Highly efficient reprogrammable mouse lines with integrated reporters to track the route to pluripotency.

Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.

Young steady-state rabbit platelets do not have an enhanced capacity to expose procoagulant phosphatidylserine.

Platelets are recognized to be physiologically and functionally heterogeneous. An example of the diversity in reactivity is the formation of a distinct subpopulation of procoagulant phosphatidylserine (PS)-exposing platelets upon activation. Platelet age has been proposed as a determinant of platelet function, and it has been reported that young platelets are more reactive in exposing PS; using the same methodology of thiazole orange (TO) staining to distinguish young and old platelets, the percentages of procoagulant platelets produced by thrombin plus collagen activation of platelets from healthy controls were examined by flow cytometry. The procoagulant subpopulation formed by TO-positive platelets (with high TO fluorescence), purported to be young reticulated platelets, was observed to be significantly larger than that formed by TO-negative platelets (with low TO fluorescence), purported to be older platelets. However, it was noted that TO fluorescence in the total platelet population was unimodal and increased with platelet size, assessed by forward scatter. This observation raised the concern that TO-positive platelets are not necessarily the youngest platelets in the condition of steady-state platelet production. Thus, to unequivocally determine whether platelet age is a factor in procoagulant platelet formation, a different approach to identify young, steady-state platelets was employed. Rabbits were injected with biotin to label >95% of circulating platelets in vivo; 24 hours post-biotinylation, the non-biotinylated platelets in the circulation, detected flow cytometrically, are the youngest, newly-formed platelets. It was demonstrated that these youngest platelets were not larger in size than older, biotinylated platelets, and that they did not have an enhanced capacity to expose PS.

Prevalence of blaZ gene types and the cefazolin inoculum effect among methicillin-susceptible Staphylococcus aureus blood isolates and their association with multilocus sequence types and clinical outcome.

Cefazolin treatment failures have been described for bacteraemia caused by methicillin-susceptible Staphylococcus aureus (MSSA) with type A ß-lactamase and inoculum effect (InE). We investigated the prevalence of blaZ (ß-lactamase) gene types and a cefazolin InE among MSSA blood isolates in South Korea and evaluated their association with specific genotypes. The clinical impact of the cefazolin InE was also evaluated. A total of 220 MSSA isolates were collected from a prospective cohort study of S. aureus bacteraemia. A pronounced InE with cefazolin was defined as a ≥4-fold increase in the minimum inhibitory concentration (MIC) between a standard and high inoculum, resulting in a non-susceptible MIC. Sequencing of blaZ and multilocus sequence typing (MLST) were performed. Clinical outcomes were assessed in 77 patients treated with cefazolin. The blaZ gene was detected in 92 % of the 220 MSSA isolates. Type C ß-lactamase was the most common (53 %), followed by type B (20 %) and type A (17 %). Certain genotypes were significantly associated with specific ß-lactamase types (notably, ST30 and type A ß-lactamase). A pronounced cefazolin InE was observed in 13 % of isolates. Most of these (79 %) expressed type A ß-lactamase and ST30 was the predominant (55 %) clone amongst them. Cefazolin treatment failure was not observed in patients infected with strains exhibiting a pronounced InE. These strains had no impact on other clinical outcomes. In conclusion, the prevalence of a pronounced InE with cefazolin could be dependent upon distributions of MSSA genotypes. Cefazolin can likely be used for the treatment of MSSA bacteraemia (except endocarditis), without consideration of an InE.

Ectopic Expression of Neurod1 Is Sufficient for Functional Recovery following a Sensory-Motor Cortical Stroke.

Stroke is the leading cause of adult disability worldwide. The majority of stroke survivors are left with devastating functional impairments for which few treatment options exist. Recently, a number of studies have used ectopic expression of transcription factors that direct neuronal cell fate with the intention of converting astrocytes to neurons in various models of brain injury and disease. While there have been reports that question whether astrocyte-to-neuron conversion occurs in vivo, here, we have asked if ectopic expression of the transcription factor Neurod1 is sufficient to promote improved functional outcomes when delivered in the subacute phase following endothelin-1-induced sensory-motor cortex stroke. We used an adeno-associated virus to deliver Neurod1 from the short GFAP promoter and demonstrated improved functional outcomes as early as 28 days post-stroke and persisting to at least 63 days post-stroke. Using Cre-based cell fate tracking, we showed that functional recovery correlated with the expression of neuronal markers in transduced cells by 28 days post-stroke. By 63 days post-stroke, the reporter-expressing cells comprised ~20% of all the neurons in the perilesional cortex and expressed markers of cortical neuron subtypes. Overall, our findings indicate that ectopic expression of Neurod1 in the stroke-injured brain is sufficient to enhance neural repair.

