Morphological dependent effect of cell-free formed supramolecular fibronectin on cellular activities.

Fibrillar fibronectin (FFN), an active form of fibronectin (FN), plays important roles in various cellular processes. Our goal is to investigate effect of FFN morphology on cellular behaviors. Plasma FN at two concentrations was cross-linked into FFN by dialysis against 2 M urea followed by morphological analysis under Scanning Electron Microscopy. To evaluate effect of FFN morphology, fibroblasts were cultured on FN or different FFNs. Fibroblast behaviors including adhesion, spreading, and migration were evaluated. Our data showed that FN fibrillogenesis was dependent on FN concentration. At high concentrations (0.75 mg/mL), large FFN approximately 2.167 + 0.875 µm in diameter were formed with attached nodular structures and rough surface. In contrast, smooth surface FFN fibrils with diameter of 1.886 + 0.412 µm were formed from FN at 0.25 mg/mL. Cellular assays revealed morphological dependent biological effects of different FFNs. Fibroblast separately adhered to native FN and remained spherical while on FFN, cells attached with higher quantity and showed spreading morphology. A synergistic ligand interaction of integrin α5ß1 and αvß3 was observed in cell adhering on FFN. Cell migration results showed that large FFN decreased migration rate while small FFN did not. Taken together, our data draws new attention towards controlling biological function of FN by its fibrillar structure.

Serum metabolic signature of binge-like palatable food consumption in female rats by nuclear magnetic resonance spectroscopy.

Maladaptive eating behavior is a growing public health problem and compulsively eating excessive food in a short time, or binge eating, is a key symptom of many eating disorders. In order to investigate the binge-like eating behavior in female rats, induced by intermittent food restrictions/refeeding and frustration stress, we analyzed for the first time the metabolic profile obtained from serum of rats, through nuclear magnetic resonance (NMR) spectroscopy. In this experimental protocol, rats were exposed to chow food restricting/refeeding and frustration stress manipulation. This stress procedure consists of 15 min exposure to the odor and sight of a familiar chocolate paste, without access to it, just before offering the palatable food. In this model, a "binge-eating episode" was considered the significantly higher palatable food consumption within 2 h in restricted and stressed rats (R + S) than in the other three experimental groups: rats with no food restriction and no stress (NR + NS), only stressed rats (NR + S) or only restricted rats (R + NS). Serum samples from these four different rat groups were collected. The statistical analysis of the 1 H NMR spectral profiles of the four sets of samples pointed to O- and N-acetyl glycoproteins as the main biomarkers for the discrimination of restriction effects. Other metabolites, such as threonine, glycine, glutamine, acetate, pyruvate and lactate, showed trends that may be useful to understand metabolic pathways involved in eating disorders. This study suggested that NMR-based metabolomics is a suitable approach to detect biomarkers related to binge-eating behavior.

An ingestible self-orienting system for oral delivery of macromolecules.

Biomacromolecules have transformed our capacity to effectively treat diseases; however, their rapid degradation and poor absorption in the gastrointestinal (GI) tract generally limit their administration to parenteral routes. An oral biologic delivery system must aid in both localization and permeation to achieve systemic drug uptake. Inspired by the leopard tortoise's ability to passively reorient, we developed an ingestible self-orienting millimeter-scale applicator (SOMA) that autonomously positions itself to engage with GI tissue. It then deploys milliposts fabricated from active pharmaceutical ingredients directly through the gastric mucosa while avoiding perforation. We conducted in vivo studies in rats and swine that support the applicator's safety and, using insulin as a model drug, demonstrated that the SOMA delivers active pharmaceutical ingredient plasma levels comparable to those achieved with subcutaneous millipost administration.

Delivery-associated presence of supramolecular fibronectin-fibrin complexes in puerperal and cord plasma.

