Biomimetic carriers mimicking leukocyte plasma membrane to increase tumor vasculature permeability.

Recent advances in the field of nanomedicine have demonstrated that biomimicry can further improve targeting properties of current nanotechnologies while simultaneously enable carriers with a biological identity to better interact with the biological environment. Immune cells for example employ membrane proteins to target inflamed vasculature, locally increase vascular permeability, and extravasate across inflamed endothelium. Inspired by the physiology of immune cells, we recently developed a procedure to transfer leukocyte membranes onto nanoporous silicon particles (NPS), yielding Leukolike Vectors (LLV). LLV are composed of a surface coating containing multiple receptors that are critical in the cross-talk with the endothelium, mediating cellular accumulation in the tumor microenvironment while decreasing vascular barrier function. We previously demonstrated that lymphocyte function-associated antigen (LFA-1) transferred onto LLV was able to trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Herein, we provide a more comprehensive analysis of the working mechanism of LLV in vitro in activating this pathway and in vivo in enhancing vascular permeability. Our results suggest the biological activity of the leukocyte membrane can be retained upon transplant onto NPS and is critical in providing the particles with complex biological functions towards tumor vasculature.

Biomimetic proteolipid vesicles for targeting inflamed tissues.

A multitude of micro- and nanoparticles have been developed to improve the delivery of systemically administered pharmaceuticals, which are subject to a number of biological barriers that limit their optimal biodistribution. Bioinspired drug-delivery carriers formulated by bottom-up or top-down strategies have emerged as an alternative approach to evade the mononuclear phagocytic system and facilitate transport across the endothelial vessel wall. Here, we describe a method that leverages the advantages of bottom-up and top-down strategies to incorporate proteins derived from the leukocyte plasma membrane into lipid nanoparticles. The resulting proteolipid vesicles-which we refer to as leukosomes-retained the versatility and physicochemical properties typical of liposomal formulations, preferentially targeted inflamed vasculature, enabled the selective and effective delivery of dexamethasone to inflamed tissues, and reduced phlogosis in a localized model of inflammation.

Leptin promotes a proinflammatory lipid profile and induces inflammatory pathways in human SZ95 sebocytes.

BACKGROUND: Leptin, the adipocyte-secreted hormone that regulates weight, is known to link lipid metabolism with inflammation in various cell types. However, its role in human sebocytes has not yet been investigated. OBJECTIVES: The purpose of this study was to investigate the effects of leptin in human sebaceous gland biology. METHODS: Expression of the long form of the leptin receptor (Ob-Rb) was detected by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunochemistry. Lipid analysis was by high-performance thin-layer chromatography, gas chromatography-mass spectrometry and time-of-flight mass spectrometer mass detection. Lipid bodies were visualized by BODIPY staining using fluorescent microscopy and measured by flow cytometry. Interleukin (IL)-6 and IL-8 mRNA levels were assessed by real-time qRT-PCR and their release was evaluated by enzyme-linked immunosorbent assay. Cyclooxygenase (COX)-2 and 5-lipooxygenase (LOX) protein expression and phosphorylation of p65 and signal transducer and activator of transcription (STAT)-3 were determined by Western blot analysis. RESULTS: Expression of Ob-Rb was detected in human sebaceous glands and in cultured human SZ95 sebocytes. The treatment of SZ95 sebocytes with leptin led to enlarged intracellular lipid bodies, increased ratios of unsaturated/saturated fatty acids and decreased vitamin E levels. Further supporting a proinflammatory role, leptin induced COX-2 and 5-LOX expression in SZ95 sebocytes and augmented the production of IL-6 and IL-8 cytokines. On leptin treatment, the STAT-3 and nuclear factor-κB pathways were activated, indicating that these known leptin signalling pathways are active in human sebocytes. CONCLUSIONS: Our findings suggest that leptin signalling may be involved in the proinflammatory regulation of sebaceous lipid metabolism and the induction of inflammatory enzymes and cytokines.

Haemodilution-induced profibrinolytic state is mitigated by fresh-frozen plasma: implications for early haemostatic intervention in massive haemorrhage.

BACKGROUND: Fibrinolysis contributes to coagulopathy after major trauma and surgery. We hypothesized that progressive haemodilution is responsible, at least in part, for increased fibrinolytic tendency of blood clot. METHODS: The study was performed in two parts. First, whole blood (WB) samples collected from six healthy, consented volunteers were diluted in vitro with either saline or fresh-frozen plasma (FFP) to 40% and 15% of baseline. We quantified factor levels related to coagulation and fibrinolysis, and measured endogenous thrombin generation in undiluted control plasma samples and in samples diluted with saline or FFP. Additionally, thromboelastometry was used to assess susceptibility to fibrinolysis after adding tissue plasminogen activator in undiluted WB samples and in samples diluted with saline before and after substitution of fibrinogen or FFP. Secondly, as a model of in vivo haemodilution, we evaluated the same parameters before and after operation in nine consented patients undergoing off-pump coronary artery bypass surgery. RESULTS: The dilution with saline caused dose-dependent decreases in plasma levels of coagulation and antifibrinolytic factors, and in thrombin generation. In FFP-supplemented samples, factor levels and thrombin generation were maintained within normal ranges. Fibrinolytic tendency was significantly higher after haemodilution with saline independent of fibrinogen substitution compared with FFP. Similarly, increased tendency for fibrinolysis was also observed in the in vivo haemodilution. CONCLUSIONS: We demonstrated in vitro and in vivo that progressive haemodilution decreases endogenous antifibrinolytic proteins including alpha(2)-antiplasmin and thrombin-activatable fibrinolysis inhibitor, resulting in increased fibrinolytic tendency. Therefore, early fluid replacement therapy with FFP might be advantageous after massive haemorrhage.

