Nanosilver-loaded PMMA bone cement doped with different bioactive glasses - evaluation of cytocompatibility, antibacterial activity, and mechanical properties.

Nanosilver-loaded PMMA bone cement (BC-AgNp) is a novel cement developed as a replacement for conventional cements. Despite its favorable properties and antibacterial activity, BC-AgNp still lacks biodegradability and bioactivity. Hence, we investigated doping with bioactive glasses (BGs) to create a new bioactive BC characterized by time-varying porosity and gradual release of AgNp. The BC Cemex was used as the base material and modified simultaneously with the AgNp and BGs: melted 45S5 and 13-93B3 glasses with various particle sizes and sol-gel derived SiO2/CaO microparticles. The effect of BG addition was examined by microscopic analysis, an assessment of setting parameters, wettability, FTIR and UV-VIS spectroscopy, mechanical testing, and hemo- and cytocompatibility and antibacterial efficiency studies. The results show that it is possible to incorporate various BGs into BC-AgNp, which leads to different properties depending on the type and size of BGs. The smaller particles of melted BGs showed higher porosity and better antibacterial properties with the moderate deterioration of mechanical properties. The sol-gel derived BGs, however, displayed a tendency for agglomeration and random distribution in BC-AgNp. The BGs with greater solubility more efficiently improve the antibacterial properties of BC-AgNp. Besides, the unreacted MMA monomer release could negatively influence the cellular response. Despite that, cements doped with different BGs are suitable for medical applications.

The urine podocin/creatinine ratio as a novel biomarker of cardiorenal syndrome in dogs due to degenerative mitral valve disease.

Dysfunction of heart leads inevitable to the dysfunction of kidney which is termed as the cardiorenal syndrome (CRS). Previous studies have confirmed existence of CRS in dogs with degenerative mitral valve disease (DMVD). The goal of the study was to assess the usefulness of commercial test to measure podocyturia in dogs and test the urine podocine/creatinine ratio (UPoC) as an early marker of kidney injury. Urine podocine/creatinine ratio was calculated because numbers of podocytes is dependent on the urine concentration. Fifty dogs was divided into three groups: fifteen healthy (control group), twenty nine with DMVD class C-chronic according to ACVIM (heart group) and six with chronic kidney disease (kidney group). Each dog underwent a clinical examination: electrocardiography, echocardiography, chest radiograph, abdominal ultrasound, blood haematological and biochemical analysis including symmetric dimethylarginine (SDMA) and cystatin C (Cyst C), routine urine analysis and analysis of podocytes using an ELISA test. UPoC was calculated. Mean value ± standard deviation for UPoC was respectively 9.7 ± 4.8 x 10-10 for control group, 49.0 ± 80.0 x 10-10 for heart group, 33.7 ± 18.0 x 10-10 for kidney group. The UPoC in the heart and kidney group was significantly higher than in the control group (P < 0.0001, sensivity 0.83, specyfity 0.20). Commercial ELISA tests may be used to assess podocyturia in dogs. An UPoC increase exceeding 12.93 x 10-10 indicates glomerular damage in DMVD dogs. Based on UPoC, 79.3% of dogs with C-chronic stage of DMVD developed CRS.

The anti-inflammatory action of the analgesic kyotorphin neuropeptide derivatives: insights of a lipid-mediated mechanism

Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential stretched conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same

Experimental verification of SMC with moving switching lines applied to hoisting crane vertical motion control.

In this paper we propose sliding mode control strategies for the point-to-point motion control of a hoisting crane. The strategies employ time-varying switching lines (characterized by a constant angle of inclination) which move either with a constant deceleration or a constant velocity to the origin of the error state space. An appropriate design of these switching lines results in non-oscillatory convergence of the regulation error in the closed-loop system. Parameters of the lines are selected optimally in the sense of two criteria, i.e. integral absolute error (IAE) and integral of the time multiplied by the absolute error (ITAE). Furthermore, the velocity and acceleration constraints are explicitly taken into account in the optimization process. Theoretical considerations are verified by experimental tests conducted on a laboratory scale hoisting crane.

Coronary artery calcification, common carotid artery intima-media thickness and aortic pulse wave velocity in patients on peritoneal dialysis.

