The activity of monocyte-derived macrophages after stimulation with platelet-rich and platelet-poor concentrates. Study on an ovine model of insertion of a tibial implant coated with silicon-doped diamond-like carbon.

Introduction: Macrophages are crucial immune cells that play a role in tissue repair and can exhibit pro- or anti-inflammatory behaviour based on environmental stimulation. Their functional phenotype can be affected by platelet-derived products as determined by those products' composition. When the inflammatory response caused by implantation is excessive, it can lead to rejection of the implant. Therefore, a thorough evaluation of implant haemocompatibility is necessary to minimise undesirable consequences. Material and Methods: In an in vitro study, monocyte-derived macrophages (MDMs) were obtained from the whole blood of sheep after a silicon-doped diamond-like carbon-coated implant insertion. These MDMs were then exposed to autologous platelet-derived products for functional marker analysis. Results: Platelet-poor plasma (PPP) and pure platelet-rich plasma (P-PRP) stimulation increased arginase-1 activity, while leukocyte-rich PRP stimulation produced a mixed response involving higher O2- (6.49 ± 2.43 nM vs non-stimulated 3.51 ± 1.23 nM, P-value < 0.05) and NO (3.28 ± 1.38 µM vs non-stimulated 2.55 ± 0.32µM, P-value < 0.05) generation. Conclusion: Using PPP and P-PRP stimulation in post-implantation procedures may contribute to the polarisation of macrophages towards the M2-like pro-resolving phenotype, thereby accelerating wound healing. This would also prevent implant degradation due to an excessive inflammatory process.

Adzuki and Mung Bean Sprouts Enriched with Probiotic Lactiplantibacillus plantarum 299v Improve Body Mass Gain and Antioxidant Status and Reduce the Undesirable Enzymatic Activity of Microbiota in Healthy Rats.

Introducing and establishing new food requires a detailed evaluation of its safety, nutritional value and functionality, thus the control and probiotic-rich adzuki and mung bean sprouts were studied in an in vivo rats model. However, the total feed intake did not differ significantly between the groups, the highest body weight gain and body weight change were recorded in the control AIN diet. At the same time, the addition of legume sprouts caused a reduction of these parameters (up to 25% in the variant with probiotic-rich adzuki bean sprouts). There was no significant effect on serum morphology, except white blood cells (ca. 20% reduction in the control sprout-supplemented diets). Serum and liver antiradical properties were significantly elevated by consuming mung bean sprouts (no effect of the probiotics). The faecal lactic acid bacteria were already increased by the control sprouts (a 2.8- and 2.1-fold increase for adzuki and mung bean sprouts, respectively). The probiotic-rich sprouts further improved this parameter. The diets enriched with mung bean sprouts significantly decreased the urease (by ca. 65%) and ß-glucuronidase activities (by ca. 30%). All the tested diets caused also a significant reduction of faecal tryptophanase activity (the effect was intensified by Lactiplantibacillus plantarum 299v). The functional components did not affect negatively the nutritional parameters and blood morphological characteristics. They improved also the antioxidant potential and significantly decreased the activities of colon cancer-related enzymes (urease and tryptophanase). The results confirmed that these new probiotic carriers may be a valuable, safe and functional element of a healthy diet.

Metabolic Imbalances and Bone Remodeling Agents in Adolescent Idiopathic Scoliosis: A Study in Postmenarcheal Girls.

