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.

Streptococcal Arginine Deiminase Inhibits T Lymphocyte Differentiation In Vitro.

Pathogenic microbes use arginine-metabolizing enzymes as an immune evasion strategy. In this study, the impact of streptococcal arginine deiminase (ADI) on the human peripheral blood T lymphocytes function in vitro was studied. The comparison of the effects of parental strain (Streptococcus pyogenes M49-16) with wild type of ArcA gene and its isogenic mutant with inactivated ArcA gene (Streptococcus pyogenes M49-16delArcA) was carried out. It was found that ADI in parental strain SDSC composition resulted in a fivefold decrease in the arginine concentration in human peripheral blood mononuclear cell (PBMC) supernatants. Only parental strain SDSCs suppressed anti-CD2/CD3/CD28-bead-stimulated mitochondrial dehydrogenase activity and caused a twofold decrease in IL-2 production in PBMC. Flow cytometry analysis revealed that ADI decreased the percentage of CM (central memory) and increased the proportion of TEMRA (terminally differentiated effector memory) of CD4+ and CD8+ T cells subsets. Enzyme activity inhibited the proliferation of all CD8+ T cell subsets as well as CM, EM (effector memory), and TEMRA CD4+ T cells. One of the prominent ADI effects was the inhibition of autophagy processes in CD8+ CM and EM as well as CD4+ CM, EM, and TEMRA T cell subsets. The data obtained confirm arginine's crucial role in controlling immune reactions and suggest that streptococcal ADI may downregulate adaptive immunity and immunological memory.

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.

Regulated Arginine Metabolism in Immunopathogenesis of a Wide Range of Diseases: Is There a Way to Pass between Scylla and Charybdis?

More than a century has passed since arginine was discovered, but the metabolism of the amino acid never ceases to amaze researchers. Being a conditionally essential amino acid, arginine performs many important homeostatic functions in the body; it is involved in the regulation of the cardiovascular system and regeneration processes. In recent years, more and more facts have been accumulating that demonstrate a close relationship between arginine metabolic pathways and immune responses. This opens new opportunities for the development of original ways to treat diseases associated with suppressed or increased activity of the immune system. In this review, we analyze the literature describing the role of arginine metabolism in the immunopathogenesis of a wide range of diseases, and discuss arginine-dependent processes as a possible target for therapeutic approaches.

Recombinant Human Interleukin-2 Corrects NK Cell Phenotype and Functional Activity in Patients with Post-COVID Syndrome.

Post-COVID syndrome develops in 10-20% of people who have recovered from COVID-19 and it is characterized by impaired function of the nervous, cardiovascular, and immune systems. Previously, it was found that patients who recovered from infection with the SARS-CoV-2 virus had a decrease in the number and functional activity of NK cells. The aim of the study was to assess the effectiveness of recombinant human IL-2 (rhIL-2) administered to correct NK cell phenotype and functional activity in patients with post-COVID syndrome. Patients were examined after 3 months for acute COVID-19 of varying severity. The phenotype of the peripheral blood NK cells was studied by flow cytometry. It was found that disturbances in the cell subset composition in patients with post-COVID syndrome were characterized by low levels of mature (p = 0.001) and cytotoxic NK cells (p = 0.013), with increased release of immature NK cells (p = 0.023). Functional deficiency of NK cells in post-COVID syndrome was characterized by lowered cytotoxic activity due to the decreased count of CD57+ (p = 0.001) and CD8+ (p < 0.001) NK cells. In the treatment of patients with post-COVID syndrome with recombinant IL-2, peripheral blood NK cell count and functional potential were restored. In general, the effectiveness of using rhIL-2 in treatment of post-COVID syndrome has been proven in patients with low levels of NK cells.

Features of Peripheral Blood Th-Cell Subset Composition and Serum Cytokine Level in Patients with Activity-Driven Ankylosing Spondylitis.

