Chitosan-Based Dressing as a Sustained Delivery System for Bioactive Cytokines.

Wounds represent a common occurrence in human life. Consequently, scientific investigations are underway to advance wound healing methodologies, with a notable focus on dressings imbued with biologically active compounds capable of orchestrating the wound microenvironment through meticulously regulated release mechanisms. Among these bioactive agents are cytokines, which, when administered to the wound milieu without appropriate protection, undergo rapid loss of their functional attributes. Within the context of this research, we present a method for fabricating dressings enriched with G-CSF (granulocyte colony-stimulating factor) or GM-CSF (granulocyte-macrophage colony-stimulating factor), showcasing both biological activity and protracted release dynamics. Based on Ligasano, a commercial polyurethane foam dressing, and chitosan crosslinked with TPP (sodium tripolyphosphate), these dressings are noncytotoxic and enable cytokine incorporation. The recovery of cytokines from dressings varied based on the dressing preparation and storage techniques (without modification, drying, freeze-drying followed by storage at 4 °C or freeze-drying followed by storage at 24 °C) and cytokine type. Generally, drying reduced cytokine levels and their bioactivity, especially with G-CSF. The recovery of G-CSF from unmodified dressings was lower compared to GM-CSF (60% vs. 80%). In summary, our freeze-drying approach enables the storage of G-CSF or GM-CSF enriched dressings at 24 °C with minimal cytokine loss, preserving their biological activity and thus enhancing future clinical availability.

Looking into the Eyes-In Vitro Models for Ocular Research.

Animal research undoubtedly provides scientists with virtually unlimited data but inflicts pain and suffering on animals. Currently, legislators and scientists alike are promoting alternative in vitro approaches allowing for an accurate evaluation of processes occurring in the body without animal sacrifice. Historically, one of the most infamous animal tests is the Draize test, mainly performed on rabbits. Even though this test was considered the gold standard for around 50 years, the Draize test fails to mimic human response mainly due to human and rabbit eye physiological differences. Therefore, many alternative assays were developed to evaluate ocular toxicity and drug effectiveness accurately. Here we review recent achievements in tissue engineering of in vitro 2D, 2.5D, 3D, organoid and organ-on-chip ocular models, as well as in vivo and ex vivo models in terms of their advantages and limitations.

Encapsulation of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in liposomes prepared by thin film hydration and their transfer to mesenchymal stem cells and cord blood hematopoietic stem cells.

Introduction: Cytokines are important immune modulator factors controlling homeostasis of the body and are involved in tissue regeneration after wound healing. The encapsulation of cytokines in liposomes has many advantages potentially useful for their transfer to the cells. Liposomes protect cytokines from neutralization, improving their pharmacokinetics or biologic activity in vivo. They are targeted to specific cell types and may delay the release of cytokines, allowing their sustained paracrine delivery. Their physicochemical characteristics such as size, shape, charge, and stability are important parameters improving bio-distribution and prolonged pharmacokinetics of encapsulated cytokines. Material and methods: We developed an efficient protocol for the encapsulation of two types of cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), in liposomes that can be stored long term in the active state. Results: This method allows for the encapsulation of 12-13% of the total amount of cytokines and 50% of encapsulated cytokines are entrapped in liposomes of more than ≤ 600 nm in diameter. We show that in the studied cell lines the liposome-encapsulated cytokines do not affect cell morphology, proliferation or mortality. Conclusions: The G-CSF or GM-CSF can be delivered to the cells in working concentrations through the encapsulation in the liposomes. Before the clinical application, the efficiency of these liposomes should be confirmed by an in vivo study.

Effect of Different Wavelengths of Laser Irradiation on the Skin Cells.

The invention of systems enabling the emission of waves of a certain length and intensity has revolutionized many areas of life, including medicine. Currently, the use of devices emitting laser light is not only an indispensable but also a necessary element of many diagnostic procedures. It also contributed to the development of new techniques for the treatment of diseases that are difficult to heal. The use of lasers in industry and medicine may be associated with a higher incidence of excessive radiation exposure, which can lead to injury to the body. The most exposed to laser irradiation is the skin tissue. The low dose laser irradiation is currently used for the treatment of various skin diseases. Therefore appropriate knowledge of the effects of lasers irradiation on the dermal cells' metabolism is necessary. Here we present current knowledge on the clinical and molecular effects of irradiation of different wavelengths of light (ultraviolet (UV), blue, green, red, and infrared (IR) on the dermal cells. .

