelet aggregation responses, and circulating microparticle (MP) levels were recorded. All patients underwent carotid artery intima-media thickness (IMT) measurement as outcome for subclinical atherosclerosis. In univariate analyses, systolic and diastolic blood pressure, creatinine, and age were significantly associated with higher IMT (ρ values 0.37, 0.38, 0.35, and 0.054, respectively [P < 0.02 for all]). In a multiple regression model, greater number of relapses, older age at the onset of disease, and higher diastolic blood pressure were found to be associated with higher IMT (P values 0.003, <0.001, and 0.031, respectively). MP counts and platelet reactivity correlated well with disease activity in GPA. Furthermore, MPs were found to activate vascular endothelial cells and platelets in vitro. The cumulative burden of systemic inflammation in GPA correlated with the development of subclinical atherosclerosis. The correlation with subclinical atherosclerosis could be because of glucocorticoid use and not the inflammatory process in GPA, giving the inherent bias that exits with the use of glucocorticoid with each relapse. The findings of increased levels of circulating leukocyte-derived MPs and enhanced platelet reactivity during relapse suggest possible roles for MPs and platelets in disease pathogenesis and support a growing literature that links inflammation, atherosclerosis, and platelet activation. This hypothesis is further substantiated by our demonstration that MPs isolated from plasma of GPA patients can activate platelets and vascular endothelial cells.

Micropartículas de pequeño tamaño como indicadores del estado agudo en la insuficiencia cardiaca sistólica

Introduction and objectivesMicroparticles are markers for cell activation and apoptosis and could provide valuable information that is not available from clinical data. This study assesses the clinical and biological relationship of small-sized microparticles in different forms of ischemic systolic heart failure and their relation to markers of inflammation and repair. MethodsWe compared 49 patients with acute heart failure, 39 with stable heart failure and 25 patients with stable coronary artery disease. Small-size microparticles counts were determined by high-resolution flow cytometry. Moreover, 3 different monocyte subpopulations and their expression of inflammatory and adhesive scavenger receptors were analyzed using a conventional flow cytometer. ResultsEndothelial CD144+ microparticle counts were decreased in heart failure groups (P=.008). Annexin V-binding microparticle counts were found increased in heart failure (P=.024) and in patients with lower functional class (P=.013). Platelet CD42b+ microparticle counts positively correlated with left ventricular ejection fraction (P=.006), and annexin V-binding microparticle counts with interleukin-6 levels in stable heart failure (P=.034). Annexin V-binding microparticle counts in the acute status strongly correlated with Toll-like receptor-4 expression on all monocyte subsets (all P<.01). Three months after admission with acute heart failure, annexin V-binding microparticle counts were positively correlated with receptors for interleukin-6, CD163 and CD204 (all P<.05). ConclusionsAnnexin V-binding microparticle counts constitute valuable hallmarks of acute decompensated state in systolic heart failure. The observed relationship between small-size annexin V-binding microparticles and scavenger receptors supports their involvement in the progression of the acute response to injury, and thus their contribution to the pathogenesis of acute decompensated heart failure.Full English text available from:www.revespcardiol.org/en

Small-size Microparticles as Indicators of Acute Decompensated State in Ischemic Heart Failure

Introduction and objectivesMicroparticles are markers for cell activation and apoptosis and could provide valuable information that is not available from clinical data. This study assesses the clinical and biological relationship of small-sized microparticles in different forms of ischemic systolic heart failure and their relation to markers of inflammation and repair. MethodsWe compared 49 patients with acute heart failure, 39 with stable heart failure and 25 patients with stable coronary artery disease. Small-size microparticles counts were determined by high-resolution flow cytometry. Moreover, 3 different monocyte subpopulations and their expression of inflammatory and adhesive scavenger receptors were analyzed using a conventional flow cytometer. ResultsEndothelial CD144+ microparticle counts were decreased in heart failure groups (P=.008). Annexin V-binding microparticle counts were found increased in heart failure (P=.024) and in patients with lower functional class (P=.013). Platelet CD42b+ microparticle counts positively correlated with left ventricular ejection fraction (P=.006), and annexin V-binding microparticle counts with interleukin-6 levels in stable heart failure (P=.034). Annexin V-binding microparticle counts in the acute status strongly correlated with toll-like receptor-4 expression on all monocyte subsets (all P<.01). Three months after admission with acute heart failure, annexin V-binding microparticle counts were positively correlated with receptors for interleukin-6, CD163 and CD204 (all P<.05). ConclusionsAnnexin V-binding microparticle counts constitute valuable hallmarks of acute decompensated state in systolic heart failure. The observed relationship between small-size annexin V-binding microparticles and scavenger receptors supports their involvement in the progression of the acute response to injury, and thus their contribution to the pathogenesis of acute decompensated heart failure.

