Time-dependent effects of ATP and its degradation products on inflammatory markers in human blood ex vivo

We recently reported that adenosine 5′-triphosphate (ATP) modulates cytokine release in lipopolysaccharide (LPS)-phytohemagglutinin (PHA)-stimulated blood. ATP inhibited tumor necrosis factor-alpha (TNF-) release via activation of the P2Y₁₁ receptor and increased interleukin (IL)-10 release via stimulation of the P2Y₁₂ receptor. Because ATP is known to be broken down by various ecto-enzymes, we determined the degradation profile of ATP in time in LPS-PHA-stimulated blood. ATP slowly metabolized with 14% remaining after 6 h. Simultaneously, adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP) and hypoxanthine were formed. Subsequently, we investigated the time-dependent effects of ATP and its metabolites on inflammatory markers. Results showed that ATP decreased the rise in concentrations of TNF-, interferon-gamma (IFN-γ) and IL-1β, but increased concentrations of IL-8 and IL-10. Metabolites of ATP showed either no, similar or opposite effects on cytokine release, compared to ATP. In conclusion, ATP has rapid immunomodulatory effects on a variety of cytokines in stimulated whole blood that persist until 24 h.     

Clinical evaluation of platelet antagonists on the ex vivo inhibitory effects of platelet aggregation

It is necessary in clinical to establish the effects of new substances for the primary and secondary prevention of thrombotic illnesses. We measured maximum aggregation after addition of collagen (F.C. 1.7 micrograms/ml) or ADP (F.C. 1.7 mumol/l) in 41 patients without drug treatment, 91 with ticlopidine, 22 with aspirin (ASA); 82-750 mg, 13 with ASA 81 mg, 20 with ticlopidine 100 mg and ASA 81 mg, 13 with cilostazol, 25 with flurbiprofen, 19 with nifedipine and evaluated the ex vivo effect of platelet antagonists. ASA inhibited remarkably collagen-induced platelet aggregation compared with other drugs, but there was significant (p less than 0.01 for 0.5 mumol/l ADP; p less than 0.02 for 1.0 mumol/l ADP) increase of primary aggregation induced by low dose of ADP in 14 healthy volunteers. While ticlopidine only significantly reduced ADP-induced platelet aggregation and we couldn't find dose dependency of ticlopidine (100-300 mg/day) on inhibitory effects of ADP or collagen-induced platelet aggregation. Our results indicate that the administration of one tablet of aspirin for pediatrics (aspirin 81 mg/tab) together with ticlopidine 100 mg is suitable for reduction of platelet aggregation.     

A model of human whole blood lymphokine release for in vitro and ex vivo use

Endotoxin (lipopolysaccharide, LPS) inducible cytokine release by human whole blood is increasingly used to model inflammatory responses in vitro, to detect the presence of pyrogenic contaminations as well as to monitor disease states or immunomodulatory treatments ex vivo. However, the LPS-stimulated blood model primarily allows the assessment of monocyte responses. Here, a whole blood model was established which allows assessment of lymphocyte responses. Four different superantigens, namely staphylococcal enterotoxin A and B (SEA, SEB), toxic shock syndrome toxin-1 (TSST-1) or streptococcal exotoxin A (SPEA) were tested with respect to the induction of lymphokine release. All superantigens were capable of inducing significant amounts of the lymphokines interferon-gamma (IFNgamma), interleukin 2 (IL-2), IL-4, IL-5, IL-13 and tumor necrosis factor beta (TNFbeta) after 72 h of incubation. Concentration-dependencies and kinetics were determined. Blood from 160 healthy donors was used to assess the variability of SEB-inducible lymphokine release. Interindividual differences were more pronounced compared to LPS-inducible monokine release. However, the individual response was maintained when blood from six donors was tested once a week for 8 weeks, suggesting that the individual response represents a donor characteristic. The model appears to be suitable for the evaluation of immunomodulatory agents in vitro as well as ex vivo.     

