Cytotoxicity and Degradation Resistance of Cryo- and Hydrogels Based on Carboxyethylchitosan at Different pH Values.

Background: The use of chitosan-based gels is still limited due to their restricted solubility in acid solutions, where the molecules have a positive charge. The functionalization of chitosan makes it possible to significantly expand the possibilities of using both the polymer itself and hydrogels based on its derivatives. Objective: To evaluate the effect of the conditions for the production of cryo- and hydrogels based on carboxyethylchitosan (CEC) crosslinked with glutaraldehyde on gel swelling and its resistance to degradation depending on pH and cytotoxic effects and to test the hypothesis that the amount of crosslinking agent during synthesis may affect the cytotoxicity of the gel. Methods: Gels' swelling values and degradation resistance were determined using the gravimetric method. The cytotoxic effect was evaluated during the co-cultivation of gels in the presence of human fibroblasts using light optical microscopy and flow cytometry. Results: All CEC-based cryogels had a higher equilibrium swelling value and degradation time than the CEC hydrogel in the pH range from 4.6 to 8.0. This demonstrates the superiority of cryogels relative to CEC-based hydrogels in terms of swelling potential and degradation resistance, while an increase in the number of crosslinks with glutaraldehyde contributes to longer swelling of the cryogel. The positive control (intact fibroblasts) and all gel samples were statistically identical in the number of viable cells. On the third day, the viability of the fibroblast cells was consistently high (above 95%) and did not differ between all tested CEC-based gels. And in general, the cell morphology analysis results corresponded with the results obtained in the flow cytometry-based cytotoxicity test. We also did not find proof in our experiment to support our hypothesis that the amount of crosslinking agent during synthesis may affect the cytotoxicity of the material.

In Vivo Studies of Medical Implants for Maxillofacial Surgery Produced from Nanostructured Titanium.

This paper presents the results of comprehensive in vivo studies into the osseointegration behavior of medical implants for maxillofacial surgery produced from nanostructured grade 4 titanium. Special attention is given to the phenomenology of bone tissue formation with consideration of its surface relief features and to evaluating the quantitative parameters of the morphological indicators of osteoblast and endothelial cells in the osseointegration zone. These parameters were compared with their measurement data for standard factory-made implants, and considerable acceleration in the fixation of nanotitanium implants due to osseointegation was found. The obtained results indicate a better osseointegration of implants made of nanotitanium in comparison to similar standard products.

A Carboxyethylchitosan Gel Cross-Linked with Glutaraldehyde as a Candidate Carrier for Biomedical Applications.

To date, few publications describe CEC's properties and possible applications-thus, further evaluation of these properties is a point of interest. The present in vitro model study aimed to evaluate a carboxyethylchitosan (CEC) gel with a degree of substitution of 1, cross-linked with glutaraldehyde at a polymer:aldehyde molar ratio of 10:1, as a potential carrier for delivering bacteriophages to various pH-fixed media (acidic, alkaline), and including gastrointestinal tract (GIT) variable medium. A quantitative analysis of bacteriophages released from the gel was performed using photon correlation spectrophotometry, and phage activity after emission into medium was evaluated using the spot test. The results showed that the CEC gel's maximum swelling ratios were at a nearly neutral alkaline pH. Increasing temperature enhances the swelling ratio of the gel independent from pH, up to 1127% at 37 °C and alkaline pH. The UV and photon correlation spectrophotometry showed equal gel release kinetics in both fixed media with acidic (pH = 2.2) and alkaline (pH = 7.4) pH environments at 37 °C, with the maximum release within two hours. However, phage lytic activity in the spot test during this simulation was absent. At the same time, we obtained an opaque phage lytic activity in the alkaline pH-fixed medium for at least three hours. Phages released from the tested CEC gel in different pHs suggest that this gel could be used for applications that require fast release at the treatment site both in acidic and alkaline pH. Such treatment sites could be a wound or even soil with mild acidic or alkaline pH. However, such CEC gel is not suitable as a delivery system to the GIT because of possible transported acid-sensitive agent (such as phages) release and destruction already in the stomach.

Delivery of B. subtilis into Animal Intestine Using Chitosan-Derived Bioresorbable Gel Carrier: Preliminary Results.

The oral delivery of bacteria in the human intestine is of great interest because of its potential to correct the gut microbiota and treat inflammatory bowel diseases. The aim of this study was to evaluate sodium N-(2-sulfonatoethyl)chitosan gel cross-linked with glutaraldehyde as a delivery carrier for probiotic bacteria to the gut using in vitro and in vivo experiments. The bacterial test strain was B. subtilis 20. The cytotoxicity of the gel was evaluated via cell culture using flow cytometry and light microscopy. The gel as a delivery system was assessed by the dye release in medium with different pH levels in vitro, and by bacterial titer monitoring in mouse feces using the microbiology method in vivo. Results of an in vitro experiment showed that tested gel has no cytotoxicity. The use of gel as a carrier for bacterial delivery into the intestine was more effective than oral gavage of bacterial suspension. Therefore, gel delivery of bacteria decreased the titer level by up to two times. However, a gavage of bacterial suspension decreased the titer level by over 200 times. Tested gel has the potential to be a carrier for the safe delivery of bacteria to the intestine through the stomach, reducing the rate of the elimination of probiotic bacteria from the intestine.