Altered profile of circulating microparticles in rheumatoid arthritis patients.

Microparticles (MPs) could be considered biomarkers of cell damage and activation as well as novel signalling structures. Since rheumatoid arthritis (RA) is characterized by immune and endothelial activation, the main aim of the present study was to analyse MP counts in RA patients. Citrated-blood samples were obtained from 114 RA patients, 33 healthy controls (HC) and 72 individuals with marked cardiovascular (CV) risk without autoimmune manifestations (CVR). MPs were analysed in platelet-poor plasma (PPP) and different subsets were identified by their surface markers: platelet- (CD41+), endothelial- (CD146+), granulocyte- (CD66+), monocyte- (CD14+) and Tang- (CD3+CD31+) derived. Disease activity score (DAS28), clinical and immunological parameters as well as traditional CV risk factors (diabetes, hypertension, dyslipidaemia and obesity) were registered from clinical records and all data were integrated using Principal Component Analysis (PCA). Absolute MP number was increased in RA patients compared with HC and positively correlated with traditional CV risk factors, similar to that of CVR subjects. In addition, frequency of the different MP subsets was different in RA patients and significantly associated with disease features. Moreover, in vitro assays revealed that MPs isolated from RA patients were able to promote endothelial activation and exhibited detrimental effects on human microvascular endothelial cells (HMEC-I) endothelial cell functionality. Circulating MPs from RA patients displayed quantitative and qualitative alterations that are the result of both disease-specific and traditional CV risk factors. Accordingly, this MP pool exhibited in vitro detrimental effects on endothelial cells, thus supporting their role as biomarkers of vascular damage.

Procoagulant Platelet-Derived Microparticles Are Lower in Calreticulin-Than in-JAK2-Mutated Essential Thrombocythemia