A novel dermal substitute based on biofunctionalized electrospun PCL nanofibrous matrix

In this study, nanofibrous matrices of polycaprolactone (PCL) and PCL/collagen with immobilized epidermal growth factor (EGF) were successfully fabricated by electrospinning for the purpose of damaged skin regeneration. Nanofiber diameters were found to be 284 ± 48 nm for PCL and 330 ± 104 nm for PCL/collagen matrices. The porosities were calculated as 85% for PCL and 90% for PCL/collagen matrices. The covalent immobilization of EGF onto the nanofibrous matrices was verified by the increase of surface atomic nitrogen ratio from 1.0 to 2.4% for PCL and from 3.7 to 4.7% for PCL/collagen. Moreover, EGF immobilization efficiencies of PCL and PCL/collagen matrices were determined as 98.5 and 99.2%, respectively. Human dermal keratinocytes (HS2) were cultivated on both neat and EGF immobilized PCL and PCL/collagen matrices to investigate the effects of matrix chemical composition and presence of EGF on cell proliferation and differentiation. EGF immobilized PCL/collagen matrices exerted early cell spreading and rapid proliferation. Statistically high expression levels of loricrin in HS2 cells cultivated on EGF immobilized PCL/collagen matrices were (p < 0.001) regarding superior differentiation ability of these cells compared to HS2 cells cultured on neat PCL and PCL/collagen matrices. In conclusion, this novel EGF immobilized PCL/collagen nanofibrous matrix could potentially be considered as an alternative dermal substitutes and wound healing material for skin tissue engineering applications.     

Cytotoxicity evaluation of three light-cured dentin adhesive materials on human gingival fibroblasts,ex vivo

PurposeTo evaluate the cytotoxic effects of three current light-cured dentin adhesives, in both uncured and post-cured conditions, on human gingival fibroblasts.Material and methodsThe materials tested were Heliobond, Adper Single Bond 2 and Xeno V, which are characterized by various compositions and application procedures. Each agent, in volumes of 5 and 10 μL, was tested after polymerization, and those unpolymerized were diluted in DMEM to 10^?3 and 10^?5. The cytotoxicity of the adhesives was assessed on the basis of a test of cell viability in a culture of human gingival fibroblasts, with the use of tetrazolic salt (MTT assay).ResultsThe results showed that, among the adhesive/bonding systems tested, Xeno V was the least cytotoxic. There were statistically significant differences in cell survival between polymerized Xeno V, Adper Single Bond 2 and Heliobond in the amount of 5 μL as well as between the Xeno V and Adper Single Bond 2 in 10^?5 dilutions. The tested adhesives were more toxic in the polymerized form than in the dilutions. Samples of 10 μL resulted in a lower survival percentage of fibroblasts compared to 5 μL.ConclusionAll the tested adhesives demonstrated cytopathic effects towards human gingival fibroblasts, but varied in their cytotoxicity. This has clinical implications. Dentists should follow the rules of adhesive application, precisely dose them and not allow direct contact with the gums as, even after polymerization, adhesive agents exhibit potential cytotoxic activity.     

Porphyromonas gingivalis-induced inflammatory mediator profile in an ex vivo human whole blood model

Periodontitis is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. Leukocytes play a major role in the host response to Porphyromonas gingivalis, a major aetiological agent of chronic periodontitis. Secretion of high levels of inflammatory mediators, including cytokines and prostaglandins, by leucocytes is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of an ex vivo whole blood model to P. gingivalis stimulation. The production of interleukin-1 beta (IL-1beta), IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), IFN-gamma-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), Regulated on Activation Normal T cell Expressed and Secreted (RANTES) and prostaglandin E2 (PGE2) were quantified by enzyme-linked immunosorbent assays. P. gingivalis induced the secretion of the pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 and IFN-gamma, the chemokines IL-8, RANTES and MCP-1 and the inflammatory mediator PGE2 in an ex vivo human whole blood model. The secretion levels were dependent on the strain and the infectious dose used. While the mediator profiles were comparable between six healthy subjects, a high interindividual variability in the levels of secreted mediators was observed. This study supports the view that P. gingivalis, by inducing high levels of inflammatory mediators from a mixed leucocyte population, can contribute to the progression of periodontitis.     

Transplantation of ex vivo expanded cord blood.