Objective: The variable fibronectin (FN) molecular forms are known to be engaged in coagulation and fibrinolysis pathways as well as tissue remodeling and repair processes. Some of them seem to be indispensable molecules within intensive biological processes associated with delivery. The aim of the study was to evaluate the FN molecular status in maternal and cord plasma after vaginal birth and cesarean section (C-section). Materials and methods: The study included nonpregnant women's plasma samples (n = 31) and puerperal and cord plasma samples collected from 49 mothers who delivered healthy newborns at term by vaginal birth (n = 25) and C-section (n = 24). The maternal and cord plasma FN concentrations and presence and relative ratios of different FN-fibrin complexes were determined by ELISA and sodium dodecyl sulfate (SDS) -agarose immunoblotting, respectively. Results: FN concentration in puerperal plasma after vaginal birth (232.08 ± 71.8 mg/L) and C-section (228.17 ± 71.2 mg/L) was significantly higher than in the plasma of nonpregnant women (190.00 ± 48.75 mg/L). In contrast, FN concentration in cord plasma of the C-section group (101.95 ± 30.3 mg/L) was significantly lower than that of the vaginal birth group (121.80 ± 22.2 mg/L). Immunoblotting of puerperal and cord plasma distinguished the most abundant dimeric plasma FN form, the 220-280-kDa FN degradation products and 750-1900-kDa FN-fibrin complexes, which occurred more frequently and in higher amounts in puerperal and cord plasma groups than the nonpregnant women group, although independently of the mode of delivery. Conclusions: Occurrence and relative amount of delivery-associated FN-fibrin complexes in both puerperal and cord plasmas might be bound with the physiological adaptive mechanisms reducing the risk of hemorrhage and intensive remodeling and repair processes after delivery.

A method for determining polysaccharide content in biological samples.

Difficulties in the determination of polysaccharide content in biological samples has been one of the bottlenecks limiting the development of polysaccharides pharmacokinetics, greatly due to the complicated chemical structure of these compounds. In this study, we established a simple, reproducible and reliable method for the determination of polysaccharide content in biological samples. Polysaccharides were replaced by fluorescein isothiocyanate (FITC) and iodine to generate a complex. The iodine content in the complex was measured by inductively coupled plasma-mass spectrometry (ICP-MS) to indirectly reflect polysaccharide content. We investigated the stability of the complex, carried on methodological validation and proceeded to determine the polysaccharide content in rat blood and organs after oral administration of iodine substituted polysaccharides using ICP-MS. The results showed that the iodine complex was stable in vivo and in vitro. In the bioavailability of polysaccharides, after 1h, the absorption rate of polysaccharides in blood was the highest, reaching 5.84%. After 3h, the bioaccessibility was differently distributed in various organs, reaching 1% on average across organs.

The Application of a Modified d-ROMs Test for Measurement of Oxidative Stress and Oxidized High-Density Lipoprotein.

Reactive oxygen species (ROS) are involved in the initiation and progression of atherosclerosis. ROS-derived hydroperoxides, as an indicator of ROS production, have been measured by using the diacron reactive oxygen metabolites (d-ROMs) test, which requires iron-containing transferrin in the reaction mixture. In this study we developed a modified d-ROMs test, termed the Fe-ROMs test, where iron ions were exogenously added to the reaction mixture. This modification is expected to exclude the assay variation that comes from different blood iron levels in individuals. In addition, this Fe-ROMs test was helpful for determining the class of plasma lipoproteins that are hydroperoxidized. Low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and high-density lipoprotein (HDL) were purified by use of an LDL/VLDL purification kit and the dextran sulfate-Mg2+ precipitation method, respectively; their hydroperoxide contents were assessed by performing the Fe-ROMs test. The majority of the hydroperoxides were detected only in the HDL fraction, not in the LDL/VLDL. Further detailed analysis of HDLs by size-exclusion high-performance liquid chromatography revealed that the hydroperoxide-containing molecules were small-sized HDLs. Because HDL was shown to be the principal vehicle for the plasma hydroperoxides, this Fe-ROMs test is a beneficial method for the assessment of oxidized-HDL levels. Indeed, Fe-ROMs levels were strongly associated with the levels of oxidized HDL, which were determined by performing the malondialdehyde-modified HDL enzyme immunoassay. In conclusion, the Fe-ROMs test using plasma itself or the HDL fraction after dextran sulfate-Mg2+ precipitation is useful to assess the functionality of HDL, because the oxidation of HDL impairs its antiatherogenic capacity.