Evaluating Germplasm of Cultivated Oat Species from the VIR Collection under the Russian Northwest Conditions.

Oat is one of the most widespread and important cereal crops in the global agricultural production. Searching for new high-yielding and nutritious forms continues to be relevant, especially under the global trend of climate change, when most local oat cultivars may become economically inefficient. Spring oat accessions from VIR collection served as the material for this study; their origin is diverse, as they came from 11 countries. The basic nutritional value (the content of protein, oil, starch, and ß-glucans) and characters important for breeding (plant height, panicle length, number of spikelets, number of grains per panicle, 1000 grain weight, and grain yield) were analyzed in 49 accessions of the cultivated covered oat species: Avena sativa L., A. strigosa Schreb., A. abyssinica Hochst., and A. byzantina Coch., grown under the conditions of the Russian Northwest (Leningrad Province) for two years. Variability parameters, interspecific and intervarietal differences, and the effect of weather conditions were assessed. Sources of useful agronomic traits were identified; they can be used to expand the range of the source material for the development of new high-yielding and highly nutritious oat cultivars adapted to local cultivation conditions. It is demonstrated that the VIR collection has a great potential for contemporary food and feed production and for the breeding of new oat cultivars for various purposes. Thus, the contribution of Nikolai Vavilov to the plant genetic resources investigation for the benefit of humanity is invaluable.

New Insights into the Genomic Structure of Avena L.: Comparison of the Divergence of A-Genome and One C-Genome Oat Species.

We used next-generation sequencing analysis of the 3'-part of 18S rDNA, ITS1, and a 5'-part of the 5.8S rDNA region to understand genetic variation among seven diploid A-genome Avena species. We used 4−49 accessions per species that represented the As genome (A. atlantica, A. hirtula, and wiestii), Ac genome (A. canariensis), Ad genome (A. damascena), Al genome (A. longiglumis), and Ap genome (A. prostrata). We also took into our analysis one C-genome species, A. clauda, which previously was found to be related to A-genome species. The sequences of 169 accessions revealed 156 haplotypes of which seven haplotypes were shared by two to five species. We found 16 ribotypes that consisted of a unique sequence with a characteristic pattern of single nucleotide polymorphisms and deletions. The number of ribotypes per species varied from one in A. longiglumis to four in A. wiestii. Although most ribotypes were species-specific, we found two ribotypes shared by three species (one for A. damascena, A. hirtula, and A. wiestii, and the second for A. longiglumis, A. atlantica, and A. wiestii), and a third ribotype shared between A. atlantica and A. wiestii. A characteristic feature of the A. clauda ribotype, a diploid C-genome species, is that two different families of ribotypes have been found in this species. Some of these ribotypes are characteristic of Cc-genome species, whereas others are closely related to As-genome ribotypes. This means that A. clauda can be a hybrid between As- and C-genome oats.

Regulatory T Cells Fail to Suppress Fast Homeostatic Proliferation In Vitro.

Homeostatic proliferation (HP) is a physiological process that reconstitutes the T cell pool after lymphopenia involving Interleukin-7 and 15 (IL-7 and IL-15), which are the key cytokines regulating the process. However, there is no evidence that these cytokines influence the function of regulatory T cells (Tregs). Since lymphopenia often accompanies autoimmune diseases, we decided to study the functional activity of Tregs stimulated by HP cytokines from patients with rheumatoid arthritis as compared with that of those from healthy donors. Since T cell receptor (TCR) signal strength determines the intensity of HP, we imitated slow HP using IL-7 or IL-15 and fast HP using a combination of IL-7 or IL-15 with anti-CD3 antibodies, cultivating Treg cells with peripheral blood mononuclear cells (PBMCs) at a 1:1 ratio. We used peripheral blood from 14 patients with rheumatoid arthritis and 18 healthy volunteers. We also used anti-CD3 and anti-CD3 + IL-2 stimulation as controls. The suppressive activity of Treg cells was evaluated in each case by the inhibition of the proliferation of CD4+ and CD8+ cells. The phenotype and proliferation of purified CD3+CD4+CD25+CD127lo cells were assessed by flow cytometry. The suppressive activity of the total pool of Tregs did not differ between the rheumatoid arthritis and healthy donors; however, it significantly decreased in conditions close to fast HP when the influence of HP cytokines was accompanied by anti-CD3 stimulation. The Treg proliferation caused by HP cytokines was lower in the rheumatoid arthritis (RA) patients than in the healthy individuals. The revealed decrease in Treg suppressive activity could impact the TCR landscape during lymphopenia and lead to the proliferation of potentially self-reactive T cell clones that are able to receive relatively strong TCR signals. This may be another explanation as to why lymphopenia is associated with the development of autoimmune diseases. The revealed decrease in Treg proliferation under IL-7 and IL-15 exposure can lead to a delay in Treg pool reconstitution in patients with rheumatoid arthritis in the case of lymphopenia.