The causes and mechanisms underlying adolescent idiopathic scoliosis (AIS) remain unclear, and the available information regarding metabolic imbalances in AIS is still insufficient. This investigation aimed to evaluate the concentrations of specific bone remodeling-related agents in postmenarcheal girls diagnosed with AIS. The study encompassed thirty-six scoliosis patients and eighteen age-matched healthy individuals assigned to the control group. The patients underwent clinical and radiological examinations to assess the degree of the spinal deformity, type of curvature, and skeletal maturity. Blood and urine samples were collected from all participants and serological markers were measured using an enzyme-linked immunosorbent assay. Our study results demonstrated that the balance of phosphate-calcium and parathormone levels seems normal in individuals with AIS. Furthermore, no statistically significant differences were observed in the content of Klotho protein, osteocalcin, osteoprotegerin, C-terminal telopeptide of type I collagen (CTX), sclerostin, and alkaline phosphatase. Nevertheless, the serum levels of vitamin D (25-OH-D) were lowered, while N-terminal propeptide of type I procollagen (PINP), and fibroblast growth factor-23 (FGF23) were increased in the AIS group, with p-values of 0.044, 0.001, and 0.022, respectively. This finding indicates the potential involvement of these factors in the progression of AIS, which necessitates further studies to uncover the fundamental mechanisms underlying idiopathic scoliosis.

Analysis of In Vitro Leukocyte Responses to Biomaterials in the Presence of Antimicrobial Porcine Neutrophil Extract (AMPNE).

Implant insertion can evoke excessive inflammation which disrupts the healing process and potentially leads to complications such as implant rejection. Neutrophils and macrophages play a vital role in the early inflammatory phase of tissue repair, necessitating the study of cellular responses in host-implant interactions. In order to deepen the knowledge about these interactions, the response of neutrophils and macrophages to contact with selected biomaterials was examined in vitro on the basis of secretory response as well as reactive oxygen species/reactive nitrogen species (ROS/RNS) generation. Porcine neutrophils exposed to hydroxyapatite (HA) released more enzymes and generated higher levels of ROS/RNS compared to the control group. The addition of AMPNE diminished these responses. Although the results from porcine cells can provide valuable preliminary data, further validation using human cells or clinical studies would be necessary to fully extrapolate the findings to human medicine. Our study revealed that human neutrophils after contact of with HA increased the production of nitric oxide (NO) (10.00 ± 0.08 vs. control group 3.0 ± 0.11 µM, p < 0.05), while HAP or FAP did not elicit a significant response. Human macrophages cultured with HA produced more superoxide and NO, while HAP or FAP had a minimal effect, and curdlan reduced ROS/RNS generation. The addition of AMPNE to cultures with all biomaterials, except curdlan, reduced neutrophil activity, regardless of the peptides' origin. These results highlight the potential of antimicrobial peptides in modulating excessive biomaterial/host cell reactions involving neutrophils and macrophages, enhancing our understanding of immune reactions, and suggesting that AMPNE could regulate leukocyte response during implantation.

Native Pig Neutrophil Products: Insights into Their Antimicrobial Activity.

Cationic antimicrobial peptides are molecules with potential applications for treating infections due to their antimicrobial and immunomodulatory properties. The aim of this work was to explore the antimicrobial activity and mechanisms of action of a porcine neutrophil cathelicidin mixture (MPPN). Gram-positive and Gram-negative bacteria were used to determine the minimum inhibitory concentration (MIC) and experiments of both time-kill kinetics and effects on growth curves were performed. Planar black lipid bilayer conductance was measured to analyze the interaction of MPPN with lipid bilayers. Visualization of bacterial surfaces and membrane alterations was achieved using atomic force microscopy and transmission electron microscopy. The effects on the activity of efflux pumps (EPs) were studied with an intracellular accumulation of acridine orange (AO) assay. In E. coli, MPPN behaves as a bactericide at high concentrations and as a bacteriostatic at lower concentrations. The bacteriostatic effect was also observed for slightly shorter periods in S. enterica. The mixture was not active on S. aureus. The increase in AO accumulation in the presence of MPPN indicates that, at least in E. coli, the mixture causes inhibition of the EP function. Observed and detected variable conductance events demonstrate a strong MPPN effect on lipid bilayers. Damage to the structure of treated E. coli indicates that MPPN induces alterations in the bacterial surface. The use of AMPs capable of inhibiting EP can be seen as a good tool to combat antimicrobial resistance since they could be used alone or in combination with other conventional antibiotics to which bacteria have become resistant.