Children were less frequently infected with SARS-CoV-2 than adults during 2020 COVID-19 pandemic in Warsaw, Poland.

Clinical data suggest that during the current COVID-19 pandemic, children are less prone than adults to SARS-CoV-2 infection. Our purpose was to determine the frequency of SARS-CoV-2 in children vs. adults during the 2020 pandemic in Warsaw, Poland, and to investigate whether RSV and/or influenza A/B infections were associated with SARS-CoV-2 infections. We present results of RT-PCR tests for SARS-CoV-2 performed in Warsaw, Poland. Some of the pediatric subjects were also PCR-tested for RSV, and A and B influenza. We compared the test results from the four groups of symptomatic and asymptomatic subjects: 459 symptomatic pediatric patients (children 0-18 years old), 1774 symptomatic adults, 445 asymptomatic children, and 239 asymptomatic adults. 3.26% (15/459) of symptomatic pediatric patients were positive for SARS-CoV-2 in contrast to 5.58% (99/1774) of symptomatic adults (p = 0.0448). There were no SARS-CoV-2 positive cases in the group of asymptomatic children (0/445) and two positive cases in the group of asymptomatic adults (2/239), i.e., 0.83%. In the group of symptomatic pediatric patients, 17.14% (6/35) (p = 0.0002) were positive for RSV, 8.16% (4/49) were positive for influenza A, and 2.04% (1/49), thus 10.20% (5/49) (p = 0.0176) for influenza A/B. Children were less prone to SARS-CoV-2 infection than the adults during the COVID-19 pandemic in Warsaw. Higher percentage of symptomatic children was infected with RSV or influenza A/B than with SARS-CoV-2. This suggests a necessity for the testing for all these viruses for an early identification and isolation of SARS-CoV-2-positive patients for an ensuing 2020 autumn return of COVID-19.

Effect of Vitamin D Treatment on Dynamics of Stones Formation in the Urinary Tract and Bone Density in Children with Idiopathic Hypercalciuria.

Vitamin D supplementation in patients with urolithiasis and hypercalciuria is considered to be unsafe. We analyzed the impact of vitamin D supplementation on selected health status parameters in children with idiopathic hypercalciuria. The study included 36 children with urolithiasis resulting from excessive calcium excretion. The level of calcium and 25(OH)D (hydroxylated vitamin D - calcidiol) in serum, urinary calcium excretion and the presence of stones in urinary tract were assessed prospectively. Blood and urine samples were collected at the time when the patient was qualified for the study and every three months up to 24 month of vitamin D intake at a dose of 400 or 800 IU/day. At time zero and at 12, and 24 months of vitamin D supplementation, densitometry was performed. Supplementation with vitamin D caused a statistically significant increase in the concentration of 25(OH)D in serum. There were no significant changes in calcium concentration in serum, excretion of calcium in urine but also in bone density. There was no significant increase in the risk of formation or development of stones in the urinary tract. Supplementation with vitamin D (400-800 IU/day) in children with idiopathic hypercalciuria significantly increases 25(OH)D concentration, does not affect calciuria, but also does not improve bone density.

A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture.

An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 µM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80-200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 µM and below was not cytotoxic to the keratinocytes, and 150 µM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.

Supplementation of Plants with Immunomodulatory Properties during Pregnancy and Lactation-Maternal and Offspring Health Effects.

A pregnant woman's diet consists of many products, such as fruits, vegetables, cocoa, tea, chocolate, coffee, herbal and fruit teas, and various commercially available dietary supplements, which contain a high number of biological active plant-derived compounds. Generally, these compounds play beneficial roles in women's health and the development of fetus health. There are, however, some authors who report that consuming excessive amounts of plants that contain high concentrations of polyphenols may negatively affect the development of the fetus and the offspring's health. Important and problematic issues during pregnancy and lactation are bacterial infections treatment. In the treatment are proposals to use plant immunomodulators, which are generally considered safe for women and their offspring. Additional consumption of biologically active compounds from plants, however, may increase the risk of occurrences to irreversible changes in the offspring's health. Therefore, it is necessary to carry out safety tests for immunomodulators before introducing them into a maternal diet. Here, we present data from animal experiments for the four most-studied plants immunomodulators genus: Rhodiola, Echinacea, Panax, and Camellia, which were used in maternal nutrition.

Vitamin B6 improves blood parameters in rats fed a protein-deficient diet and subjected to moderate, long-term exercise.

