Immunological Profile and Markers of Endothelial Dysfunction in Elderly Patients with Cognitive Impairments.

The process of aging is accompanied by a dynamic restructuring of the immune response, a phenomenon known as immunosenescence. Further, damage to the endothelium can be both a cause and a consequence of many diseases, especially in elderly people. The purpose of this study was to carry out immunological and biochemical profiling of elderly people with acute ischemic stroke (AIS), chronic cerebral circulation insufficiency (CCCI), prediabetes or newly diagnosed type II diabetes mellitus (DM), and subcortical ischemic vascular dementia (SIVD). Socio-demographic, lifestyle, and cognitive data were obtained. Biochemical, hematological, and immunological analyses were carried out, and extracellular vesicles (EVs) with endothelial CD markers were assessed. The greatest number of significant deviations from conditionally healthy donors (HDs) of the same age were registered in the SIVD group, a total of 20, of which 12 were specific and six were non-specific but with maximal differences (as compared to the other three groups) from the HDs group. The non-specific deviations were for the MOCA (Montreal Cognitive Impairment Scale), the MMSE (Mini Mental State Examination) and life satisfaction self-assessment scores, a decrease of albumin levels, and ADAMTS13 (a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13) activity, and an increase of the VWF (von Willebrand factor) level. Considering the significant changes in immunological parameters (mostly Th17-like cells) and endothelial CD markers (CD144 and CD34), vascular repair was impaired to the greatest extent in the DM group. The AIS patients showed 12 significant deviations from the HD controls, including three specific to this group. These were high NEFAs (non-esterified fatty acids) and CD31 and CD147 markers of EVs. The lowest number of deviations were registered in the CCCI group, nine in total. There were significant changes from the HD controls with no specifics to this group, and just one non-specific with a maximal difference from the control parameters, which was α1-AGP (alpha 1 acid glycoprotein, orosomucoid). Besides the DM patients, impairments of vascular repair were also registered in the CCCI and AIS patients, with a complete absence of such in patients with dementia (SIVD group). On the other hand, microvascular damage seemed to be maximal in the latter group, considering the biochemical indicators VWF and ADAMTS13. In the DM patients, a maximum immune response was registered, mainly with Th17-like cells. In the CCCI group, the reaction was not as pronounced compared to other groups of patients, which may indicate the initial stages and/or compensatory nature of organic changes (remodeling). At the same time, immunological and biochemical deviations in SIVD patients indicated a persistent remodeling in microvessels, chronic inflammation, and a significant decrease in the anabolic function of the liver and other tissues. The data obtained support two interrelated assumptions. Taking into account the primary biochemical factors that trigger the pathological processes associated with vascular pathology and related diseases, the first assumption is that purine degradation in skeletal muscle may be a major factor in the production of uric acid, followed by its production by non-muscle cells, the main of which are endothelial cells. Another assumption is that therapeutic factors that increase the levels of endothelial progenitor cells may have a therapeutic effect in reducing the risk of cerebrovascular disease and related neurodegenerative diseases.

Development and Bioactivity of Zinc Sulfate Cross-Linked Polysaccharide Delivery System of Dexamethasone Phosphate.

Improving the biopharmaceutical properties of glucocorticoids (increasing local bioavailability and reducing systemic toxicity) is an important challenge. The aim of this study was to develop a dexamethasone phosphate (DexP) delivery system based on hyaluronic acid (HA) and a water-soluble cationic chitosan derivative, diethylaminoethyl chitosan (DEAECS). The DexP delivery system was a polyelectrolyte complex (PEC) resulting from interpolymer interactions between the HA polyanion and the DEAECS polycation with simultaneous incorporation of zinc ions as a cross-linking agent into the complex. The developed PECs had a hydrodynamic diameter of 244 nm and a ζ-potential of +24.4 mV; the encapsulation efficiency and DexP content were 75.6% and 45.4 µg/mg, respectively. The designed DexP delivery systems were characterized by both excellent mucoadhesion and prolonged drug release (approximately 70% of DexP was released within 10 h). In vitro experiments showed that encapsulation of DexP in polysaccharide nanocarriers did not reduce its anti-inflammatory activity compared to free DexP.