The Isolation and Quantitation of Fetuin-A-Containing Calciprotein Particles from Biological Fluids.

Multiple overlapping systemic and local inhibitory networks have evolved to prevent the unwanted deposition of mineral at ectopic sites. Fetuin-A is a liver-derived glycoprotein abundant in plasma that binds and stabilizes nascent mineral ion nuclei to form soluble colloidal high molecular weight complexes, called calciprotein particles (CPP). The binding of fetuin-A to mineral retards crystal ripening and precipitation from the aqueous phase, thereby facilitating the regulated clearance of mineral debris from the extracellular fluid. However, persistent disturbances in this humoral homeostatic system, as frequently seen in patients with Chronic Kidney Disease, may lead to the accumulation and aggregation of these nanoparticles in extraosseous tissues like the vasculature, driving inflammatory cascades, aberrant tissue remodeling, and functional impairment. Consistent with this conceptual framework, higher circulating CPP levels are associated with reduced renal function, increments in systemic inflammatory markers, derangements in bone morphogenetic cytokines, higher vascular calcification scores, aortic stiffening and an increased risk of death. This chapter describes optimized sample collection and preparative procedures for the isolation and enrichment of CPP from biological fluids. Methods for CPP quantitation are critically reviewed and detailed.

Elevated uptake of plasma macromolecules by regions of arterial wall predisposed to plaque instability in a mouse model.

Atherosclerosis may be triggered by an elevated net transport of lipid-carrying macromolecules from plasma into the arterial wall. We hypothesised that whether lesions are of the thin-cap fibroatheroma (TCFA) type or are less fatty and more fibrous depends on the degree of elevation of transport, with greater uptake leading to the former. We further hypothesised that the degree of elevation can depend on haemodynamic wall shear stress characteristics and nitric oxide synthesis. Placing a tapered cuff around the carotid artery of apolipoprotein E -/- mice modifies patterns of shear stress and eNOS expression, and triggers lesion development at the upstream and downstream cuff margins; upstream but not downstream lesions resemble the TCFA. We measured wall uptake of a macromolecular tracer in the carotid artery of C57bl/6 mice after cuff placement. Uptake was elevated in the regions that develop lesions in hyperlipidaemic mice and was significantly more elevated where plaques of the TCFA type develop. Computational simulations and effects of reversing the cuff orientation indicated a role for solid as well as fluid mechanical stresses. Inhibiting NO synthesis abolished the difference in uptake between the upstream and downstream sites. The data support the hypothesis that excessively elevated wall uptake of plasma macromolecules initiates the development of the TCFA, suggest that such uptake can result from solid and fluid mechanical stresses, and are consistent with a role for NO synthesis. Modification of wall transport properties might form the basis of novel methods for reducing plaque rupture.

Identification of soluble supramolecular FN-fibrin complexes in human plasma.

SDS-agarose FN immunoblotting of 257 normal and pathological human plasma samples revealed the ladder pattern of multiple plasma FN bands which corresponded to FN monomer and dimer, and 5 FN-fibrin bands with increasing molecular masses. The FN-fibrin bands of about 750 kDa, 1000 kDa, 1300 kDa, 1600 kDa, and 1900 kDa appeared more frequently and in significantly higher relative amounts in the pathological samples (P < 0.000) than in relatively healthy individuals. The revealing of high-molecular FN-fibrin complexes by SDS-agarose FN immunobloting might have the potential to become a laboratory biomarker of some diseases in which the coagulation system is triggered.