Thrombin generation and fibrinolysis in anti-factor IX treated blood and plasma spiked with factor VIII inhibitor bypassing activity or recombinant factor VIIa.

Activated prothrombin complex concentrates (aPCC) and recombinant activated factor VIIa (rFVIIa) are two important therapies in haemophilia patients with inhibitors and improve clot stability. We hypothesized that potential differences in procoagulant and fibrinolytic actions of aPCC and rFVIIa may lie in the clot stability against fibrinolytic activation. We used thrombin generation, fluorescence detection and thromboelastometry in anti-factor IXa (FIXa) aptamer-treated whole blood (WB) and plasma to evaluate: (i) generation of thrombin and activated factor X (FXa) and (ii) viscoelastic properties of blood clots in the presence of tissue plasminogen activator (tPA) after addition of aPCC (0.4 U mL(-1)) or rFVIIa (60 nm). Peak thrombin generation increased from 85 +/- 19 nm in aptamer-treated plasma to 276 +/- 83 nm and 119 +/- 22 nm after addition of aPCC and rFVIIa respectively (P < 0.001). FXa activity increased within 20 min by 87 +/- 6% and by 660 +/- 97% after addition of aPCC and rFVIIa respectively (P < 0.001). TPA-induced lysis time increased from 458 +/- 378 s in aptamer-treated WB to 1597 +/- 366 s (P = 0.001) and 1132 +/- 214 s (P = 0.075), after addition of aPCC and rFVIIa respectively. In this haemophilia model using the anti-FIXa aptamer, the larger amount of thrombin was generated with aPCC compared with rFVIIa, while FXa generation was more rapidly increased in the presence of rFVIIa. Furthermore, clot formation in anti-FIXa aptamer-treated WB was less susceptible to tPA-induced fibrinolysis after adding aPCC compared with rFVIIa.

Finding the optimal concentration range for fibrinogen replacement after severe haemodilution: an in vitro model.

BACKGROUND: Replacement of fibrinogen is presumably the key step in managing dilutional coagulopathy. We performed an in vitro study hypothesizing that there is a minimal fibrinogen concentration in diluted whole blood above which the rate of clot formation approaches normal. METHODS: Blood samples from six healthy volunteers were diluted 1:5 v/v with saline keeping haematocrit at 24% using red cell concentrates. We measured coagulation factors and thrombin generation in plasma at baseline and after dilution. Thromboelastometry was used to evaluate (i) speed and quality of clot formation in diluted samples supplemented with fibrinogen 50-300 mg dl(-1) and (ii) clot resistance to fibrinolysis. Diluted and undiluted samples with no added fibrinogen served as controls. RESULTS: Coagulation parameters and platelets were reduced by 74-85% after dilution. Peak thrombin generation was reduced by 56%. Adding fibrinogen led to a concentration-dependent improvement of all thromboelastometric parameters. The half maximal effective concentration (EC50) for fibrinogen replacement in haemodiluted blood was calculated to be 125 mg dl(-1). Adding tissue plasminogen activator, 0.15 microg ml(-1), led to a decrease of clot firmness and lysis time. CONCLUSIONS: The target plasma concentration for fibrinogen replacement was predicted by these in vitro results to be greater than 200 mg dl(-1) as only these concentrations optimized the rate of clot formation. This concentration is twice the level suggested by the current transfusion guidelines. Although improved, clots were prone to fibrinolysis indicating that the efficacy of fibrinogen therapy may be influenced by co-existing fibrinolytic tendency occurring during dilutional coagulopathy.

Localization and quantitation of eotaxin mRNA in human nasal polyps.

Nasal Polyps (NPs) are the most common mass lesions found in the nose. NPs cause airway obstruction, prevent normal sinus function, and can lead to infection of the eye, facial bones and central nervous system. The predominant cell type inhabiting NPs is the eosinophil, and the chemokine eotaxin is believed to play an important role in NP eosinophilia. The objective of this study was to localize and quantitate expression of eotaxin mRNA in human NPs. Total RNA was isolated from NPs that were collected from 5 patients who had undergone polypectomy. Portions of these polyps were also fixed in formalin, embedded in paraffin, and sectioned onto slides for use in in situ hybridization. Total RNA from one patient was used in a reverse transcriptase polymerase chain reaction using eotaxin specific primers to generate a human eotaxin cDNA. The eotaxin cDNA was cloned and used to generate probes for Northern blot analyses and for use in in situ hybridization (ISH). Eotaxin mRNA was detected by Northern analyses in all patient samples, though the relative expression level in each patient varied. ISH localized the expression of eotaxin mRNA specifically in eosinophils in 2 of the 3 patients in the study for whom the embedded polyp tissue appeared sufficiently well preserved for mRNA localization. Our findings suggest that eosinophilia in NPs is likely a self-amplification process whereby increasing numbers of eosinophils are recruited to enter the polyp as a result of production of eotaxin by eosinophils already within the polyp.