Umbilical cord blood (CB) from unrelated donors is increasingly used to restore hematopoiesis after myeloablative therapy. CB transplants are associated with higher rates of delayed and failed engraftment than are bone marrow transplants, particularly for adult patients. We studied the ex vivo expansion of CB in an attempt to improve time to engraftment and reduce the graft failure rate in the recipients. In this feasibility study, 37 patients (25 adults, 12 children) with hematologic malignancies (n = 34) or breast cancer (n = 3) received high-dose therapy followed by unrelated allogeneic CB transplantation. A fraction of each patient's CB allograft was CD34-selected and cultured ex vivo for 10 days prior to transplantation in defined media with stem cell factor, granulocyte colony-stimulating factor, and megakaryocyte growth and differentiation factor. The remainder of the CB graft was infused without further manipulation. Two sequential cohorts of patients were accrued to the study. The first cohort had 40% and the second cohort had 60% of their CB graft expanded. Patients received a median of 0.99 x 10(7) total nucleated cells (expanded plus unexpanded) per kilogram. The median time to engraftment of neutrophils was 28 days (range, 15-49 days) and of platelets was 106 days (range, 38-345 days). All evaluable patients who were followed for 28 days or longer achieved engraftment of neutrophils. Grade III/IV acute GVHD was documented in 40% and extensive chronic GVHD in 63% of patients. At a median follow-up of 30 months, 13 (35%) of 37 of patients survived. This study demonstrates that the CD34 selection and ex vivo expansion of CB prior to transplantation of CB is feasible. Additional accrual will be required to assess the clinical efficacy of expanded CB progenitors.     

In vitro and ex vivo effect of tiaprofenic acid on human peripheral blood mononuclear cells.

The effect of the non-steroidal anti-inflammatory drug (NSAID) tiaprofenic acid on different human immune parameters was investigated in vitro or following in vivo administration in healthy adult volunteers. Results from the in vitro study demonstrated an increased mitogen-induced blastogenesis and interleukin 2 (IL-2) production together with a reduced polyclonal immunoglobulin (Ig) secretion in the presence of the drug. Results from the ex vivo study showed that treatment with tiaprofenic acid had no significant effects on the immune parameters investigated, i.e. unstimulated and mitogen-induced proliferation and IL-2 production, spontaneous and stimulated Ig synthesis, lymphocyte subpopulations, serum Ig and complement levels.     

A comparison of ex vivo cytokine production in venous and capillary blood

We performed a randomized study of the immunological effects of an early measles vaccine given at 4.5 months of age and aimed to obtain venous samples from the infants at baseline and 6 weeks later. If this was not feasible, a capillary sample was obtained. We analysed baseline samples from the first 50 children enrolled in the study to investigate the potential differences in ex vivo cytokine production between venous blood and capillary blood. We also obtained paired venous and capillary blood samples from 11 adult volunteers. Whole blood was stimulated with lipopolysaccharide (LPS) [a Toll-like receptor (TLR)-4 ligand], (S)-(2, 3-bis (palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, trihydrochloride (PAM3Cys) (a TLR-2 ligand), phytohaemagglutinin (PHA) or purified protein derivative (PPD). Cytokine concentrations in the supernatants were assessed by a multiplexed assay and were compared between venous and capillary samples in both infants and adults. The production of both the pro- and the anti-inflammatory cytokines, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10, was higher in cultures of capillary blood compared with venous blood. This was found in non-stimulated control samples as well as in blood stimulated with PAM3Cys and PPD. Adults produced more IL-5 in venous blood than in capillary blood upon PHA stimulation. We found no other difference in the levels of IL-5 or IFN-gamma between venous and capillary blood. In capillary blood we found sex differences in response to PHA but this was not the case in venous blood. We found significant differences in the production of cytokines between venous and capillary blood. Such differences should be taken into account when setting up immuno-epidemiological studies.     

Stroma from different developmental stages of fetal liver has different effects on human mononuclear fraction of umbilical cord blood during ex vivo co-culture

Fetal liver (FL) has transient and intense hematopoietic activity during gestation. It has been shown that hematopoietic activity in FL begins at embryonic day (E)11, is maximal at E16 and rapidly ceases thereafter. This development of FL is simultaneous with transition of several hematopoietic features. Stromal cells (SCs) appear to regulate hematopoiesis in FL. To compare in vitro hematopoietic support ability of FL SCs from different developmental stages, we established three mouse primary FL SCs from different gestation days and compared their capacity in supporting the growth and differentiation of human umbilical cord blood, by flow cytometry and long-term culture-initiating cell (LTC-IC) assays. Results showed that expansion of hematopoietic progenitor in an E15.5 FL SC microenvironment was superior to that in E12.5 and E18.5 FL SC microenvironments. Co-culture on E15.5 FL SCs resulted in a 3.04-fold increase in early CD34⁺/CD38^– progenitors and a 6.4-fold increase in LTC-ICs after 2 weeks. However, the comparison of CD2⁺/CD19^– cell output and CD19⁺ cell output in different conditions did not attain statistical significance. These results show that murine E15.5 FL SCs are most effective in sustaining stem cell function along with greatest expansion of total nuclear cells of human umbilical cord blood (hUCB). We conclude that the change in SC function is responsible for the transition of hematopoietic function in FL.     