Evaluation of the Immunosafety of Cucurbit[n]uril on Peripheral Blood Mononuclear Cells In Vitro.

Cucurbiturils (CB[n]s) are nanoscale macrocyclic compounds capable of encapsulating a molecule or part of a molecule by forming host-guest complexes. Integration of drugs with CB[n] is used for the following purposes: controlling clearance; protection of the drug from biodegradation; targeted delivery to specific organs, tissues, or cells; reduction of toxicity; and improving solubility. One of the major problems encountered in the application of new drug delivery systems is lack of knowledge of their biological properties. CB[n], unlike many other often toxic nanoparticles, has extremely low toxicity, even at high doses. However, many aspects of the biological actions of these nanoscale cavitands remain unclear, including the immunotropic properties. In this study, we investigated the immunotoxicity and immunomodulation properties of CB[n]. It was found that CB[7] and CB[6] did not decrease the viability of mononuclear cells at all tested concentrations from 0.1-1 mM. Overall, the results indicated an immunomodulatory effect of different concentrations of CB[n]. In the case of a longer cultivation time, CB[n] had an immunostimulating effect, which was indicated by an enhancement of the proliferative activity of cells and increased expression of HLA-DR on lymphocytes.

IgG abzymes with peroxidase and oxidoreductase activities from the sera of healthy humans.

We present the evidence showing that small fractions of electrophoretically homogeneous immunoglobulin G (IgGs) from the sera of healthy humans and their Fab and F(ab)2 fragments oxidize 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2 O2 and through an oxidoreductase activity in the absence of H2 O2 . During purification on protein G-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me(2+) ions. After extensive dialysis of purified Abs against agents chelating metal ions, the relative peroxidase activity decreased dependently of IgG analyzed from 100 to ~10-85%, while oxidoreductase activity from 100 to 14-83%. Addition of external metal ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. Chromatography of the IgGs on Chelex non-charged with Cu(2+) ions results in the adsorption of a small IgG fraction bound with metal ions (~5%), while Chelex charged with Cu(2+) ions bind additionally ~38% of the total IgGs. Separation of Abs on both sorbents results in IgG separation to many different subfractions demonstrating various affinities to the chelating resin and different levels of the specific oxidoreductase and peroxidase activities. In the presence of external Cu(2+) ions, the specific peroxidase activity of several IgG subfractions achieves 20-27 % as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of these fractions is ~4-6-fold higher than that for HRP. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activities of human IgGs could also play an important role in the protection of organisms from oxidative stress and toxic compounds.

Electrocardiographic diagnosis of left ventricular hypertrophy on the basis of dipole electrocardiotopography method.

PURPOSE: The aim of this study was to verify a new criterion of hypertension-related left ventricular hypertrophy (LVH) on the basis of the dipole electrocardiotopographic technique, which provides intelligible pictorial patterns and efficient quantitative parameters for recognition of pathologic states of the heart. METHODS: Standard 12-lead and vectorcardiographic measurements with the McFee-Parungao lead system were carried out for 289 echocardiographically accessed healthy subjects and hypertensive patients with LVH. The proposed parameter index of left ventricular hypertrophy for recognition of LVH is a product of the following 3 characteristics: maximum magnitude of depolarization vector, total duration of ventricular depolarization, and local spatial integral of activation duration. This parameter was compared with several other parameters commonly used for LVH diagnosis. The diagnostic efficiencies of these parameters were statistically estimated and compared using the receiver operating characteristics (ROC curves), area under the ROC curve, and partial areas under the ROC curve (pAUC). The area under the ROC curve values were compared by the standard nonparametric method, and pAUC comparison was performed by bootstrap methodology. RESULTS: The ROC curves display the definite advantage of the proposed criterion, especially for the specificities greater than 0.7. Statistical comparative analysis showed that the corresponding pAUC for the index of left ventricular hypertrophy is greater than for R(x) + S(z), Cornell product, and Sokolow-Lyon criteria at confidence level > or = .95. CONCLUSION: The dipole electrocardiotopography-based diagnostic criterion of LVH is demonstrated to be statistically more accurate as compared to other criteria studied. The advantage of this criterion suggests that with the use of decartograms, it is possible to reveal some additional information not accessible for other diagnostic tools. Decartograms help to visualize and quantify the redistribution of activation duration over the myocardium, thus providing a valuable instrument for heuristic analysis and dynamic observation of the heart state affected by LVH.