Molecular lipids identify cardiovascular risk and are efficiently lowered by simvastatin and PCSK9 deficiency.

CONTEXT: Coronary artery disease (CAD) is among the leading causes of mortality and morbidity worldwide. Traditional risk markers explain only a proportion of total cardiovascular risk. Thus, development and improvement of early diagnostic strategies and targeted initiation of preventive measures would be of great benefit. OBJECTIVE: We aimed to identify molecular lipids that are associated with fatal outcome of CAD patients. Furthermore, the effect of different lipid-lowering drugs on novel risk lipids was evaluated. METHODS: Serum samples of 445 CAD subjects participating in a long-term follow-up of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study were analyzed. In addition, samples obtained from a separate randomized parallel three-group study of subjects treated with simvastatin (n=24), ezetimibe (n=24), or their combination (n=24) were studied. Furthermore, samples from the LURIC participants with a loss-of-function mutation (R46L) in the PCSK9 gene (n=19) were analyzed and compared with major allele carriers (n=868). RESULTS: Distinct ceramide species were significantly associated with the fatal outcome of CAD patients. Simvastatin lowered plasma ceramides broadly by about 25%, but no changes in ceramides were observed in the ezetimibe group. PCSK9 deficiency was significantly associated (-13%) with lowered low-density lipoprotein cholesterol accompanied by a significant 20% reduction in CAD outcome risk-related ceramides. CONCLUSIONS: These data suggest that distinct ceramides associate significantly with CAD outcome independently of traditional risk factors and that the mechanism of lipid lowering is important.

Therapeutic plasma exchange: a technical and operational review.

Therapeutic plasma exchange (TPE) is an extracorporeal blood purification technique designed for the removal of large molecular weight substances. Examples of these substances include pathogenic autoantibodies, immune complexes, cryoglobulins, myeloma light chains, endotoxin and cholesterol containing lipoproteins. The basic premise of the treatment is that removal of these substances will allow for the reversal of the pathologic processes related to their presence. This review will cover the techniques for performing TPE, the kinetics of the removal of large molecules from the plasma and the benefits and risks of the different types of replacement fluids.

Capillary viscometer for fully automated measurement of the concentration and shear dependence of the viscosity of macromolecular solutions.

The construction and operation of a novel viscometer/rheometer are described. The instrument is designed to measure the viscosity of a macromolecular solution while automatically varying both solute concentration and shear rate. Viscosity is calculated directly from Poiseuille's law, given the measured difference in pressure between two ends of a capillary tube through which the solution is flowing at a known rate. The instrument requires as little as 0.75 mL of a solution to provide a full profile of viscosity as a function of concentration and shear rate, and it can measure viscosities as high as 500 cP and as low as 1 cP, at shear rates between 10 and 2 × 10(3) s(-1). The results of control experiments are presented to document the accuracy and precision of measurement at both low and high concentration of synthetic polymers and proteins.

The proteomic profile of circulating pentraxin 3 (PTX3) complex in sepsis demonstrates the interaction with azurocidin 1 and other components of neutrophil extracellular traps.

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (~200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (K(D) = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.

Evidence for a macromolecular complex in poor prognosis CLL that contains CD38, CD49d, CD44 and MMP-9.

Progressive chronic lymphocytic leukaemia is characterized by the accumulation of neoplastic B-cells in the tissues and correlates with the expression of prognostic biomarkers, such as CD38, CD49d and matrix metalloproteinase-9 (MMP9), which are involved in migration and tissue invasion. In this study we investigated the physical relationship between these molecules and demonstrated that CD38, CD49d, MMP9 and CD44 were physically associated in a supramolecular cell surface complex. Our findings provide a molecular basis for the correlation between expression of these proteins and prognosis and, as the complex is not present in normal B-cells, suggest a novel leukaemia-specific therapeutic target.