Combined Use of Antimicrobial Peptides with Antiseptics against Multidrug-Resistant Bacteria: Pros and Cons.

Antimicrobial peptides (AMPs) are acknowledged as a promising template for designing new antimicrobials. At the same time, existing toxicity issues and limitations in their pharmacokinetics make topical application one of the less complicated routes to put AMPs-based therapeutics into actual medical practice. Antiseptics are one of the common components for topical treatment potent against antibiotic-resistant pathogens but often with toxicity limitations of their own. Thus, the interaction of AMPs and antiseptics is an interesting topic that is also less explored than combined action of AMPs and antibiotics. Herein, we analyzed antibacterial, antibiofilm, and cytotoxic activity of combinations of both membranolytic and non-membranolytic AMPs with a number of antiseptic agents. Fractional concentration indices were used as a measure of possible effective concentration reduction achievable due to combined application. Cases of both synergistic and antagonistic interaction with certain antiseptics and surfactants were identified, and trends in the occurrence of these types of interaction were discussed. The data may be of use for AMP-based drug development and suggest that the topic requires further attention for successfully integrating AMPs-based products in the context of complex treatment. AMP/antiseptic combinations show promise for creating topical formulations with improved activity, lowered toxicity, and, presumably, decreased chances of inducing bacterial resistance. However, careful assessment is required to avoid AMP neutralization by certain antiseptic classes in either complex drug design or AMP application alongside other therapeutics/care products.

Succinyl Chitosan-Colistin Conjugates as Promising Drug Delivery Systems.

The growth of microbial multidrug resistance is a problem in modern clinical medicine. Chemical modification of active pharmaceutical ingredients is an attractive strategy to improve their biopharmaceutical properties by increasing bioavailability and reducing drug toxicity. Conjugation of antimicrobial drugs with natural polysaccharides provides high efficiency of these systems due to targeted delivery, controlled drug release and reduced toxicity. This paper reports a two-step synthesis of colistin conjugates (CT) with succinyl chitosan (SucCS); first, we modified chitosan with succinyl anhydride to introduce a carboxyl function into the polymer molecule, which was then used for chemical grafting with amino groups of the peptide antibiotic CT using carbodiimide chemistry. The resulting polymeric delivery systems had a degree of substitution (DS) by CT of 3-8%, with conjugation efficiencies ranging from 54 to 100% and CT contents ranging from 130-318 µg/mg. The size of the obtained particles was 100-200 nm, and the ζ-potential varied from -22 to -28 mV. In vitro release studies at pH 7.4 demonstrated ultra-slow hydrolysis of amide bonds, with a CT release of 0.1-0.5% after 12 h; at pH 5.2, the hydrolysis rate slightly increased; however, it remained extremely low (1.5% of CT was released after 12 h). The antimicrobial activity of the conjugates depended on the DS. At DS 8%, the minimum inhibitory concentration (MIC) of the conjugate was equal to the MIC of native CT (1 µg/mL); at DS of 3 and 5%, the MIC increased 8-fold. In addition, the developed systems reduced CT nephrotoxicity by 20-60%; they also demonstrated the ability to reduce bacterial lipopolysaccharide-induced inflammation in vitro. Thus, these promising CT-SucCS conjugates are prospective for developing safe and effective nanoantibiotics.

Synthesis and Characterization of Novel Succinyl Chitosan-Dexamethasone Conjugates for Potential Intravitreal Dexamethasone Delivery.