Rationale: Essential Thrombocythemia (ET) is a Philadelphia-negative myeloproliferative neoplasm characterized by an increased risk of thrombosis. Previous reports suggest a role for microparticles (MP) in the pathogenesis of thrombosis in ET. MPs are small plasma membrane vesicles bearing potent procoagulant proteins and phospholipids. They are released into the circulation by various blood and endothelial cells after cellular activation and/or apoptosis. The recently described somatic calreticulin (CALR) exon 9 mutations in almost 20% of ET defines a lower-risk thrombosis ET subtype. We extensively studied phenotype and procoagulant activity of plasma MPs in order to assess MP contribution to the thrombotic risk in CALR versus JAK2 (V617F) mutated ET patients.Patients and methods: We analyzed MP count, phenotype and procoagulant activity in 45 JAK2 V617F+ and 15 CALR+ consecutive and newly diagnosed ET patients in accordance to WHO criteria recruited between november 2010 and april 2013. After given informed consent, blood samples were obtained in all patients before initiating any cytoreductive therapy. Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-Committee and each test was performed in duplicate . Using flow cytometry (FC500 flow-cytometer, Beckman-Coulter), MPs were characterized and measured in platelet-free plasma samples. MPs were characterized by their size and co-expression of bound Annexin V and the following cell-specific monoclonal antibodies: CD41 (Platelet-MP, PMP), CD235a (Red cell-MP, RMP), CD14 (Monocytes-MP, MoMP), CD11b (Granulocytes-MP, GMP), CD144 (Endothelial-MP, EMP), CD62P and CD41 (P-Selectin+ PMP), CD142 and CD41 (Tissue Factor (TF)+ PMP, TF+PMP). MP-associated procoagulant activity was also measured using a-thrombin generation assay (Zymuphen MP-activity). Statistical analysis was performed with SPSS software. Results are expressed as median [interquartile range].Results: Patients characteristics: The platelet count (109/L) was higher in CALR+ than in JAK2+ patients (866 [666 – 918], 659 [571 – 807] respectively, p= 0.049), and all other clinical and hematological characteristics were also distributed in agreement with previous reports. Furthermore, CALR+ patients were preferentially distributed in the lower risk categories of the IPSET-thrombosis and IPSET-survival scores than in JAK2+ patients (p<0.00001 and p= 0.04 respectively).The main results are summarized in table 1. MP count and MP/Platelets ratio were significantly lower in CALR+ than in JAK2+ patients. PMP count, PMP percentage of all MPs and PMP/Platelets ratio were lower in CALR+ than in JAK2+ patients. Furthermore, CALR+ patients had lower PMP surface P-Selectin. MP-associated procoagulant activity/Platelets ratio was significantly lower in CALR+ than in JAK2+ patients. There were no significant differences in TF-carrying PMP or in other cell-derived MP (RMP, MoMP, GMP, EMP).Conclusion: Our results demonstrate a decreased in circulating procoagulant PMP in CALR+ ET-patients compared to JAK2+ patients and lower platelet activation in CALR+ ET patients as measured by P-Selectin expression on PMP. Thus, as CALR+ patients have lower thrombotic-risk according to the literature and the IPSET-survival and IPSET-thrombosis scores observed in our study, the lower level of procoagulant PMP could account, at least in part, for a lower thrombotic risk of CALR+ ET patients.Table 1main resultsCALR +JAK2 V617F +p valueMP (/µL)3289 [1662-4240]4961 [2697-6687]0.04MP/Platelets3.6 [2.8-5.3]6.9 [4.7-9.6]0.01PMP (/µL)3100 [2068-3887]5702 [3423-10257]0.004PMP/Platelets3.38 [2.97-6.33]7.55 [5.12-12.66]0.002% PMP90 [84-92]93 [90-96]0.03MP-activity(nM)/platelet0.015 [0.008-0.028]0.029 [0.019-0.041]0.05P-Selectin+ PMP (/µL)195 [152-219]747 [383-965]0.001RMP (/µL)4 [1.7-8.2]4 [2.5-9.7]nsMoMP (/µL)43 [27-52]74.5 [22-135]nsGMP (/µL)31.5 [24-48]30 [11-55]nsEMP (/µL)99.4 [32-110]95.6 [51-140]nsTF+ PMP (/µL)22.3 [9.8-115]27.1 [20.8-94.4]ns DisclosuresNo relevant conflicts of interest to declare.

The impact of predive exercise on repetitive SCUBA diving.

SCUBA diving frequently involves repetitive exposures. The goal of this study was to see how exercise impacts microparticles (MPs), endothelial function and venous gas emboli (VGE) throughout multiple dives. Sixteen divers in two groups (G1 and G2) each completed six dives, three preceded by exercise (EX) and three as controls (CON). Blood for MP analysis was collected before and after each dive. VGE were monitored via transthoracic echocardiography 30, 60 and 90 min after surfacing. Exercise before diving consisted of 60-min running including eight, 3-min intervals at 90% VO2max. Exercise did not have a significant impact on VGE. There was no significant difference in MP counts between EX and CON. Both groups experienced a significant decrease in MP counts in the last three dives compared to the first three (G1 P = 0·0008, G2 P = 0001). Other indices of neutrophil/platelet interaction (dual-positive CD63/41 and CD62/41) show a significant increase (P = 0·004 and 0·0001) in G2. Both groups experienced a significant decrease in MPs at all measurements in the second series of dives compared to the first, regardless of the placement of exercise. Whether this is related to an effect of suppression of MPs or exercise timing is not clear.