A novel ovine ex vivo arteriovenous shunt model to test vascular implantability

The major objective of successful development of tissue-engineered vascular grafts is long-term in vivo patency. Optimization of matrix, cell source, surface modifications, and physical preconditioning are all elements of attaining a compatible, durable, and functional vascular construct. In vitro model systems are inadequate to test elements of thrombogenicity and vascular dynamic functional properties while in vivo implantation is complicated, labor-intensive, and cost-ineffective. We proposed an ex vivo ovine arteriovenous shunt model in which we can test the patency and physical properties of vascular grafts under physiologic conditions. The pressure, flow rate, and vascular diameter were monitored in real-time in order to evaluate the pulse wave velocity, augmentation index, and dynamic elastic modulus, all indicators of graft stiffness. Carotid arteries, jugular veins, and small intestinal submucosa-based grafts were tested. SIS grafts demonstrated physical properties between those of carotid arteries and jugular veins. Anticoagulation properties of grafts were assessed via scanning electron microscopy imaging, en face immunostaining, and histology. Luminal seeding with endothelial cells greatly decreased the attachment of thrombotic components. This model is also suture free, allowing for multiple samples to be stably processed within one animal. This tunable (pressure, flow, shear) ex vivo shunt model can be used to optimize the implantability and long-term patency of tissue-engineered vascular constructs.     

Relevance of ex vivo blood lymphocyte assay for in vivo lymphocyte function

Determinations of in vitro proliferative and secretory activities of peripheral blood cells are used widely for research in clinical immunology but, to our knowledge, have not been evaluated as to their power to reflect in vivo activities quantitatively. Here, we addressed this question by quantitatively correlating the in vitro secretion of interleukin (IL)-5 by peripheral blood cells to the in vivo activity of IL-5 as reflected by peripheral-blood eosinophil counts. Studying 458 humans exposed to transmission of the nematode Onchocerca volvulus, IL-5 was measured in the supernatants of 0.02-ml whole-blood cells cultured in the presence of O. volvulus extract or mitogen. O. volvulus-reactive IL-5 secretion was correlated significantly to blood eosinophilia in a quantitative manner explaining 15.1% (95% CI 8.3-19.9%) of the variability of eosinophil counts. Interestingly, correlations were obtained only if parasite counts were included in the calculation using multiple regression analysis. The results show that in vitro assays of minute amounts of blood lymphocytes may quantitatively reflect activities of the entire lymphocyte population in vivo.     

Megakaryothrombopoiesis during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO-induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO-induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum-free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8 N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P-selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC-1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid-Schiff (PAS)-positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.     

Chromosome analysis after ex vivo expansion of CD34(+) cells from human cord blood

Ex vivo expansion of cord blood hematopoietic progenitors is an attractive way to prepare a sufficient number of transplantable cells for cord blood transplantation in adult patients. The expanded cells need to have genetic stability. Karyotypic analysis of the expanded cells from cord blood CD34(+) cells by 7-day culture with stem cell growth factor, interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor, and erythropoietin was performed. Several-fold increases in total cell number and CFU-GM were 186.7 +/- 62.1 and 27.1 +/- 9.4 (mean +/- standard deviation of data from 6 samples), respectively. Karyotypes were analyzed in 600 expanded cells in total, and all of them showed normal karyotypes. This observation suggests that multifactor supported expansion of cord blood cells may not induce karyotypic abnormality, although a limited number of ex vivo expanded cells were tested.     