An increasing body of evidence suggests that atherosclerosis in patients with uremia differs from that found in general population in terms of advancement and localization of vascular lesions. It has also been suggested that different non-invasive techniques of vascular system evaluation are designed to show different types of lesions (i.e. vascular calcification, stiffness or 'classical' atherosclerosis). The aim of the study was to search for possible associations between results obtained with three different non-invasive methods of vascular system assessment in three different vascular sites in patients treated with peritoneal dialysis (PD). 61 patients (28 F, 33 M), mean age of 50.4+/-13.6 years, on maintenance PD for a median period of 10 months (range 1-96 months) were included. Coronary artery disease (CAD) was present in 21 subjects. In all subjects coronary artery calcification score (CaSc) using multi-row spiral computed tomography (MSCT), aortic pulse wave velocity (AoPWV) and ultrasound-based common carotid artery intima-media thickness (CCA-IMT) were performed as methods for assessing coronary calcium burden, arterial stiffness and atherosclerosis, respectively. Median value of CaSc equaled 11.5 Agatston units (range 0-5502.8 units). Median AoPWV was 10.4 m/s (range 7.56-18.1 m/s), and median CCA-IMT-0.6 mm (range 0.3-1.0 mm). In 16 patients (26.2%) at least one plaque in at least one common carotid artery was found on ultrasound. CaSc correlated with AoPWV (R=0.32, p<0.01) and with CCA-IMT (R=0.35, p<0.005), whereas no association was found between AoPWV and CCA-IMT. AoPWV, but not CaSc nor IMT correlated with blood pressure. The values of CCA-IMT and AoPWV increased together with consecutive Agatston categories (with p<0.001 for differences in AoPWV and p<0.05 for CCA-IMT). Patients with at least one plaque found in at least one CCA and patients with CAD were characterized with significantly higher values of CaSc, IMT and PWV, when compared to plaque-free and CAD- negative subjects, respectively. Association between CaSc and both IMT and PWV may suggest that the mechanism of three assessed vascular pathologies may be based, to some extent, on the process of pathologic calcium-phosphate deposition. Lack of correlation found between PWV and IMT may suggest that aortic stiffness and carotid atherosclerosis may partially differ in their pathologic background and/or are dissociated in time.

Effects of particulate and soluble (1-3)-beta-glucans on Ca2+ influx in NR8383 alveolar macrophages.

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.

Activation of rat macrophages by Betafectin PGG-glucan requires cross-linking of membrane receptors distinct from complement receptor three (CR3).

PGG-glucan (Betafectin) is a soluble, highly purified yeast (1,3)-beta-glucan with broad anti-infective and immunomodulatory activities. These studies evaluated the ability of PGG-glucan to directly elicit O2- and tumor necrosis factor alpha (TNF-alpha) production by rat leukocytes in vitro. Particulate beta-glucan stimulated O2- production by the rat NR8383 alveolar macrophage cell line and resident rat peritoneal macrophages, but soluble PGG-glucan did not. In contrast, presentation of PGG-glucan to cells after covalent immobilization to a plastic surface caused a direct stimulation of O2- and TNF-alpha production. The O2- response of rat leukocytes to immobilized PGG-glucan was inhibited by soluble PGG-glucan, indicating that cellular responses to both immobilized and soluble PGG-glucan occur via common cell surface receptors. Because complement receptor type three (CR3) has been proposed as a beta-glucan receptor on human leukocytes, NR8383 cells were evaluated for the presence of CR3. Indirect immunofluorescence and flow cytometric analysis showed that despite being responsive to both particulate and immobilized beta-glucans, NR8383 cells expressed no detectable CR3. These results indicate that the beta-glucan receptors on NR8383 cells are not CR3 and suggest that physical presentation plays an important role in inducing pro-inflammatory leukocyte responses to PGG-glucan.

The concentration of cotinine in urine, colostrum and amniotic fluids within the system mother-baby.

The findings obtained by the authors of the thesis submit the new cognitive values to the diagnosis of pathology of pregnancy i.e. the influence of nicotine on the organisms of a mother and a new-born child, estimated by the assay of cotinine, the most important metabolite of nicotine. The authors lay a particular stress on the "colostrum-milk way" in the mother-child relationship and this is that needs to be emphasized in this thesis.

Average quantitative concentration of cotinine within the system pregnant woman-baby.