Vitamin B6 is necessary for many enzymatic pathways (glucose and lipid metabolism, DNA/RNA synthesis, or modulation of gene expression) and affects immune cell function and blood-forming processes. We hypothesised that supplementing a protein-deficient diet with vitamin B6 may reduce the negative impact of protein malnutrition. Here, we evaluated the effect of moderate, long-term exercise (ninety days) on selected blood parameters in rats fed a normal diet, a protein-deficient diet, or a protein-deficient diet supplemented with vitamin B6. Selected haematological, immunological, and biochemical parameters were examined. A protein-deficient diet lasting 90 days caused significant reduction in body mass, increased activity of aminotransferases (asparagine and alanine), an increased percentage of innate cells in the blood, and decreased haemoglobin concentration in the blood. Adding vitamin B6 significantly increased body and muscle mass, decreased liver parameters, and caused normalisation of haemoglobin concentration and the proportion of white blood cells in the blood. These results indicate that vitamin B6 supplementation significantly improves the health of protein-malnourished rats and paves the way for the development of novel anti-malnutrition therapies.

Diamond Nanoparticles Downregulate Expression of CycD and CycE in Glioma Cells.

Our previous studies have shown that diamond nanoparticles (NDs) exhibited antiangiogenic and proapoptotic properties in vitro in glioblastoma multiforme (GBM) cells and in tumors in vivo. Moreover, NDs inhibited adhesion, leading to the suppression of migration and invasion of GBM. In the present study, we hypothesized that the NDs might also inhibit proliferation and cell cycle in glioma cells. Experiments were performed in vitro with the U87 and U118 lines of GBM cells, and for comparison, the Hs5 line of stromal cells (normal cells) after 24 h and 72 h of treatment. The analyses included cell morphology, cell death, viability, and cell cycle analysis, double timing assay, and gene expression (Rb, E2F1, CycA, CycB, CycD, CycE, PTEN, Ki-67). After 72 h of ND treatment, the expression level of Rb, CycD, and CycE in the U118 cells, and E2F1, CycD, and CycE in the U87 cells were significantly lower in comparison to those in the control group. We observed that decreased expression of cyclins inhibited the G1/S phase transition, arresting the cell cycle in the G0/G1 phase in glioma cells. The NDs did not affect the cell cycle as well as PTEN and Ki-67 expression in normal cells (Hs5), although it can be assumed that the NDs reduced proliferation and altered the cell cycle in fast dividing cells.

Long-term supplementation of Rhodiola kirilowii extracts during pregnancy and lactation does not affect mother health status.

Antibiotics treatment during pregnancy and lactation is problematic. The alternative to the antibiotic treatment is the use of plant-derived supplements, which stimulate immune system to prevent and eliminate bacterial infection. Here, we evaluated the effect of long-term use of Rhodiola kirilowii on the health of mouse mothers. Pregnant mice were fed daily, for whole pregnancy and for 28 days after giving birth, with Rhodiola kirilowii water (RKW) or hydroalcoholic extract (RKW-A) (at 20 mg of extracts/kg). The control group received sterile water. There was no significant change in the total body weight and selected organs weight and in the status of macroscopically evaluated liver, spleen, kidney, brain, and eyes, between the Rhodiola kirilowii groups and the control group. There was also no change in hematological parameters and components of adaptive immunity (level of CD3+, CD4+, CD8+, CD19+, CD335+ cells). Mice fed with RKW extracts exhibited lower percentage of oxidative burst in the granulocytes. In contrast, the supplementation with RKW-A extract caused increase in the percentage of granulocytes in the blood and the percentage of monocytes with oxidative burst. Other studied components of innate immunity were unaffected. Minor effect on the innate immunity and lack of side effects on hematological parameters and components of immunological system of mouse mothers indicates that both water and 50% hydroalcoholic extracts of Rhodiola kirilowii (in concentration 20 mg/kg per day) could be used as an immunostimulators during pregnancy and nursing. However, to fully assess the effects of Rhodiola kirilowii extracts on the mother and offspring health, further studies in mouse and large animal models and clinical studies in humans are necessary.

Macrophage functions in wound healing.

Macrophages play a crucial role in regeneration and consecutive phases of wound healing. In this review, we summarise current knowledge on the ontogeny, origin, phenotypical heterogeneity, and functional exchangeability of macrophages participating in these processes. We also describe the genetic, pharmacologic, and bioengineering methods for manipulation of macrophage phenotype and functions and their potential for development of the novel, clinically applicable therapies.

Effects of genistein on insulin pathway-related genes in mouse differentiated myoblast C2C12 cell line: evidence for two independent modes of action.