Th cells may exhibit pathological activity depending on the regulatory and functional signals sensed under a wide range of immunopathological conditions, including ankylosing spondylitis (AS). The relationship between Th cells and cytokines is important for diagnoses and for determining treatment. Accordingly, the aim of this study was to investigate the relationship between Th-cell subset composition and serum cytokine profile for patients with activity-driven AS. In our study, patients were divided into two groups according to disease activity: low-activity AS (ASDAS-CRP < 2.1) and high-activity AS (ASDAS-CRP > 2.1). The peripheral blood Th cell subset composition was studied by flow cytometry. Using multiplex analysis, serum cytokine levels were quantified and investigated. It was found that only patients with high-activity AS had reduced central memory (CM) Th1 cells (p = 0.035) but elevated numbers of CM (p = 0.014) and effector memory (EM) Th2 cells (p < 0.001). However, no activity-driven change in the Th17 cell subset composition was observed in AS patients. Moreover, low-AS activity patients had increased numbers of Tfh17 EM cells (p < 0.001), whereas high-AS activity was associated with elevated Tfh2 EM level (p = 0.031). The serum cytokine profiles in AS patients demonstrated that cues stimulating cellular immunity were increased, but patients with high-AS activity reveled increased IL-5 level (p = 0.017). Analyzing the data obtained from AS patients allowed us to conclude that Th cell subset differentiation was mainly affected during the CM stage and characterized the IL-23/IL-17 regulatory axis, whereas increased humoral immunity was observed in the high-AS activity group.

New Application of the Commercially Available Dye Celestine Blue B as a Sensitive and Selective Fluorescent "Turn-On" Probe for Endogenous Detection of HOCl and Reactive Halogenated Species.

Hypochlorous acid (HOCl) derived from hydrogen peroxide and chloride anion by myeloperoxidase (MPO) plays a significant role in physiological and pathological processes. Herein we report a phenoxazine-based fluorescent probe Celestine Blue B (CB) that is applicable for HOCl detection in living cells and for assaying the chlorinating activity of MPO. A remarkable selectivity and sensitivity (limit of detection is 32 nM), along with a rapid "turn-on" response of CB to HOCl was demonstrated. Furthermore, the probe was able to detect endogenous HOCl and reactive halogenated species by fluorescence spectroscopy, confocal microscopy, and flow cytometry techniques. Hence, CB is a promising tool for investigating the role of HOCl in health and disease and for screening the drugs capable of regulating MPO activity.

Heterogenous CD8+ T Cell Maturation and 'Polarization' in Acute and Convalescent COVID-19 Patients.

BACKGROUND: The adaptive antiviral immune response requires interaction between CD8+ T cells, dendritic cells, and Th1 cells for controlling SARS-CoV-2 infection, but the data regarding the role of CD8+ T cells in the acute phase of COVID-19 and post-COVID-19 syndrome are still limited. METHODS: . Peripheral blood samples collected from patients with acute COVID-19 (n = 71), convalescent subjects bearing serum SARS-CoV-2 N-protein-specific IgG antibodies (n = 51), and healthy volunteers with no detectable antibodies to any SARS-CoV-2 proteins (HC, n = 46) were analyzed using 10-color flow cytometry. RESULTS: Patients with acute COVID-19 vs. HC and COVID-19 convalescents showed decreased absolute numbers of CD8+ T cells, whereas the frequency of CM and TEMRA CD8+ T cells in acute COVID-19 vs. HC was elevated. COVID-19 convalescents vs. HC had increased naïve and CM cells, whereas TEMRA cells were decreased compared to HC. Cell-surface CD57 was highly expressed by the majority of CD8+ T cells subsets during acute COVID-19, but convalescents had increased CD57 on 'naïve', CM, EM4, and pE1 2-3 months post-symptom onset. CXCR5 expression was altered in acute and convalescent COVID-19 subjects, whereas the frequencies of CXCR3+ and CCR4+ cells were decreased in both patient groups vs. HC. COVID-19 convalescents had increased CCR6-expressing CD8+ T cells. Moreover, CXCR3+CCR6- Tc1 cells were decreased in patients with acute COVID-19 and COVID-19 convalescents, whereas Tc2 and Tc17 levels were increased compared to HC. Finally, IL-27 negatively correlated with the CCR6+ cells in acute COVID-19 patients. CONCLUSIONS: We described an abnormal CD8+ T cell profile in COVID-19 convalescents, which resulted in lower frequencies of effector subsets (TEMRA and Tc1), higher senescent state (upregulated CD57 on 'naïve' and memory cells), and higher frequencies of CD8+ T cell subsets expressing lung tissue and mucosal tissue homing molecules (Tc2, Tc17, and Tc17.1). Thus, our data indicate that COVID-19 can impact the long-term CD8+ T cell immune response.