The findings presented by the authors of this report contribute to the diagnosis of pathology of pregnancy i.e. they assess the influence of nicotine on the organisms of a mother and a new-born child, estimated by concentration of cotinine, the most important metabolite of nicotine. The mean proportional share of cotinine in the fluids and organs in the pregnant women smoking actively was as follows: urine 72.1%, amniotic fluids 14.3%, colostrum 8.9% and placenta 4.7%. The authors pay a particular attention to the "colostrum-milk way" in the mother-child relationship what is emphasized in this report.

The Effect of the zeta Potential on the Stability of a Non-Polar Oil-in-Water Emulsion

The aim of this work was to investigate the electrophoretic properties of emulsion droplets of aliphatic hydrocarbons containing C6 to C16 carbon atoms in hydrocarbon chains. As it was observed experimentally, the emulsion droplets of hydrocarbons C9 to C16 revealed a completely different course of changes of the zeta potential due to pH than the n-alkanes of shorter hydrocarbon chains (C6 to C8). They revealed a negative value of the zeta potential, lower by almost twice. The zeta potentials of emulsion droplets of the n-alkanes, from n-nonane to n-hexadecane, do not practically depend on the length of the hydrocarbon chain but, first of all, on the pH of aqueous solutions. The cause of the origin of the negative zeta potential of n-alkane droplets lies in the selective adsorption of -OH ions, causing the gathering of the excessive negative charge at the oil-water interface. As it was assumed, the stability of oil-in-water emulsion droplets in the solution, i.e., the time of collection of liquid hydrocarbons C6H28 on the surface of aqueous solutions, is the criterion of the life of emulsion droplets of n-alkanes. The observations indicate a close correlation between the emulsion droplets stability of the tested aliphatic hydrocarbons in water and the value changes of the zeta potential.

Chemical denaturation and modification of ovalbumin alters its dependence on ubiquitin conjugation for class I antigen presentation.

Class I presentation of microinjected native OVA by a temperature-sensitive ubiquitin conjugation mutant, ts85, but not wild-type murine cells, was markedly inhibited following incubation at a nonpermissive temperature. In contrast, the nonpermissive temperature did not affect class I presentation of a minimal OVA peptide expressed in the cytosol. Therefore, these results provide a second example in which a temperature sensitive mutation in the ubiquitin conjugation pathway inhibits MHC class I presentation of native OVA. Surprisingly, incubation at the nonpermissive temperature did not inhibit class I presentation of chemically denatured and alkylated OVA microinjected into the cytosol of mutant cells. Similarly, the presentation of endogenously synthesized OVA (which is expressed from a recombinant vaccinia virus and, presumably, is misfolded in the cytosol) was also not inhibited in both mutant cell lines. Methylation of the lysine groups in denatured OVA, which blocks ubiquitin conjugation, reduced but did not eliminate the presentation of denatured OVA, providing evidence for both ubiquitin-dependent and ubiquitin-independent pathways for class I presentation. In contrast, a proteasome inhibitor blocked class I presentation of all forms of OVA, while a control peptide aldehyde was not inhibitory. These results indicate that modification of the structure of a protein can influence its requirements for ubiquitin conjugation for efficient class I presentation, with the key alteration possibly being the loss of proper conformation. However, regardless of the form of the Ag, the proteasome appears to be required for generating peptides from both endogenously synthesized and microinjected OVA for class I presentation.

Rate of antigen degradation by the ubiquitin-proteasome pathway influences MHC class I presentation.

The effect on MHC class I Ag presentation of enhancing a protein's rate of degradation by the ubiquitin-proteasome pathway was investigated. In extracts of mouse B-lymphoblasts and reticulocytes, as in rabbit reticulocytes, proteins with acidic or basic N-termini are conjugated to ubiquitin and degraded by the 26S proteasome very rapidly. We found that the rate of MHC class I presentation of microinjected beta-galactosidase was enhanced when this antigenic protein was modified with such a destabilizing amino-terminal residue. This enhanced presentation was inhibited by blocking potential ubiquitination sites on the protein through methylation of amino groups and by peptide aldehyde inhibitors of the proteasome. Furthermore, in B lymphoblast cell extracts, the rapid degradation of these beta-galactosidase constructs required ATP and ubiquitin and was blocked by inhibitors of proteasomes. Their rates of degradation in extracts correlated with their rates of class I Ag presentation in vivo. These results indicate that ubiquitin conjugation is a key rate-limiting step in Ag presentation and provide further evidence for a critical role of ubiquitin and the 26S proteasome in generating MHC class I-presented peptides.