Formation of microparticles in the injured brain of patients with severe isolated traumatic brain injury.

The potential pathophysiological role of circulating microparticles (MPs) has been recognized in various conditions, such as cardiovascular and thrombotic diseases. Traumatic brain injury (TBI) has a complex pathophysiology that involves coagulopathy and inflammation. We investigated endothelial-, platelet-, and leukocyte-derived microparticles (EMPs, PMPs, and LMPs, respectively) in 16 patients with severe isolated TBI. Arterial and cerebrovenous samples were taken repeatedly, during 1-72 h after injury. Subpopulations of MPs, exposing tissue factor (TF) and P-selection, were also studied. MP counts in cerebrovenous samples, irrespective of cellular origin, were higher in TBI cases, compared to healthy controls (peak levels of EMPs were approximately 7 times higher, PMPs 1.4 times higher, and LMPs 2 times higher, respectively; p<0.001 for all). MP counts declined sharply from high levels shortly after the trauma toward slightly elevated levels 72 h later. EMPs and PMPs exposing TF, as well as PMPs exposing P-selection, showed a transcranial gradient with higher concentration in cerebrovenous, compared to arterial, samples. In contrast, LMPs exposing TF were higher in arterial samples, suggesting accumulation of LMPs in the brain. We conclude that the pattern of circulating MPs is altered after TBI. PMPs exposing P-selection and EMPs exposing TF seem to be generated in the injured brain, whereas LMPs exposing TF are accumulated. The pathophysiological significance of these changes in MP pattern in TBI should be further investigated. Including MPs exposing brain-specific antigens in the assessment of brain injury would give further information of origin and likely give additional information of the size of the injury, given that the MP phenotypes investigated in the present study are not brain-specific markers.

Exercise before Scuba Diving Ameliorates Decompression-Induced Neutrophil Activation

The goals of this study were to investigate the difference in responses between a scuba dive preceded by aerobic exercise (EX) and a nonexercise control dive (CON) and to further evaluate the potential relation between venous gas emboli (VGE) and microparticles (MP). We hypothesized that exercise would alter the quantity and subtype of annexin V-positive MP and VGE. Nineteen divers performed two dives to 18 m seawater for 41 min separated by at least 3 d, one of which was preceded by 60 min of treadmill interval exercise. Blood was obtained before exercise, before diving, and 15 min, 2 h, 4 h, and 24 h after surfacing. Intravascular bubbles were quantified by transthoracic echocardiography at 15, 40, 80, and 120 min. The median VGE remained unchanged between the two dives; however, there was a significant increase in VGE in the exercise dive at 40 and 80 min at rest. MP were significantly elevated by approximately 2 times at all time points after CON compared with those after EX. Markers of neutrophil and platelet activation were elevated by both dives, and these elevations were attenuated in the EX dive. We conclude that some of the differences observed between the EX and CON related to MP and platelet and neutrophil activation provide additional insight into the potential protective benefits of exercise; however, further study is needed to understand the mechanism and true potential of these benefits.

Method validation for preparing urine samples for downstream proteomic and metabolomic applications.

Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4°C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. The optimal protocol was a 20-min, 12,000 g centrifugation at 4°C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4°C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4°C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation.

The influence of body mass index, age and sex on inflammatory disease risk in semi-captive Chimpanzees.