Integrating single-cell and spatially resolved transcriptomic strategies to survey the astrocyte response to stroke in male mice.

Astrocytes, a type of glial cell in the central nervous system (CNS), adopt diverse states in response to injury that are influenced by their location relative to the insult. Here, we describe a platform for spatially resolved, single-cell transcriptomics and proteomics, called tDISCO (tissue-digital microfluidic isolation of single cells for -Omics). We use tDISCO alongside two high-throughput platforms for spatial (Visium) and single-cell transcriptomics (10X Chromium) to examine the heterogeneity of the astrocyte response to a cortical ischemic stroke in male mice. We show that integration of Visium and 10X Chromium datasets infers two astrocyte populations, proximal or distal to the injury site, while tDISCO determines the spatial boundaries and molecular profiles that define these populations. We find that proximal astrocytes show differences in lipid shuttling, with enriched expression of Apoe and Fabp5. Our datasets provide a resource for understanding the roles of astrocytes in stroke and showcase the utility of tDISCO for hypothesis-driven, spatially resolved single-cell experiments.

BRCA1 gene therapy reduces systemic inflammatory response and multiple organ failure and improves survival in experimental sepsis.

Sepsis-related complications and mortality remain a major clinical problem. Increased cell death and unresolved cellular repair have been implicated as key upstream mediators of sepsis-induced organ dysfunction and death. We hypothesised that gene therapy with BRCA1, a critical regulator of DNA damage repair and cell survival, would attenuate the sequelae of sepsis and peritonitis in mice subjected to caecal ligation and perforation (CLP) and thioglycollate stimulation. C57Bl/6J mice underwent sham or CLP surgery 3 days following treatment with either human BRCA1 adenovirus (AdBRCA1) or the adeno-CMV-null vector (Adnull). The 24-h post-CLP mortality was 2.8% vs 17.9% (P<0.001) and the median post-CLP survival was 50.5 vs 33 h (P<0.05) for AdBRCA1- vs Adnull-treated mice, respectively. AdBRCA1 therapy blunted CLP-associated cardiac, pulmonary, hepatic and renal dysfunction and also reduced CLP-elicited double strand breaks and apoptosis in the liver. BRCA1 gene therapy was associated with lower CLP-evoked cardiac and hepatic superoxide generation that in the liver was in part due to improved reactive oxygen species removal. CLP also elevated mesenteric arteriolar and serum intercellular adhesion molecule-1, both of which were partially abrogated with AdBRCA1 administration. Thioglycollate-challenged AdBRCA1-treated mice displayed reduced peritoneal neutrophil recruitment and dampened cytokine elaboration relative to their Adnull-treated counterparts. Taken together, we report a novel role of BRCA1 gene therapy in limiting systemic inflammation, multiple-organ failure and mortality in experimental sepsis.

mAb therapy controls CNS-resident lyssavirus infection via a CD4 T cell-dependent mechanism.

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre-symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti-lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11-dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell-dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ-binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization-independent T cell-mediated effects, even against an established CNS infection by a lethal neurotropic virus.

Platelet apoptosis in paediatric immune thrombocytopenia is ameliorated by intravenous immunoglobulin.

