Macrophage-Agglutinating Factor Produced In Vitro by BCG-Sensitized Lymphocytes

Supernatant fluids from cultures of BCG-sensitized rabbit lymph node and spleen cells contained a factor that strongly agglutinated normal rabbit alveolar macrophages within 3 min at room temperature. In contrast, fluids from nonsensitized cell cultures did not agglutinate normal rabbit alveolar macrophages. This factor was designated macrophage-agglutinating factor (MAgF) because it is similar to the previously described factor found in lung lavages of rabbits exhibiting a BCG-induced pulmonary delayed hypersensitivity reaction. The kinetics of MAgF production in vitro by sensitized lymph node cells and its inhibition by puromycin and actinomycin D suggest active synthesis; sensitized spleen cells exhibited kinetics resembling release rather than synthesis. Studies on purified lymphocyte and macrophage populations from sensitized spleen and lymph nodes indicated that lymphocytes are responsible for MAgF production. However, MAgF production was not induced in normal cells incubated in vitro with concanavalin A or phytohemagglutinin. Fractionation of cell culture supernatant fluids in Sephadex G-100 or Ultrogel AcA-34 clearly separated MAgF from migration inhibition factor; MAgF was present in the void volume of the eluates, suggesting a molecular weight of over 400,000, whereas migration inhibition factor was recovered in the same peak as albumin. The role of MAgF in vivo is unknown, but it is postulated that it may cause the adherence of macrophages during granuloma formation.     

In Vitro Platelet Adhesion of PNaAMPS/PAAm and PNaAMPS/PDMAAm Double‐Network Hydrogels

Nowadays, tough double‐network (DN) hydrogels have attracted great attention owing to their excellent mechanical properties and good biocompatibility, which give them the potential to be used as blood‐contacting soft tissue prostheses and medical devices. However, the study of platelet adhesion behavior on the surface of DN hydrogels has not been reported yet. In this work, the human platelet adhesion on the surface of poly (sodium 2‐acrylamido‐2‐methyl‐propanesulfonate) PNaAMPS/poly acrylamide (PAAm) and PNaAMPS/poly (N,N′‐dimethylacrylamide) (PDMAAm) DN hydrogels is investigated under static conditions in vitro. The numbers of adherent platelets on PNaAMPS/PAAm and PNaAMPS/PDMAAm hydrogels are 16 ± 7 and 9 ± 8 cells per 10⁴ μm², respectively, which are far less than 297 ± 41 cells per 10⁴ μm² on polyethylene terephthalate (PET) and 187 ± 26 cells per 10⁴ μm² on negatively charged PNaAMPS (4 mol%) hydrogel. The results indicate the excellent antiplatelet performance of DN hydrogels. Moreover, the platelet adhesion mechanism is also discussed. The platelet adhesion is affected by the chemical component, zeta potential, and serum proteins adsorption of the hydrogel.

     

Natural Variability of In Vitro Adherence to Fibrinogen and Fibronectin Does Not Correlate with In Vivo Infectivity of Staphylococcus aureus