The development of intravitreal glucocorticoid delivery systems is a current global challenge for the treatment of inflammatory diseases of the posterior segment of the eye. The main advantages of these systems are that they can overcome anatomical and physiological ophthalmic barriers and increase local bioavailability while prolonging and controlling drug release over several months to improve the safety and effectiveness of glucocorticoid therapy. One approach to the development of optimal delivery systems for intravitreal injections is the conjugation of low-molecular-weight drugs with natural polymers to prevent their rapid elimination and provide targeted and controlled release. This study focuses on the development of a procedure for a two-step synthesis of dexamethasone (DEX) conjugates based on the natural polysaccharide chitosan (CS). We first used carbodiimide chemistry to conjugate DEX to CS via a succinyl linker, and we then modified the obtained systems with succinic anhydride to impart a negative ζ-potential to the polymer particle surface. The resulting polysaccharide carriers had a degree of substitution with DEX moieties of 2-4%, a DEX content of 50-85 µg/mg, and a degree of succinylation of 64-68%. The size of the obtained particles was 400-1100 nm, and the ζ-potential was -30 to -33 mV. In vitro release studies at pH 7.4 showed slow hydrolysis of the amide and ester bonds in the synthesized systems, with a total release of 8-10% for both DEX and succinyl dexamethasone (SucDEX) after 1 month. The developed conjugates showed a significant anti-inflammatory effect in TNFα-induced and LPS-induced inflammation models, suppressing CD54 expression in THP-1 cells by 2- and 4-fold, respectively. Thus, these novel succinyl chitosan-dexamethasone (SucCS-DEX) conjugates are promising ophthalmic carriers for intravitreal delivery.

Mucoadhesive cholesterol-chitosan self-assembled particles for topical ocular delivery of dexamethasone.

The topical application of ophthalmic drugs is a convenient and safe mode of drug administration. However, the bioavailability of topical drugs in the eye is low due to eye barriers and the rapid removal of the drug from the conjunctival surface by the tear fluid. The aim of this study was to obtain dexamethasone-loaded mucoadhesive self-assembled particles based on a conjugate of succinyl cholesterol with chitosan (SC-CS) for potential use as a topical ocular formulation. SC-CS was obtained via a carbodiimide-mediated coupling reaction (degree of substitution DS 1.2-5.8%). SC-CS in the DS range of 1.2-3.0% can self-organize in solution to form positively charged particles (ζ-potential 20-37 mV) of submicron size (hydrodynamic diameter 700-900 nm). The SC-CS particles show good mucoadhesiveness, which decreases with increasing DS. The obtained particles can encapsulate 159-170 µg/mg dexamethasone; they release about 50% of drug in 2 h, and the cumulative drug release reached 95% in 24 h. A cell model confirmed that dexamethasone-loaded SC-CS particles are non-cytotoxic and exhibit a comparable anti-inflammatory activity to that of pure dexamethasone. Testing the osmotic resistance of erythrocytes showed that both dexamethasone-loaded and non-loaded SC-CS particles have greater membrane-stabilizing ability than that of dexamethasone.

Synthetic hydrosilicate nanotubes induce low pro-inflammatory and cytotoxic responses compared to natural chrysotile in lung cell cultures.

Asbestos (Mg-hydrosilicate; chrysotile) is known to cause pleural diseases, pulmonary fibrosis and lung cancers, via mechanisms strongly depending on diameter-length ratio and possibly metal content. A critical question is whether synthetic hydrosilicate nanotubes (NTs) of short length possess little toxic potential compared to chrysotile. Five Mg- and two NiNTs of different lengths were assessed for cytotoxicity and pro-inflammatory responses in THP-1 macrophages and human bronchial epithelial lung cells (HBEC3-KT), in comparison with chrysotile. NT lengths/diameters were characterized by TEM, surface areas by BET- and BJH analysis, and chemical composition by XRD. The different Mg- and NiNTs induced little cytotoxicity in both cell models, in contrast to chrysotile that induced marked cytotoxicity. The two longest synthetic MgNTs, with median lengths of 3 and 5 µm, induced increased levels of pro-inflammatory cytokines in THP-1 macrophages, but much less than chrysotile (median length 15 µm) and silica nanoparticles (Si10). The shortest NTs did not induce any increase in cytokines. In HBEC3-KT cells, all synthetic NTs induced no or only small changes in cytokine responses, in contrast to chrysotile and Si10. The synthetic NTs induced lower TGF-ß responses than chrysotile in both cell models. In conclusion, the pro-inflammatory responses were associated with the length of synthetic hydrosilicate NTs in THP-1 macrophages, but not in HBEC3-KT cells. Notably, the shortest NTs showed no or little pro-inflammatory activity or cytotoxicity in both cell models. Such a safety by design approach is important for development of new materials being candidates for various new products.

Tumor necrosis factor α stimulates endogenous apolipoprotein A-I expression and secretion by human monocytes and macrophages: role of MAP-kinases, NF-κB, and nuclear receptors PPARα and LXRs.