To evaluate the role of intravenous immunoglobulin (IVIg) in platelet apoptosis in paediatric immune thrombocytopenia, we investigated the platelets of 20 paediatric patients with acute immune thrombocytopenia (ITP), before and after IVIg treatment. Healthy children with platelet counts in the normal range and children with thrombocytopenia due to chemotherapy were enrolled as controls. All ITP patients presented with platelet counts <20 × 10(9) /l and bleeding symptoms. Markers of apoptosis, including activated caspase-3, -8 and -9, phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), as well as platelet-derived microparticle formation, were analysed by flow cytometry. After IVIg treatment, platelet counts increased to >20 × 10(9) /l in all patients. ITP patients had significantly increased proportions of platelets with activated caspase-3, -8 and -9, with PS exposure, and with decreased ΔΨm, and demonstrated increased microparticle formation. Except for ΔΨm, these markers for apoptosis were reduced by IVIg treatment. Platelets of children with thrombocytopenia after chemotherapy also demonstrated increased microparticle formation and decreased ΔΨm, but no activation of caspases 3, 8 and 9 or PS exposure. In conclusion, in acute paediatric ITP, enhanced platelet apoptosis is seen at diagnosis that normalizes after IVIg treatment.

Clinical significance of Staphylococcus aureus bacteremia in patients with liver cirrhosis.

Patients with liver cirrhosis (LC) have impaired immunity and thus are predisposed to infections. Few studies have attempted to evaluate Staphylococcus aureus bacteremia (SAB) in LC patients. Therefore, this study prospectively evaluated the clinical characteristics and outcomes of 642 episodes of SAB from August 1, 2008 to September 31, 2010. Of 642 patients with SAB, 109 (17.0 %) were classified as LC patients whereas the remaining 533 (83.0 %) were classified as non-LC patients. The 30-day mortality rate of LC patients was significantly higher than that of patients with other diseases (32 % vs. 22 %, respectively; P = 0.047). The 30-day mortality rates of patients with MSSA bacteremia and MRSA bacteremia were not significantly different among LC patients (35.1 % with MSSA vs. 26.9 % with MRSA; P = 0.41). A univariate analysis of the 30-day mortality rate of LC patients with SAB for survivors and non-survivors showed that rapidly fatal or ultimately fatal according to the criteria of McCabe and Jackson (OR 5.0; 95 % CI 1.60-15.65), septic shock at initial presentation (OR 3.5; 95 % CI 1.18-10.39) and Child-Pugh class C (OR 2.8; 95 % CI 1.20-6.59) were associated with increased mortality. In contrast, the removal of the eradicable focus was associated with decreased mortality (OR 0.14; 95 % CI 0.04-0.52). Disease severity and liver dysfunction may be useful for predicting the prognosis of SAB in LC patients.

Isolation and Characterization of Cross-Reactive Human Monoclonal Antibodies That Potently Neutralize Australian Bat Lyssavirus Variants and Other Phylogroup 1 Lyssaviruses.

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.

Phosphatidylserine exposure and other apoptotic-like events in Bernard-Soulier syndrome platelets.

In the Bernard-Soulier syndrome (BSS), the giant platelets are said to have increased phosphatidylserine (PS) surface exposure in the resting state and shortened survival in the circulation. When normal platelets are activated, they undergo many biochemical and morphological changes, some of which are apoptotic. Herein, we investigated apoptotic-like events in BSS platelets upon activation, specifically, PS exposure, microparticle (MP) formation, cell shrinkage, and loss of mitochondrial inner membrane potential (DeltaPsi(m)). Platelets from two unrelated BSS patients were examined in whole blood; agonists used were collagen, thrombin, PAR1- or PAR4-activating peptides (APs), or combinations of collagen with thrombin, and the PAR-APs. Flow cytometry was used to measure PS exposure (annexin A5 binding), platelet-derived MPs (forward scatter; events <0.75 microm size), and DeltaPsi(m) (TMRM fluorescence). PS exposure was increased on resting and activated BSS platelets, and this was independent of the platelet size. MP formation by BSS platelets was generally enhanced. Cell shrinkage occurred on activation to form smaller, PS-exposing platelets in BSS and controls. A proportion of PS-exposing BSS and control platelets exhibited DeltaPsi(m) loss, but unlike controls, there was also loss of DeltaPsi(m) in the BSS platelets not exposing PS. Thus, BSS platelets undergo apoptotic-like events upon activation, with PS exposure and MP formation being enhanced. These events may play a role in the shortened survival in BSS, as well as affecting thrombin generation.

CFD modelling of flow field and particle tracking in a hydrodynamic stormwater separator.