Effect of Cholecalciferol Supplementation on the Clinical Features and Inflammatory Markers in Hospitalized COVID-19 Patients: A Randomized, Open-Label, Single-Center Study.

Recent studies showed that a low 25-hydroxyvitamin D (25(OH)D) level was associated with a higher risk of morbidity and severe course of COVID-19. Our study aimed to evaluate the effects of cholecalciferol supplementation on the clinical features and inflammatory markers in patients with COVID-19. A serum 25(OH)D level was determined in 311 COVID-19 patients. Among them, 129 patients were then randomized into two groups with similar concomitant medication. Group I (n = 56) received a bolus of cholecalciferol at a dose of 50,000 IU on the first and the eighth days of hospitalization. Patients from Group II (n = 54) did not receive the supplementation. We found significant differences between groups with the preferential increase in serum 25(OH)D level and Δ 25(OH)D in Group I on the ninth day of hospitalization (p < 0.001). The serum 25(OH)D level on the ninth day was negatively associated with the number of bed days (r = −0.23, p = 0.006); we did not observe other clinical benefits in patients receiving an oral bolus of cholecalciferol. Moreover, in Group I, neutrophil and lymphocyte counts were significantly higher (p = 0.04; p = 0.02), while the C-reactive protein level was significantly lower on the ninth day of hospitalization (p = 0.02). Patients with supplementation of 100,000 IU of cholecalciferol, compared to those without supplementation, showed a decrease in the frequencies of CD38++CD27 transitional and CD27−CD38+ mature naive B cells (p = 0.006 and p = 0.02) and an increase in the level of CD27−CD38− DN B cells (p = 0.02). Thus, the rise in serum 25(OH)D level caused by vitamin D supplementation in vitamin D insufficient and deficient patients may positively affect immune status and hence the course of COVID-19.

Vitamin D Status and Immune Response in Hospitalized Patients with Moderate and Severe COVID-19.

A low 25-hydroxyvitamin D (25(OH)D) level is considered as an independent risk factor for COVID-19 severity. However, the association between vitamin D status and outcomes in COVID-19 is controversial. In the present study we investigate the association between the serum 25(OH)D level, immune response, and clinical disease course in patients with COVID-19. A total of 311 patients hospitalized with COVID-19 were enrolled. For patients with a vitamin D deficiency/insufficiency, the prevalence of severe COVID-19 was higher than in those with a normal 25(OH)D level (p < 0.001). The threshold of 25(OH)D level associated with mortality was 11.4 ng/mL (p = 0.003, ROC analysis). The frequency of CD3+CD4+ T helper (Th) cells was decreased in patients with 25(OH)D level ≤ 11.4 ng/mL, compared to healthy controls (HCs). There were no differences in the frequency of naive, central memory (CM), effector memory (EM), and terminally differentiated effector memory Th cells in patients with COVID-19 compared to HCs. The frequency of T-follicular helpers was decreased both in patients with 25(OH)D level > 11.4 ng/mL (p < 0.001) and 25(OH)D level ≤ 11.4 ng/mL (p = 0.003) compared to HCs. Patients with 25(OH)D level > 11.4 ng/mL had an increased frequency of Th2 CM (p = 0.010) and decreased Th17 CM (p < 0.001). While the frequency of Th2 EM was significantly increased, the frequency of Th17 EM was significantly decreased in both groups compared to HCs. Thus, 25(OH)D level is an independent risk factor for the disease severity and mortality in patients with COVID-19. We demonstrate that the serum 25(OH)D level ≤ 11.4 ng/mL is associated with the stimulation of Th2 and the downregulation of Th17 cell polarization of the adaptive immunity in patients with COVID-19.

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.

Alterations in B Cell and Follicular T-Helper Cell Subsets in Patients with Acute COVID-19 and COVID-19 Convalescents.