Adherence to fibrinogen and fibronectin plays a crucial role in Staphylococcus aureus experimental endocarditis. Previous genetic studies have shown that infection and carriage isolates do not systematically differ in their virulence-related genes, including genes conferring adherence, such as clfA and fnbA. We set out to determine the range of adherence phenotypes in carriage isolates of S. aureus, to compare the adherence of these isolates to the adherence of infection isolates, and to determine the relationship between adherence and infectivity in a rat model of experimental endocarditis. A total of 133 healthy carriage isolates were screened for in vitro adherence to fibrinogen and fibronectin, and 30 isolates were randomly chosen for further investigation. These 30 isolates were compared to 30 infective endocarditis isolates and 30 blood culture isolates. The infectivities of the carriage isolates, which displayed either extremely low or high adherence to fibrinogen and fibronectin, were tested using a rat model of experimental endocarditis. The levels of adherence to both fibrinogen and fibronectin were very similar for isolates from healthy carriers and members of the two groups of infection isolates. All three groups of isolates showed a wide range of adherence to fibrinogen and fibronectin. Moreover, the carriage isolates that showed minimal adherence and the carriage isolates that showed strong adherence had the same infectivity in experimental endocarditis. Adherence was proven to be important for pathogenesis in experimental endocarditis, but even the least adherent carriage strains had the ability to induce infection. We discuss the roles of differential gene expression, human host factors, and gene redundancy in resolving this apparent paradox.Staphylococcus aureus is a human commensal, but at the same time it is one of the most important bacterial pathogens that cause community-acquired and nosocomial infections. It can produce a wide variety of diseases, from benign skin infections, such as folliculitis or furunculosis, to life-threatening conditions, like osteomyelitis, septic arthritis, sepsis, pneumonia, and endocarditis (12). About 20% of humans carry S. aureus permanently in their noses, and another 60% are intermittent carriers (16). The association between S. aureus nasal carriage and staphylococcal disease has been reported for several decades (43, 44). More recently, it has been unambiguously shown that carriers have a higher risk of infection, at least when they are hospitalized (41, 45).The pathogenicity of S. aureus involves a wide range of cell wall-associated adhesins and extracellular toxins. Surface adhesins, referred to as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), bind to the host extracellular matrix and thus promote tissue colonization and infection (30). The major MSCRAMMs involved in S. aureus pathogenesis are particular surface proteins that are covalently bound to the cell wall peptidoglycan via a conserved LPXTG motif (22). Genomic analyses indicated that the S. aureus genome contains up to 21 such LPXTG surface proteins (38). In addition to their multiplicity, these proteins often have redundant functions, as exemplified by clumping factors A and B (ClfA and ClfB), which bind fibrinogen (1, 27), and fibronectin-binding proteins A and B (FnBPA and FnBPB), which bind fibronectin (21), fibrinogen (42), and elastin (37).It has been unambiguously demonstrated that in S. aureus ClfA and FnBPA are key pathogenicity factors, at least in infective endocarditis. This has been achieved by expressing these adhesins in bacteria lacking the rest of the S. aureus surface features (34). Using a variety of truncated and chimera constructs of these proteins, Que et al. observed that fibrinogen-binding domains were necessary and sufficient for colonization of damaged valves in experimental endocarditis, but not for persistence and invasion, whereas fibronectin-binding domains of FnBPA were unable to initiate infection but mediated aortic cell invasion and microbial persistence. Thus, the two adherence functions were necessary for progressive infection (35).Despite the great effort to establish whether there are specific genetic determinants that distinguish carriage and invasive infection strains, the answer was largely negative (8, 25). Genetic studies revealed that many S. aureus wild-type strains lack some of the genes coding for LPXTG motif proteins, but there is no overall difference between carriage and infection isolates (19). However, fnbA and clfA are nearly always present in carriage and clinical isolates, confirming their pivotal role. Moreover, sequence analysis of functional regions of the two proteins has shown that there is a high degree of conservation in sporadic and epidemic isolates of S. aureus (17). On the other hand, the presence of the genes does not imply that there is efficient expression of a protein on the cell surface. It is entirely possible that either the carriage isolates express FnBPA and ClfA at a lower level or the adherence to the host matrix is less efficient. To our knowledge, a comparison of the adherence phenotypes of infection and carriage isolates has never been conducted.Here we determined the levels of adherence to fibrinogen and fibronectin of 133 carriage isolates. We compared these isolates to 30 infective endocarditis and 30 blood culture isolates. In addition, we compared the infectivities of isolates displaying extreme adherence phenotypes in a rat model of experimental endocarditis.     