The best management practices (BMPs) for control of urban stormwater pollution are evaluated to remove solid particles containing various pollutants. Currently, most storm runoff treatment devices using primary pollutant removal mechanism are applied to storm water since most pollutants in runoff are associated with the solid particulates. A hydrodynamic separator is a storm water treatment device using centrifugal motion which separates solids pollution from runoff. In this study, the velocity flow field and particle tracking of hydrodynamic separator were investigated using anthracite as a computational fluid dynamics (CFD) model particle. The Fluent 6.3.26 CFD program was used to predict the solid particles removal efficiency for various parameters such as particle size, surface loading rate, and the ratio of underflow to overflow. The velocity flow field in a hydrodynamic stormwater separator (HDS) has been simulated using CFD RNG κ-ε model. Modeling results for the removal efficiency of HDS were similar with the results obtained from experimental measurements of laboratory scale HDS. These results showed that the simulated velocity field was useful to interpret the behavior of flow in the hydrodynamic separator. The results obtained from particle tracking can be applied to predict the separation efficiency.

The gut hormone receptor GIPR links energy availability to the control of hematopoiesis.

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) conveys information from ingested nutrients to peripheral tissues, signaling energy availability. The GIP Receptor (GIPR) is also expressed in the bone marrow, notably in cells of the myeloid lineage. However, the importance of gain and loss of GIPR signaling for diverse hematopoietic responses remains unclear. METHODS: We assessed the expression of the Gipr in bone marrow (BM) lineages and examined functional roles for the GIPR in control of hematopoiesis. Bone marrow responses were studied in (i) mice fed regular or energy-rich diets, (ii) mice treated with hematopoietic stressors including acute 5-fluorouracil (5-FU), pamsaccharide (LPS), and Pam3CysSerLys4 (Pam3CSK4), with or without pharmacological administration of a GIPR agonist, and (iii) mice with global (Gipr-/-) or selective deletion of the GIPR (GiprTie2-/-) with and without bone marrow transplantation (BMT). RESULTS: Gipr is expressed within T cells, myeloid cells, and myeloid precursors; however, these cell populations were not different in peripheral blood, spleen, or BM of Gipr-/- and GiprTie2-/- mice. Nevertheless, gain and loss of function studies revealed that GIPR signaling controls the expression of BM Toll-like receptor (TLR) and Notch-related genes regulating hematopoiesis. Loss of the BM GIPR attenuates the extent of adipose tissue inflammation and dysregulates the hematopoietic response to BMT. GIPR agonism modified BM gene expression profiles following 5-FU and Pam3CSK4 whereas loss of the Gipr altered the hematopoietic responses to energy excess, two TLR ligands, and 5-FU. However, the magnitude of the cellular changes in hematopoiesis in response to gain or loss of GIPR signaling was relatively modest. CONCLUSION: These studies identify a functional gut hormone-BM axis positioned for the transduction of signals linking nutrient availability to the control of TLR and Notch genes regulating hematopoiesis. Nevertheless, stimulation or loss of GIPR signaling has minimal impact on basal hematopoiesis or the physiological response to hematopoietic stress.

A large library based on a novel (CH2) scaffold: identification of HIV-1 inhibitors.

Isolated immunoglobulin CH2 domains were proposed as scaffolds for selection of binders with potential effector functions. We tested the feasibility of this approach by constructing a large (size 5 x 10(10)) library where all amino acids in two loops (BC and FG) were mutated to four residues (Y, A, D, or S). Three binders were selected from this library by panning against a gp120-CD4 complex. The strongest binder, m1a1, recognized specifically a highly conserved CD4i epitope and inhibited to various extents seven out of nine HIV-1 isolates from different clades. The loop BC and the conformational state of the scaffold are critical for its binding. These results provide a proof of concept for the potential of CH2 as a scaffold for construction of libraries containing potentially useful binders. The newly identified HIV-1 inhibitors could be further improved to candidate therapeutics and/or used as research reagents for exploration of conserved gp120 structures.

Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens.

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.

Dynamics of antibody domains studied by solution NMR.

Information on local dynamics of antibodies is important to evaluate stability, to rationally design variants, and to clarify conformational disorders at the epitope binding sites. Such information may also be useful for improved understanding of antigen recognition. NMR can be used for characterization of local protein dynamics at the atomic level through relaxation measurements. Due to the complexity of the NMR spectra, an extensive use of this method is limited to small protein molecules, for example, antibody domains and some scFv. Here, we describe a protocol that was used to study the dynamics of an antibody domain in solution using NMR. We describe protein preparation for NMR studies, NMR sample optimization, signal assignments, and dynamics experiments.