A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation.

The degradation of most cellular proteins starts with their covalent conjugation with ubiquitin. This labels the proteins for rapid hydrolysis to oligopeptides by a (26S) proteolytic complex containing a (20S) degradative particle called the proteasome. Some system in the cytosol also generates antigenic peptides from endogenously synthesized cellular and viral proteins. These peptides bind to newly synthesized class I major histocompatibility complex molecules in the endoplasmic reticulum and peptide/class I complexes are then transported to the cell surface for presentation to cytotoxic T cells. How these peptides are produced is unknown, although a modification that promotes ubiquitin-dependent degradation of a viral protein enhances its presentation with class I13 and indirect evidence suggests a role for proteolytic particles closely resembling and perhaps identical to the proteasome. Using cells that exhibit a temperature-sensitive defect in ubiquitin conjugation, we report here that non-permissive temperature inhibited class I-restricted presentation of ovalbumin introduced into the cytosol, but did not affect presentation of an ovalbumin peptide synthesized from a minigene. These results implicate the ubiquitin-dependent proteolytic pathway in the production of antigenic peptides.

The class II MHC-restricted presentation of endogenously synthesized ovalbumin displays clonal variation, requires endosomal/lysosomal processing, and is up-regulated by heat shock.

LB27.4 cells (a B lymphoblastoid APC) were transfected with a plasmid containing an OVA cDNA. Functional analysis of six independent clones yielded three patterns of MHC-restricted presentation of the endogenously synthesized OVA. A clone displayed either: 1) strong class I and class II-restricted presentation, 2) strong class I but little or no class II-restricted presentation or, 3) only a modest class I-restricted presentation. There was no clonal variation in class II-restricted presentation of exogenous Ag or in the amount of surface class I or II molecules. Heat shock increased the presentation of endogenous but not exogenous Ag with class II. These results indicate that an endogenously synthesized Ag both constitutively and during heat shock can gain access to the class II, MHC-restricted, presentation pathway. The amount of OVA synthesized by a cell correlated with whether OVA-class II complexes were detected. However, the amount of OVA secreted into the extracellular fluid was not sufficient to sensitize APC, which suggests that endogenously synthesized OVA enters the class II pathway of Ag presentation by an intracellular route rather than by an extracellular/reuptake route. Also, the functional and quantitative analysis of the clones suggests that endogenously synthesized OVA was presented more efficiently with class I as compared to class II-MHC molecules. Leupeptin and chloroquine inhibited the class II-restricted presentation of endogenously synthesized OVA. Together these results indicate that endogenously synthesized OVA can gain access to an endosomal/lysosomal compartment via an intracellular route and be processed and presented in association with class II-MHC molecules.

Weak base amines can inhibit class I MHC-restricted antigen presentation.

This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.

Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants.

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.

Two genetically identical antigen-presenting cell clones display heterogeneity in antigen processing.

Evidence from various antigen systems suggests that antigen processing can be one factor that determines the repertoire of immunogenic peptides. Thus, processing events may account for some of the disparity between the available and expressed helper T-cell repertoires. In this report, we demonstrate that the immunodominant T-cell determinant in ovalbumin [p323-339; ovalbumin-(323-339) heptadecapeptide] is processed differently by two genetically identical antigen-presenting cell lines, M12 and A20. The ovalbumin-specific T-cell-T-cell hybridomas, DO-11.10 and 3DO-54.8, were used to detect processed antigen. These T-T hybridomas have different fine specificities for the p323-339 determinant. A20 cells presented native ovalbumin well to both T-T hybridomas, whereas M12 cells presented native ovalbumin well to 3DO-54.8 but very inefficiently to DO-11.10. M12 and A20 cells effectively stimulated both T-T hybridomas with the same concentrations of the immunogenic synthetic peptide p323-339. Therefore, M12 cells and DO-11.10 can interact with each other, and both T-T hybridomas have similar sensitivities for the same immunogenic peptide. We conclude that genetically identical antigen-presenting cells can display heterogeneity in the fine processing of an immunodominant T-cell determinant, and synthetic model peptides that represent the minimal stimulatory sequence of a T-cell determinant are not necessarily identical to the structure of in vivo processed antigen. Heterogeneity in antigen processing by individual antigen-presenting cells would serve to increase the repertoire of immunogenic peptides that are presented to T cells.