Blood Flow Patterns Spatially Associated with Platelet Aggregates in Murine Colitis

In the normal murine mucosal plexus, blood flow is generally smooth and continuous. In inflammatory conditions, such as chemically‐induced murine colitis, the mucosal plexus demonstrates markedly abnormal flow patterns. The inflamed mucosal plexus is associated with widely variable blood flow velocity as well as discontinuous and even bidirectional flow. To investigate the mechanisms responsible for these blood flow patterns, we used intravital microscopic examination of blood flow within the murine mucosal plexus during dextran sodium sulphate‐and trinitrobenzenesulfonic acid‐induced colitis. The blood flow patterns within the mucosal plexus demonstrated flow exclusion in 18% of the vessel segments (P < 0.01). Associated with these segmental exclusions was significant variation in neighboring flow velocities. Intravascular injection of fluorescent platelets demonstrated platelet incorporation into both fixed and rolling platelet aggregates. Rolling platelet aggregates (mean velocity 113 μm/sec; range, 14–186 μm/sec) were associated with reversible occlusions and flow variations within the mucosal plexus. Gene expression profiles of microdissected mucosal plexus demonstrated enhanced expression of genes for CCL3, CXCL1, CCL2, CXCL5, CCL7, CCL8, and Il‐1b (P < 0.01), and decreased expression of CCL6 (P < 0.01). These results suggest that platelet aggregation, activated by the inflammatory mileau, contributes to the complex flow dynamics observed in acute murine colitis. Anat Rec, 292:1143–1153, 2009. © 2009 Wiley‐Liss, Inc.     

红细胞生成素对血小板体外生成的影响

本文研究了红细胞生成素(EPO)在体外对巨核细胞成熟及血小板生成的影响。两种纯度的EPO在一定浓度范围内,均能使血小板数呈线性增加,但巨核细胞数保持不变。EPO-Ⅰ可使2～8倍体(N)巨核细胞减少,16～64N细胞增多,同时Ⅰ期巨核细胞比例减少,Ⅳ期细胞比例增多。两种EPO以等单位或最大效应(增加血小板数的作用)相比,EPO-Ⅰ的作用强于EPO-Ⅱ。实验表明EPO使体外血小板数升高的作用是由于促进了巨核细胞DNA合成及胞浆成熟。     

Platelet Activation by Streptococcus pyogenes Leads to Entrapment in Platelet Aggregates,from Which Bacteria Subsequently Escape

Platelet activation and aggregation have been reported to occur in response to a number of Gram-positive pathogens. Here, we show that platelet aggregates induced by Streptococcus pyogenes were unstable and that viable bacteria escaped from the aggregates over time. This was not due to differential activation in response to the bacteria compared with physiological activators. All the bacterial isolates induced significant platelet activation, including integrin activation and alpha and dense-granule release, at levels equivalent to those induced by potent physiological platelet activators that induced stable aggregates. The ability to escape the aggregates and to resist the antibacterial effects of platelets was dependent on active protein synthesis by the bacteria within the aggregate. We conclude that S. pyogenes bacteria can temporarily cover themselves with activated platelets, and we propose that this may facilitate survival of the bacteria in the presence of platelets.     

Platelet Adhesion and Fibrinogen Accretion on a Family of Elastin-Like Polypeptides

Previous work in our laboratory showed the potential of using a human recombinant elastin-like polypeptide (ELP) as a thromboresistant coating. In this work we investigate the use of three particular ELPs (ELP1, ELP2 and ELP4), that differ by molecular weight and number of repeating hydrophobic and cross-linking domains, as coatings to improve blood-contacting properties. All three ELPs were passively adsorbed on Mylar surfaces. Differences in water contact angle and surface concentration were found among the three ELP coatings, with the shortest polypeptide, ELP1, being the most hydrophilic and abundant on the surface (55°, 0.76 μg/cm²), followed by ELP2 (55°, 0.35 μg/cm²) and ELP4, the longest of the three (66°, 0.25 μg/cm²), respectively. The blood interactions of the ELP coatings were investigated by measuring fibrinogen adsorption and platelet adhesion in whole blood under laminar flow in a cone and plate viscometer configuration. In general, platelet adhesion to the ELP-coated surfaces was found to correlate with fibrinogen adsorption. Decreases in fibrinogen accretion and platelet adhesion were observed for ELP-coated compared to uncoated surfaces. The magnitude of the decreases was found to depend on the ELP sequence length, with ELP4 exhibiting the lowest levels of fibrinogen adsorption and platelet adhesion at 43 ± 24 ng/cm² and 113 ± 77 platelets/mm², respectively.     