Spontaneous decrease of CD14 cell surface expression in human peripheral blood monocytes ex vivo

In the continuous presence of bacterial lipopolysaccharide (LPS), the membrane-bound LPS receptor, CD14, exhibits a biphasic pattern of surface expression in monocytes ex vivo; an initial increase is followed by a later decrease. In the presence of LPS, ex vivo changes in monocytic CD14 cell surface expression have been consistently interpreted as the direct result of LPS exposure. There has been little consideration for the possibility of additional cell culture effects. Here, an experiment is presented to dissect the differences between LPS effects and cell culture effects on monocytic CD14 cell surface expression ex vivo. The results show that in monocytes from diluted whole blood cultures, CD14 surface expression is induced in LPS-treated samples but decreased in untreated samples. These observations suggest that the previously observed biphasic surface expression pattern of CD14 in long-term LPS-treated monocytes may be the result of a superposition of LPS-induced expression and spontaneous disappearance of CD14 from the plasma membrane. Further, these results illustrate the importance of taking cell culture conditions into account when analyzing monocyte expression of CD14.     

人NK细胞体外高效扩增的实验研究

目的:建立人NK细胞体外大量扩增的方法。方法:采用基因工程方法,在K562细胞上同时表达IL-15、IL-18、4-1BBL3种基因,构建特定的K562工程细胞作为刺激细胞。IL-15、IL-18基因分别与一段特殊的跨膜蛋白基因融合,4-1BBL直接跨膜表达,使这3种蛋白在K562细胞中表达后锚定于细胞膜表面。其次,以照射致死的该K562工程细胞作为刺激细胞,以人外周血单个核细胞(PBMC)为扩增培养对象,通过与IL-2的共刺激作用,使NK细胞在体外培养条件下得到大量的扩增。结果:经过21d的刺激培养后,CD56 CD3-细胞数量扩增了(520±75)倍。CD56 CD3-细胞的纯度从培养前占PBMC的7%±4%到扩增后占总细胞比例的93%±3%。PBMC中的T细胞基本上没有得到扩增,扩增后的细胞中CD3 细胞只占2%±1.2%。扩增的细胞具备了NK细胞的基本特征和生物学特性,除了CD56 CD3-外,还对扩增的NK细胞上NKG2D、NKp46、NKp44、NKp30、CD94、CD158b、CD158a、NKB1、NKAT2等标记进行了验证。细胞毒实验表明,在效应细胞∶靶细胞为5∶1时,扩增的NK细胞的杀伤率达到了95%±4%。结论:建立的NK细胞体外扩增方法,达到了较高的扩增水平,且扩增的细胞活性较好。本方法以PBMC为原始材料,能够实现NK细胞体外的大规模制备,这将为抗病毒与抗肿瘤的NK细胞免疫治疗奠定基础。     

Development of an ex vivo model to investigate the effects of altered haemodynamics on human bypass grafts

The insertion of vein grafts into the arterial circulation may contribute to vessel wall thickening and accelerated atherosclerosis, a common feature of late vein graft failure. We aimed to develop a model suitable for investigation of the effects of altered haemodynamics on human saphenous vein following its implantation into the arterial circulation. Segments of human saphenous vein obtained from patients undergoing coronary artery bypass surgery were sutured at each end to PTFE and placed into a flow system. Pressure and flow rates to stimulate the arterial and venous systems were achieved. A theoretical model of the flow chamber was created and computational fluid dynamics software (FLOTRAN, Swanson Analysis Systems) was used to determine the flow profile within the model. In summary, a flow model has been developed to investigate the effect of altered haemodynamics on the molecular and pathological changes that occur in vein grafts incorporated into the arterial circulation.     

Development of an ex vivo model to investigate the effects of altered haemodynamics on human bypass grafts

The insertion of vein grafts into the arterial circulation may contribute to vessel wall thickening and accelerated atherosclerosis, a common feature of late vein graft failure. We aimed to develop a model suitable for investigation of the effects of altered haemodynamics on human saphenous vein following its implantation into the arterial circulation. Segments of human saphenous vein obtained from patients undergoing coronary artery bypass surgery were sutured at each end to PTFE and placed into a flowsystem. Pressure and flowrates to stimulatethe arterial and venous systems were achieved. A theoretical model of the flowchamber was created and computational fluid dynamics software (FLOTRAN, Swanson Analysis Systems) was used to determine the flowprofile within the model. In summary, a flow model has been developed to investigate the effect of altered haemodynamics on the molecular and pathological changes that occur in vein grafts incorpor-ated into the arterial circulation.     

A novel evaluation system for whole-organ-engineered liver graft by ex vivo application to a highly reproducible hepatic failure rat model

Journal of Artificial Organs - In recent years, studies on liver graft construction using the decellularized liver as a template for transplantation therapy have attracted much attention. However,...