Obesity and ageing are emerging issues in the management of captive primates, including Chimpanzees, Pan troglodytes. Studies on humans show that obesity and old age can independently increase the risk of inflammatory-associated diseases indicated by elevated levels of pro-inflammatory cells and proteins in the blood of older or obese compared to levels in younger or non-obese individuals. In humans, sex can influence the outcomes of these risks. Health management of these problems in chimpanzee populations requires an understanding of similarities and differences of factors influencing inflammatory disease risks in humans and in chimpanzees. We examined the relationship between age, sex and Body Mass Index (BMI) with hematological biomarkers of inflammatory disease risk established for humans which include the neutrophil to lymphocyte ratio (NLR), and neutrophil, white blood cell (WBC), platelet microparticle and platelet counts. We found that higher values of NLR, neutrophil count and platelet microparticle count were associated with higher BMI values and older age indicating increased inflammation risk in these groups; a similar pattern to humans. There was a strong sex by age interaction on inflammation risk, with older males more at risk than older females. In contrast to human studies, total WBC count was not influenced by BMI, but like humans, WBC and platelet counts were lower in older individuals compared to younger individuals. Our findings are similar to those of humans and suggest that further insight on managing chimpanzees can be gained from extensive studies of ageing and obesity in humans. We suggest that managing BMI should be an integral part of health management in captive chimpanzee populations in order to partially reduce the risk of diseases associated with inflammation. These results also highlight parallels in inflammation risk between humans and chimpanzees and have implications for understanding the evolution of inflammation related diseases in apes.

Method validation for preparing serum and plasma samples from human blood for downstream proteomic, metabolomic, and circulating nucleic acid-based applications.

Formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. Serum and plasma processing protocols were validated for fitness-for-purpose in terms of key downstream endpoints, and this article demonstrates methodology for biospecimen processing method validation. Serum and plasma preparation from human blood was optimized for centrifugation conditions with respect to microparticle counts. Optimal protocols were validated for methodology and reproducibility in terms of acceptance criteria based on microparticle counts, DNA and hemoglobin concentration, and metabolomic and proteomic profiles. These parameters were also used to evaluate robustness for centrifugation temperature (4°C versus room temperature [RT]), deceleration (low, medium, high) and blood stability (after a 2-hour delay). Optimal protocols were 10-min centrifugation for serum and 20-min for plasma at 2000 g, medium brake, RT. Methodology and reproducibility acceptance criteria were met for both protocols except for reproducibility of plasma metabolomics. Overall, neither protocol was robust for centrifugation at 4°C versus RT. RT gave higher microparticles and free DNA yields in serum, and fewer microparticles with less hemolysis in plasma. Overall, both protocols were robust for fast, medium, and low deceleration, with a medium brake considered optimal. Pre-centrifugation stability after a 2-hour delay was seen at both temperatures for hemoglobin concentration and proteomics, but not for microparticle counts. We validated serum and plasma collection methods suitable for downstream protein, metabolite, or free nucleic acid-based applications. Temperature and pre-centrifugation delay can influence analytic results, and laboratories and biobanks should systematically record these conditions in the scope of accreditation.

Inflammation induced-endothelial cells release angiogenesis associated-microRNAs into circulation by microparticles

Background Endothelial cells derived microRNAs can be detected in plasma and serum and there is evidence that inflammatory disease states may affect the levels of circulating microRNAs. However, there is no direct proof that inflammation induces endothelial cells to release microRNAs into circulation. This study aimed to explore whether inflammation could induce endothelial cells to release microRNAs into circulation and to investigate whether these released microRNAs derived from endothelial cells were transported in microparticles. Methods Microparticles were isolated from human atherosclerotic plaques with an active inflammatory phenotype and normal vascular tissue. Flow cytometry and real-time PCR were used to detect the levels of microparticles and microRNAs. Human umbilical vein endothelial cells (HUVEC) was treated with tumour necrosis factor α (TNF-α, 10 ng/ml) for 24 hours, and then HUVEC and the culture medium were respectively collected. Results By comparing microparticles isolated from human atherosclerotic plaques with an active inflammatory phenotype (n=9) and those from normal vascular tissues (n=9), we found levels of annexin V+ microparticles and annexin V+ CD144+ microparticles were significantly increased in plaques and angiogenesis associated microRNAs (106b, 25, 92a and 21) were also significantly increased in microparticles from plaques. After exposure to TNF-α at a concentration of 10 ng/ml (TNF-α group, n=3) or DMEM (control group, n=3) for 24 hours, counts of microparticles and expressions of microRNAs 106b, 25, 92a and 21 in microparticles isolated from medium significantly increased. However, there were no differences in the intracellular levels of microRNAs 25, 92a or 21 isolated from HUVEC between TNF-α group and control group, while microRNA 106b decreased in TNF-α group. Conclusion Inflammation could induce endothelial cells to release angiogenesis associated microRNAs into circulation, causing higher levels of circulating endothelial cells derived microRNAs in atherosclerosis.