Structure of an isolated unglycosylated antibody C(H)2 domain.

The C(H)2 (C(H)3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein-protein interactions with another C(H)2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the C(H)2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody gamma1 C(H)2 domain was determined at 1.7 A resolution and compared with corresponding C(H)2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody C(H)2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the C(H)2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications.

Evaluation of Teeth in Line of Mandibular Fractures Treated with Stable Internal Fixation.

PURPOSE: To determine whether retention or removal of teeth in line of mandibular fractures affects the incidence of postoperative complications. To evaluate the fate of teeth retained in mandibular fractures based on location of fracture line in relation to apical foramen and lateral periodontium of retained tooth. METHOD: 51 mandibular fracture sites in 39 patients were treated by open reduction and internal fixation using miniplates. Teeth in line of fracture were removed if showed extensive caries, excessive mobility of tooth, or root fracture. Accordingly patients were divided into two groups, group I tooth removed (15 sites) and group II tooth retained (36 sites). Teeth retained in fracture line were classified based on position of the fracture in relation to apical foramen and the lateral periodontium of involved tooth estimated from preoperative panoramic radiograph into four types. Postoperative periodic follow up done for 1 year and seen for incidence of postoperative complications and fate of tooth in line of fracture. RESULTS: Out of 36 fracture sites in which teeth were retained 5 (13.89%) showed postoperative complications, and out of 15 fracture sites in which teeth were removed 3 (20%) showed postoperative complications. Out of 36 fracture sites in which tooth was retained we found in 3 cases fracture line passed through 2 adjacent teeth. Thus we had in total 39 teeth in fracture line out of them 26 required no treatment 9 required endodontic treatment and 4 teeth required extraction. Need for extraction and endodontic treatment was found more in type I and type III relation. CONCLUSION: There is an increased risk for postoperative complications when a tooth is removed, although it is not statistically significant. Evaluation of fate of retained teeth showed better prognosis of teeth in type II cases than type III and type I cases. Results of this study leads to conclusions that teeth associated with mandibular fractures need not be removed on prophylactic basis.

Analysis of procoagulant phosphatidylserine-exposing platelets by imaging flow cytometry.

BACKGROUND: Upon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane. OBJECTIVE: To evaluate procoagulant PS-exposing platelets by imaging flow cytometry. METHODS: Platelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbß3, CD62P, and PS exposure. RESULTS: A subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and αIIbß3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs. CONCLUSIONS: Innovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.

Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity.

BACKGROUND: Activated platelets express a procoagulant surface when the asymmetric distribution of membrane phospholipids is scrambled, leading to phosphatidylserine (PS) exposure. PS expression, associated with apoptosis in nucleated cells, would be expected to be reversed by aminophospholipid translocase (APLT) activity. OBJECTIVE: To determine whether the procoagulant surface of activated platelets persists after it forms; to examine whether PS expression on platelets is associated with loss of mitochondrial inner membrane potential (DeltaPsi(m)), a hallmark of apoptosis; and to investigate the role of APLT in persistence of PS expression. METHODS: Platelets were stimulated with thrombin, collagen, a combination of both, or the Ca(2+)-ionophore A23187. Up to 4 h after activation, procoagulant surface expression was measured by annexin A5 binding by flow cytometry and by a prothrombinase assay. Flow cytometry was also used to measure PS expression concurrently with DeltaPsi(m) collapse, using CMXRos. APLT activity in annexin A5-negative and -positive platelets was measured flow cytometrically as the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. RESULTS AND CONCLUSIONS: Procoagulant surface expression on activated platelets persisted in vitro for at least 4 h; if such persistence occurs in vivo, there are important implications for the propagation of thrombosis. With the physiological stimuli, only 10-20% of the activated platelets expressed PS on their surface, and of these, only a portion exhibited DeltaPsi(m) collapse, indicating that PS expression can be associated with platelet apoptosis, but can also occur independently. APLT activity was very low in the PS-expressing platelet subpopulation for up to 4 h after activation, indicating that the persistence of a procoagulant surface may be attributed, at least in part, to this reduced APLT activity.