Apolipoprotein A-I (ApoA-I) is the main structural and functional protein component of high-density lipoprotein. ApoA-I has been shown to regulate lipid metabolism and inflammation in macrophages. Recently, we found the moderate expression of endogenous apoA-I in human monocytes and macrophages and showed that pro-inflammatory cytokine tumor necrosis factor α (TNFα) increases apoA-I mRNA and stimulates ApoA-I protein secretion by human monocytes and macrophages. Here, we present data about molecular mechanisms responsible for the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. This activation depends on JNK and MEK1/2 signaling pathways in human monocytes, whereas inhibition of NFκB, JNK, or p38 blocks an increase of apoA-I gene expression in the macrophages treated with TNFα. Nuclear receptor PPARα is a ligand-dependent regulator of apoA-I gene, whereas LXRs stimulate apoA-I mRNA transcription and ApoA-I protein synthesis and secretion by macrophages. Treatment of human macrophages with PPARα or LXR synthetic ligands as well as knock-down of LXRα, and LXRß by siRNAs interfered with the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. At the same time, TNFα differently regulated the levels of PPARα, LXRα, and LXRß binding to the apoA-I gene promoter in THP-1 cells. Obtained results suggest a novel tissue-specific mechanism of the TNFα-mediated regulation of apoA-I gene in monocytes and macrophages and show that endogenous ApoA-I might be positively regulated in macrophage during inflammation.

A novel strategy for the synthesis of thermally stable and apoptosis-inducing 2,3-dihydroazetes.

A general and concise approach to thermally and hydrolytically stable alkyl 2,3-dihydroazete-2,3-di-/2,2,3-tricarboxylates from alkyl 2-bromoazirine-2-carboxylates or 4-bromo-5-alkoxyisoxazoles is reported. The synthesis involves the formation of 2-azabuta-1,3-diene by the reaction of rhodium carbenoid with isoxazole or azirine followed by cyclization/hydrodebromination cascade. The latter reaction is the first example of the selective hydrodehalogenation of a valence isomer under equilibrium conditions. In vitro cytotoxicity tests on THP-1 cell line revealed that the 2,3-dihydroazetes greatly differ in their ability to induce apoptosis and/or necrosis. To adequately describe and quantitatively assess these properties, the difference between the two areas under the curves of concentration dependency of apoptosis/necrosis induction within the concentration range was used. Trimethyl 4-phenyl-2,3-dihydroazete-2,2,3-tricarboxylate was found to display the maximal apoptotic potential coupled with high cytotoxic and minimal necrotic potential.

Endogenous apolipoprotein A-I stabilizes ATP-binding cassette transporter A1 and modulates Toll-like receptor 4 signaling in human macrophages.

Apolipoprotein A-I (ApoA-I) is the main functional protein component of human high-density lipoproteins. ApoA-I shows various anti-inflammatory and atheroprotective properties toward macrophages; however, endogenous apoA-I expression has not been investigated in macrophages. We have shown that endogenous apoA-I gene is expressed in human macrophages at both mRNA and protein levels. Endogenous ApoA-I is localized in intracellular vesicles and at the external side of the plasma membrane in association with ATP-binding cassette transporter A1 (ABCA1) and lipid rafts in macrophages. We have shown that endogenous ApoA-I stabilizes ABCA1, moreover, down-regulation of ApoA-I by siRNA results in an increase of Toll-like receptor 4 (TLR4) mRNA and membrane surface protein expression, as well as an enhancement of bacterial lipopolysaccharide (LPS)-induced expression of tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), and inducible nitric oxide synthase (NOS2) genes in human macrophages. TNF-α stimulates ApoA-I expression and secretion (1.2±0.2 vs. 4.3±0.9 ng/mg total protein) in macrophages. Obtained results suggest that endogenous ApoA-I has anti-inflammatory properties, presumably due to ABCA1 stabilization in macrophages; these results elucidate the cell type-specific mechanism of the TNF-α-mediated regulation of apoA-I gene expression in monocytes and macrophages.

Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Toll-like receptor 4 activation in human macrophages.

Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liver X receptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRß in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mLDL, C3, C3a, and TLR4 during development of atherosclerotic lesions.