INTRODUCTION: Genistein (plant isoflavone) is a well-known anti-cancer drug with estrogenic-like properties. Genistein also regulates sugar and lipid metabolism; thus, it has anti-diabetic properties. The aim of the study was to evaluate in vitro effects of genistein on glucose transport, fatty acids oxidation, activation of PKB, and expression of genes related to insulin pathway in differentiated myoblast C2C12 mouse cell line. MATERIAL AND METHODS: Differentiated myoblast C2C12 mouse cell line was used to assess the effects of different genistein concentrations on glucose transport and fatty acids oxidation measured by radioactivity technique, activation of PKB, and expression of selected genes related to insulin signaling pathway (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A, SH-PTP2) at the mRNA and protein levels. Cells were incubated with various concentrations of genistein under standard conditions for 0-48 hours. RESULTS: Genistein in low concentrations (0.1-1 µM) significantly increased glucose transport and decreased fatty acids oxidation in C2C12 cells after 48 h of incubation. High concentration of genistein (50 µM) had the opposite effect. Genistein stimulated PKB phosphorylation during the first 5-10 minutes of incubation. There was no significant impact on the protein expression of selected genes (IR-a, IR-b, IRS-1, PKB, GLUT-4, PP2A-Ca, ER-a and ER-b) after 48 h treatment. We observed inverse correlation between genistein concentration and the expression of SH-PTP2 protein. Genistein affected the expression pattern of mRNAs for genes related to the insulin pathway, however, not the expression of the encoded proteins. CONCLUSIONS: The results of this study showed that depending on the concentration and time of incubation genistein significantly affects glucose and lipid metabolism and at low concentration modifies expression pattern of a few genes in C2C12 cells.

Influence of protein deficient diet, vitamin B[sub]2[/sub] supplementation and physical training on serum composition of polyunsaturated fatty acids (PUFAs) in rats.

[b]Introduction[/b]. Prolonged shortages of protein in the diet significantly alter the composition and content of polyunsaturated fatty acids (PUFA) in tissues and body fluids. One of nutritional factors which may reduce negative effects of protein malnutrition might be vitamin B[sub]2[/sub] due to its influence on lipids metabolism. [b]Objective. [/b]The aim of the study was to investigate the influence of low protein (LP) diet enriched with vitamin B[sub]2[/sub] on the content and composition of PUFA in the blood serum of rats treated with dosed physical exercise. [b]Materials and method. [/b]The experiment was carried out for 3 months on 72 growing male Wistar rats divided into 5 groups. Animals were fed ad libitum on a diet with an energy value of 350 kcal/100 g, in which 4.5% of the energy was provided by protein. In the control diet, 20% of the energy was provided by protein. Two groups were fed the diet enriched with vitamin B2. The two groups of tested animals were trained for 5 days a week. [b]Results.[/b] LP diet caused a decrease in α-linolenic acid (ALA) after 30 days, and a decrease in docosahexaenoic acid (DHA) after 60 days of experiment, compared with rats fed the control diet. After 60 and 90 days of the experiment, a significant decrease was noted in arachidonic acid (AA) in serum of trained rats, compared with sedentary rats fed the LP diet. Physical activity increased LA (mainly on day 30), EPA (on day 90) and reduced AA content (on day 90) in serum of rats fed the LP diet. B2 supplementation in the trained LP group did not change the EPA and AA dependence; however, there was a decrease in LA content in comparison to the non-supplemented trained group. [b]Conclusions. [/b] Results of this study suggest that all investigated factors (protein deficiency, physical exercise and supplementation of vitamin B2) have significant impact on PUFA composition of serum in rats.

Different effects of feeding pregnant and lactating mice Rhodiola kirilowii aqueous and hydro-alcoholic extracts on their serum angiogenic activity and content of selected polyphenols.