Electron Capture Gas-Liquid Chromatographic Study of Metabolites Produced by Some Arthritic Transudate-Associated Organisms In Vitro and In Vivo in Rabbit Models

Computerized, frequency-pulsed, modulated electron capture gas-liquid chromatography was used to study the acid metabolites produced in vitro in fetal calf serum and in vivo in an animal chamber model. Several strains of Diplostreptococcus agalactiae, Propionibacterium acnes, Staphylococcus aureus, and Streptococcus serogroups A, B, and G were studied. All of these organisms have been reported to be associated with arthritic transudates in humans. Metabolites were detected by this method from derivatized extracts of both spent fetal calf serum and chamber fluids. Since there was little host response to the organisms cultured in the chambers, it is highly probable that the products detected represent metabolites produced in an in vivo type of environment. The metabolic patterns were reproducible and exhibited many similarities in vitro and in vivo. Production of the acids detected was reproducible, and these acids were useful identification markers. The data support published reports (J. B. Brooks, C. C. Alley, and J. A. Liddle, Anal. Chem. 46: 1930-1934, 1974; J. B. Brooks, G. Choudhary, R. B. Craven, D. Edman, C. C. Alley, and J. A. Liddle, J. Clin. Microbiol. 5:625-628, 1977; J. B. Brooks, R. B. Craven, A. R. Melton, and C. C. Alley, in H. H. Johnson and W. B. Newson, ed., Second International Symposium on Rapid Methods and Automation on Microbiology, 1976; J. B. Brooks, R. B. Craven, D. Schlossberg, C. C. Alley, and F. M. Pitts, J. Clin. Microbiol. 8:203-208, 1978; J. B. Brooks, D. S. Kellogg, C. C. Alley, H. B. Short, and H. H. Handsfield, J. Infect. Dis. 129:660-668, 1974) that bacterial metabolites might be detectable in diseased body fluids. The growth characteristics of the organisms in the animal model and fetal calf serum are discussed, and a moderately priced computer for performing data manipulations is evaluated.     

Analysis of Reparative Activity of Platelet Lysate: Effect on Cell Monolayer Recovery In Vitro and Skin Wound Healing In Vivo

Bulletin of Experimental Biology and Medicine - Platelet lysate prepared from donor platelet concentrate and pooled according to a developed technique stimulates migration of multipotent...     

Ultrastructural Changes in Platelet Aggregates of HIV Patients: A Scanning Electron Microscopy Study

Several hematological abnormalities associated with HIV have been documented, but the mechanisms responsible for the cytopenias in AIDS patients are complex and not always completely understood. Thrombocytopenia, which occurs in about 40% of patients with HIV infection, may be caused by increased peripheral platelet destruction, a defect in platelet production due to the impaired formation of platelets by HIV-infected magakaryocytes, or a combination of these. The aim of this study was to compare the morphology of the platelet aggregates in platelet-rich plasma (PRP) clots prepared from HIV patients with those of controls without HIV. These platelet aggregates were studied using the scanning electron microscope to determine the effect of the virus on platelet ultrastructure. The results showed that although the platelets do aggregate, the morphology was changed with membrane blebbing as well as torn cellular membranes. Membrane blebbing is typically associated with apoptosis. It is concluded that the altered morphology of platelet aggregates in HIV patients may be related to thrombocytopenia as a result of peripheral platelet destruction.     