High intensity cycling before SCUBA diving reduces post-decompression microparticle production and neutrophil activation.

Venous gas emboli (VGE) have traditionally served as a marker for decompression stress after SCUBA diving and a reduction in bubble loads is a target for precondition procedures. However, VGE can be observed in large quantities with no negative clinical consequences. The effect of exercise before diving on VGE has been evaluated with mixed results. Microparticle (MP) counts and sub-type expression serve as indicators of vascular inflammation and DCS in mice. The goal of the present study is to evaluate the effect of anaerobic cycling (AC) on VGE and MP following SCUBA diving. Ten male divers performed two dives to 18 m for 41 min, one dive (AC) was preceded by a repeated-Wingate cycling protocol; a control dive (CON) was completed without exercise. VGE were analyzed at 15, 40, 80, and 120 min post-diving. Blood for MP analysis was collected before exercise (AC only), before diving, 15 and 120 min after surfacing. VGE were significantly lower 15 min post-diving in the AC group, with no difference in the remaining measurements. MPs were elevated by exercise and diving, however, post-diving elevations were attenuated in the AC dive. Some markers of neutrophil elevation (CD18, CD41) were increased in the CON compared to the AC dive. The repeated-Wingate protocol resulted in an attenuation of MP counts and sub-types that have been related to vascular injury and DCS-like symptoms in mice. Further studies are needed to determine if MPs represent a risk factor or marker for DCS in humans.

Circulating microparticles reflect treatment effects and clinical status in multiple sclerosis.

To evaluate whether circulating microparticles (MPs) derived from three cell subtypes (platelets, total leukocytes or monocytes) obtained from multiple sclerosis (MS) patients were modulated depending on the clinical status and to investigate the effect of treatments on MP levels. The MP counts were assessed with flow cytometry. The platelet-derived MP level was higher in untreated MS patients than controls. Relapsing-remitting patients showed the highest levels in the three subtypes of MP while secondary progressive patients presented similar levels to those of healthy controls. Treatments had significant effects increasing the three subtypes of MP counts. We suggest that MPs play a role in MS pathogenesis, reflecting disease status with an increment of their shedding during inflammatory periods and turning to baseline during chronic progressive degeneration.

Splenectomy Is Modifying the Vascular Remodeling of Thrombosis

BackgroundSplenectomy is a clinical risk factor for complicated thrombosis. We hypothesized that the loss of the mechanical filtering function of the spleen may enrich for thrombogenic phospholipids in the circulation, thereby affecting the vascular remodeling of thrombosis.Methods and ResultsWe investigated the effects of splenectomy both in chronic thromboembolic pulmonary hypertension (CTEPH), a human model disease for thrombus nonresolution, and in a mouse model of stagnant flow venous thrombosis mimicking deep vein thrombosis. Surgically excised thrombi from rare cases of CTEPH patients who had undergone previous splenectomy were enriched for anionic phospholipids like phosphatidylserine. Similar to human thrombi, phosphatidylserine accumulated in thrombi after splenectomy in the mouse model. A postsplenectomy state was associated with larger and more persistent thrombi. Higher counts of procoagulant platelet microparticles and increased leukocyte–platelet aggregates were observed in mice after splenectomy. Histological inspection revealed a decreased number of thrombus vessels. Phosphatidylserine‐enriched phospholipids specifically inhibited endothelial proliferation and sprouting.ConclusionsAfter splenectomy, an increase in circulating microparticles and negatively charged phospholipids is enhanced by experimental thrombus induction. The initial increase in thrombus volume after splenectomy is due to platelet activation, and the subsequent delay of thrombus resolution is due to inhibition of thrombus angiogenesis. The data illustrate a potential mechanism of disease in CTEPH.