BACKGROUND: Humoral immunity requires interaction between B cell and T follicular helper cells (Tfh) to produce effective immune response, but the data regarding a role of B cells and Tfh in SARS-CoV-2 defense are still sparse. METHODS: Blood samples from patients with acute COVID-19 (n = 64), convalescents patients who had specific IgG to SARS-CoV-2 N-protein (n = 55), and healthy donors with no detectable antibodies to any SARS-CoV-2 proteins (HC, n = 44) were analyses by multicolor flow cytometry. RESULTS: Patients with acute COVID-19 showed decreased levels of memory B cells subsets and increased proportion plasma cell precursors compared to HC and COVID-19 convalescent patients, whereas for the latter the elevated numbers of virgin naïve, Bm2' and "Bm3+Bm4" was found if compared with HC. During acute COVID-19 CXCR3+CCR6- Tfh1-like cells were decreased and the levels of CXCR3-CCR6+ Tfh17-like were increased then in HC and convalescent patients. Finally, COVID-19 convalescent patients had increased levels of Tfh2-, Tfh17- and DP Tfh-like cells while comparing their amount with HC. CONCLUSIONS: Our data indicate that COVID-19 can impact the humoral immunity in the long-term.

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.

Sarcoidosis as an Autoimmune Disease.

Despite the large number of performed studies, the etiology and pathogenesis of sarcoidosis still remain unknown. Most researchers allude to the possible autoimmune or immune-mediated genesis of the disease. This review attempts an integral analysis of currently available information suggesting an autoimmune genesis of sarcoidosis and is divided into four categories: the evaluation of clinical signs described both in patients with sarcoidosis and "classic" autoimmune diseases, the role of triggering factors in the development of sarcoidosis, the presence of immunogenic susceptibility in the development of the disease, and the analysis of cellular and humoral immune responses in sarcoidosis. Studying the etiology and pathogenesis of sarcoidosis will improve diagnostic procedures as well as the prognosis and patients' quality of life.

Neutrophil activation in response to monomeric myeloperoxidase.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also regulate cellular functions via its nonenzymatic effects. Mature active MPO isolated from normal human neutrophils is a 145 kDa homodimer, which consists of 2 identical protomers, connected by a single disulfide bond. By binding to CD11b/CD18 integrin, dimeric MPO induces neutrophil activation and adhesion augmenting leukocyte accumulation at sites of inflammation. This study was performed to compare the potency of dimeric and monomeric MPO to elicit selected neutrophil responses. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. Analysis of the crucial signal transducer, intracellular Ca2+, showed that dimeric MPO induces Ca2+ mobilization from the intracellular calcium stores of neutrophils and influx of extracellular Ca2+ whereas the effect of monomeric MPO on Ca2+ increase in neutrophils was less. It was also shown that monomeric MPO was less efficient than dimeric MPO at inducing actin cytoskeleton reorganization, cell survival, and neutrophil degranulation. Furthermore, we have detected monomeric MPO in the blood plasma of patients with acute inflammation. Our data suggest that the decomposition of dimeric MPO into monomers can serve as a regulatory mechanism that controls MPO-dependent activation of neutrophils and reduces the proinflammatory effects of MPO.

Molecular targets for the therapy of cancer associated with metabolic syndrome (transcription and growth factors).

Metabolic syndrome (MS) is one of the leading risk factors for the development of cardiovascular diseases, type II diabetes mellitus and reproductive system diseases. Currently, not only cardiovascular disease and reproductive history risks related with MS are frequently discussed, but it has been also shown that MS is associated with increased risk of some common cancers (endometrial cancer, postmenopausal breast cancer, colorectal cancer, biliary tract cancers and liver cancer for men). Further studies are required to understand the mechanisms of the involvement of MS components in the pathogenesis of malignant neoplasms. Changes in the expression of transcription and growth factors in the peripheral tissues as well as in cancer tissues of patients with MS were revealed. Transcription factors (AMP-activated protein kinase-1, STAT3, sterol regulatory element-binding protein-1 and peroxisome proliferator-activated receptor-γ), leptin and adiponectin receptors seem to be the most promising molecular targets for the therapy of cancers associated with MS.

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.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.