中华眼镜蛇蛇毒因子体外凝血的机理

在以往的实验中已经证明,ＣＶＦ（中华眼镜蛇蛇毒因子）在体内具有抗血小板凝聚的作用,但在体外实验中却发现它有使血小板聚集的作用。为了探讨ＣＶＦ体外引起血小板凝聚的机理,我们用放射免疫分析法测定了ＣＶＦ作用后的血小板内ｃＡＭＰ（环磷酸腺苷）的变化。结果表明,不同剂量ＣＶＦ均可使血小板内ｃＡＭＰ含量降低。其中生理盐水对照组血小板ｃＡＭＰ含量（ｐｍｏｌ／１０＾９血小板）为２５．２±６．８,ＣＶＦ低浓度组为     

血液净化材料在体外静态接触血浆时血浆中 C3a des Arg生成的研究

用放射免疫分析法研究了五种血液净化材料：磺化聚醚砜,聚醚砜,聚砜,聚甲基丙烯酸甲酯和醋酸纤维素与人血浆在37℃温育30、60、90、120min后,血浆中补体C3激活产物C3a des Arg的生成情况。结果发现：醋酸纤维素、聚甲基丙烯酸甲酯、聚醚砜、聚砜在与血浆温育后血浆中C3a des Arg的浓度依次降低;以醋酸纤维激活补体C3的情况最严重;磺化聚醚砜的最低,并且磺化度越大,C3a des Arg的浓度越小。说明聚醚砜材料为血液净化材料有较优异的补体相容性,并且通过磺化可大大提高聚醚砜的补体相容性。放免分析法检测材料激活补体C3是一种方便可靠的检测手段,可作为开发生物材料时对有应用前景的生物材料的筛选和评价。     

Prognostic Significance of Initial Platelet Counts and Fibrinogen Level in Advanced Non-Small Cell Lung Cancer

Thrombocytosis and coagulation systems activation are commonly associated with disease progression and are suggested poor prognostic factors in patients with malignancies. This study aimed to investigate the prevalence and prognostic significance of thrombocytosis and elevated fibrinogen levels in patients with advanced non-small cell lung cancer (NSCLC). Initial platelet counts and fibrinogen levels were reviewed in 854 patients with histologically proven NSCLC. Thrombocytosis was defined as platelet counts > 450 × 10⁹/L. A serum fibrinogen level > 4.5 g/L was considered high. At the time of diagnosis, initial platelet counts and serum fibrinogen levels were evaluated before treatment. Clinicopathologic data including histological type, tumor, node, metastasis (TNM) stage, performance status, treatment method, and survival time were evaluated. Initial thrombocytosis was found in 6.9% of patients, and elevated fibrinogen levels were found in 55.1% of patients. Patients with thrombocytosis had a significantly poorer prognosis than patients with normal platelet counts (P < 0.001). In multivariate survival analysis, thrombocytosis was an independent prognostic factor (P < 0.001). An elevated serum fibrinogen level was associated with poor prognosis (P < 0.001). In conclusion, initial thrombocytosis and a high fibrinogen level are independent factors for predicting poor prognosis in patients with advanced NSCLC.

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Mechanism of Activation of ADP-Induced Platelet Aggregation under the Influence of Oxidatively Modified Fibrinogen

For evaluation of the mechanisms underlying the effect of oxidized fibrinogen on platelet aggregation we studied ADP-induced platelet aggregation in the presence of UV-oxidized fibrinogen and inhibitors of major enzymes of platelet activation. Cyclooxygenase inhibitor acetylsalicylic acid, protein kinase C inhibitor H7, and to a lesser extent, protein tyrosine kinase inhibitor genistein suppressed ADP-induced platelet aggregation. In the presence of oxidized fibrinogen the degree of suppression was lower than in the presence of nonoxidized fibrinogen. Phospholipase C inhibitor U73122 markedly suppressed platelet aggregation in the presence of oxidized and nonoxidized fibrinogen. It can be hypothesized that oxidized fibrinogen activates platelets by modulating activity of the key signal component phospholipase C.     