Angiogenesis plays an important role in many physiological processes, among them the formation of tissues and organs during embryogenesis. A lot of medicinal plants exhibit angiomodulatory properties. This creates the need for a thorough check of whether the plant extracts that we would like to give to pregnant women in order to increase their resistance to bacterial or viral infection will have negative effects on angiogenesis, and consequently on fetal development. This paper seeks to investigate the effect of serum of pregnant and nursing Balb/c mice that received aqueous (RKW) or hydro-alcoholic (RKW-A) R. kirilowii extracts (20 mg/kg), or epigallocatechin (0.2 mg/kg), on the in vitro proliferation and migration of mouse endothelial cell line Heca10. Of the 15 identified polyphenols in the extracts by HPLC, 8 were present in the sera. Chemical analysis revealed higher salidroside, kaempferol, chlorogenic acid, bFGF and VEGF concentration in RKW-A sera than in the sera of RKW group of mice. RKW-A and EGC sera did not affect migration of endothelial cells, however we noted some increase of migrating cells after RKW-sera treatment. RKW and EGC sera did not affect proliferation of endothelial cells. Sera of mothers from RKW-A group impaired the proliferation of endothelial cells in comparison to other groups. These data allow us to assume that Rhodiola kirilowii hydro-alcoholic extract (RKW-A) is potentially able to modulate pre- and post- natal angiogenesis what might influence the development of organs in progeny. Sera of RKW mothers have not harm the proliferation of endothelial cells, despite they also contain antiangiogenic catechins and salidroside. This suggests the existence in RKW-A extract and in RKW-A sera of some other, as yet unidentified substances influencing endothelial cells proliferation.

Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways.

Acetylcholine has been well known as one of the most exemplary neurotransmitters. In humans, this versatile molecule and its synthesizing enzyme, choline acetyltransferase, have been found in various non-neural tissues such as the epithelium, endothelium, mesothelium muscle, blood cells and immune cells. The non-neuronal acetylcholine is accompanied by the expression of acetylcholinesterase and nicotinic/muscarinic acetylcholine receptors. Increasing evidence of the non-neuronal acetylcholine system found throughout the last few years has indicated this neurotransmitter as one of the major cellular signaling molecules (associated e.g. with kinases and transcription factors activity). This system is responsible for maintenance and optimization of the cellular function, such as proliferation, differentiation, adhesion, migration, intercellular contact and apoptosis. Additionally, it controls proper activity of immune cells and affects differentiation, antigen presentation or cytokine production (both pro- and anti-inflammatory). The present article reviews recent findings about the non-neuronal cholinergic system in the field of immune system and intracellular signaling pathways.

The role of Chromium III in the organism and its possible use in diabetes and obesity treatment.

INTRODUCTION: Diabetes and obesity are diseases characterized by their increasing incidence every year. When comparing with healthy subjects, the serum levels of chromium (Cr) are lowered in these two diseases. Several studies conducted in laboratory animals with experimentally- induced diabetes demonstrated that supplementation with chromium ions (III) decreased glucose concentration in the blood, reduced the probability of atherosclerosis and heart attack, lowered the levels of cholesterol and low density lipoprotein (LDL). The Importance of chromium is actually challenged due to lack of clear manifestations of Cr deficiency in humans and animals. OBJECTIVE: The aim of this review was to present current knowledge about Cr its role in the organism and possible mechanisms of its action also in metabolic disorders such as diabetes or obesity. STATE OF KNOWLEDGE: In the last decade, Cr was established to be rather a beneficial than essential trace element in mammals, and has gained popularity as a nutritional supplement and a component of many multivitamin/mineral formulations, fortified food and energy drinks. Cr supplements are widespread for diabetes and obesity treatment, despite conflicting reports on its efficacy. It was suggested that Cr shows a beneficial influence upon glucose and lipid disturbances. CONCLUSIONS: The recent clinical trials provided evidence both in favor and against the importance of Cr in healthy and ill organisms. Unfortunately, also the molecular mechanism by which chromium affects glucose and lipid metabolism is still unclear. Beneficial effects of diet supplementation with different sources of Cr³âº can be potentially explained by rather pharmacological than nutritional effects.

The influence of vitamin B12 supplementation on the level of white blood cells and lymphocytes phenotype in rats fed a low-protein diet.

Protein malnutrition has a negative effect on body composition and some blood parameters, especially in the young growing organism. One of nutritional factors which could protect against negative consequences of protein deficiency may be B group vitamins. The aim of the study was to investigate the effect of vitamin B12 supplementation on the immune system in rats fed a standard and a low-protein diet. Rats were fed a control (20% of energy from protein) or a protein-deficient diet (4.5% of energy from protein). Half of animals in each group were additionally supplemented with vitamin B12 (300% of the daily intake). The white blood cells analysis and lymphocytes immunophenotyping (number and percentage) were performed. Low-protein diets caused disturbances in WBC and lymphocyte subpopulations in both short- (30-day) as well as long-term periods (90-day). Vitamin B12 supplementation significantly reduced the negative impact of protein malnutrition after 30 days, however had no effect on long-term malnutrition. Furthermore, vitamin B12 addition in rats fed a control diet did not affect the studied parameters. This observation opens the promise of use of vitamin B12 supplementation to improve immune system parameters in protein malnourished organisms.