Quantification of Thrombocyte Growth Factors in Platelet Concentrates Produced by Discontinuous Cell Separation

Platelet concentrates (PC) are increasingly used to increase bone regeneration in pre-prosthetic surgery. Although it is generally appreciated that certain growth factors (PDGF, TGF, EGF, and ECGF) are present in thrombocyte preparations, relatively little is known about these components in quantitative terms. The study reported here analysed the amounts of growth factors in PC produced under standard conditions from healthy volunteers. All the blood samples (237 in total) were analysed using Quantikine ELISA kits (R and D). The mean &#45 SD platelet count in whole blood from these donors was 262,000 &#45 58,000/ &#119 l, while in PC produced by discontinuous cell separation it was 1,419,000 &#45 333,000/ &#119 l. The mean growth factor concentrations in PC preparations in ng/ml were as follows: PDGF-AB 125 &#45 55 ng/ml; TGF- &#103 1 221 &#45 92 ng/ml; IGF-I 85 &#45 25 ng/ml; PDGF-BB 14 &#45 9 ng/ml; TGF- &#103 2 0.4 &#45 0.3 ng/ml. These growth factor concentrations typically covered a 3-10 fold range: PDGF-AB 29-277 ng/ml; PDGF-BB 2-33 ng/ml; TGF- &#103 1 32-397 ng/ml; TGF- &#103 2 0.1-1.2 ng/ml; IGF-I 40-138 ng/ml. Platelet counts in PC were slightly higher for women (Mann-Whitney Test all p <0.001 ) than for men, while the concentrations of growth factors in PC exhibited no gender-related difference of any statistical significance.     

酶组织化学方法在HA/TCP体内外生物相容性评价中的初步应用

通过研究材料与细胞 /组织相互作用后胞内酶活性的变化与材料生物相容性之间的关系 ,探讨酶组织化学法应用于材料生物相容性评价的可行性。结果发现 ,与 HA/ TCP和钛合金复合培养的成骨细胞形态学上无明显差异。但 HA/ TCP与兔成骨细胞复合培养初期可导致细胞 NADH、SDH、L DH和 CCO酶活性的一过性下降 ,而钛合金对复合培养细胞的酶活性没有明显影响 ,提示 HA/ TCP材料溶出物对细胞有轻微的损伤。两种材料体内植入后引起的组织学变化过程相似 ,主要表现为损伤引起的急性炎症过程。酶组织化学检测发现 ,术后 10 d内植入体周围组织四种酶活性均明显下降 ,15 - 30 d内逐渐恢复正常。但 HA/ TCP组酶活性的恢复略滞后于钛合金组 ,也表明材料溶出物对细胞有一定损伤。体内外实验均发现酶学指标能更灵敏地反映材料对细胞的作用 ,酶组织化学法可望应用于材料生物相容性评价。     

低切应力对体外培养动脉壁细胞PDGF表达的影响

为了研究切应力对完整血管的生物学作用 ,探讨应力引起血管重建过程中生长因子的变化 ,用血管应力培养系统体外培养猪颈总动脉 ,通过免疫组织化学、抗体夹心法酶联免疫吸附测定实验 (EL ISA)和计算机图像分析等方法 ,观察了低切应力作用下动脉壁细胞 PDGF- A、PDGF- B表达变化情况。结果显示 ,低切应力作用下体外培养动脉 EC中 PDGF- B及 VSMC中 PDGF- A的合成与分泌皆增加。PDGF表达上升 ,促进了 VSMC表型转换与增殖。在低切应力引起的血管重建中 ,这一机制可能起着重要作用。