Hydroxyapatite or Fluorapatite-Which Bioceramic Is Better as a Base for the Production of Bone Scaffold?-A Comprehensive Comparative Study.

Hydroxyapatite (HAP) is the most common calcium phosphate ceramic that is used in biomedical applications, e.g., as an inorganic component of bone scaffolds. Nevertheless, fluorapatite (FAP) has gained great attention in the area of bone tissue engineering in recent times. The aim of this study was a comprehensive comparative evaluation of the biomedical potential of fabricated HAP- and FAP-based bone scaffolds, to assess which bioceramic is better for regenerative medicine applications. It was demonstrated that both biomaterials had a macroporous microstructure, with interconnected porosity, and were prone to slow and gradual degradation in a physiological environment and in acidified conditions mimicking the osteoclast-mediated bone resorption process. Surprisingly, FAP-based biomaterial revealed a significantly higher degree of biodegradation than biomaterial containing HAP, which indicated its higher bioabsorbability. Importantly, the biomaterials showed a similar level of biocompatibility and osteoconductivity regardless of the bioceramic type. Both scaffolds had the ability to induce apatite formation on their surfaces, proving their bioactive property, that is crucial for good implant osseointegration. In turn, performed biological experiments showed that tested bone scaffolds were non-toxic and their surfaces promoted cell proliferation and osteogenic differentiation. Moreover, the biomaterials did not exert a stimulatory effect on immune cells, since they did not generate excessive amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS), indicating a low risk of inflammatory response after implantation. In conclusion, based on the obtained results, both FAP- and HAP-based scaffolds have an appropriate microstructure and high biocompatibility, being promising biomaterials for bone regeneration applications. However, FAP-based biomaterial has higher bioabsorbability than the HAP-based scaffold, which is a very important property from the clinical point of view, because it enables a progressive replacement of the bone scaffold with newly formed bone tissue.

Wound-Related Complication in Growth-Friendly Spinal Surgeries for Early-Onset Scoliosis-Literature Review.

BACKGROUND: The treatment for early-onset scoliosis (EOS) is one of the most challenging for pediatric orthopedics. Surgical treatment is often necessary, and wound problems and surgical site infections (SSIs) are common, with potentially severe complications in these patients. The aim of the study was to review current literature according to this complication. METHODS: PubMed, Cochrane Library, and Embase were systematically searched for relevant articles by two independent reviewers in January 2022. Every step of the review was done according to PRISMA guidelines. RESULTS: A total of 3579 articles were found. Twenty four articles were included in this systematic review after applying our inclusion and exclusion criteria. EOS surgery has a varying but high rate of wound-related problems (on average, 15.5%). CONCLUSION: The literature concerning the definitions, collection, and interpretation of data regarding EOS wound-related complications is often difficult to interpret. This causes problems in the comparison and analysis. Additionally, this observation indicates that data on the incidence of SSI may be underestimated. Awareness of the high rate of SSI of EOS surgery is crucial, and an optimal strategy for prevention should become a priority.

Osteoarthritis: Pathogenesis, Animal Models, and New Regenerative Therapies.

Osteoarthritis (OA) is a chronic, progressive, multifactorial disease resulting in a progressive loss of articular cartilage structure and function that is most common in middle-aged and older patients. OA is involved in the loss of extracellular matrix and cartilage as well as cell number decreases within the matrix, especially in the further stages of the disease. The immune system plays a pivotal role in the pathomechanism of this condition. Both humoral and cellular mediators contribute to cartilage destruction, abnormal bone remodeling, synovitis, and joint effusion. The increasing prevalence of this disease has led to a growing interest in using animal models as the primary way to broaden the knowledge of the pathogenesis of OA and possible therapies at each stage of disease development. This review aims to describe the signs, pathogenesis, and classification of OA as well as discuss the advantages and disadvantages of some animal models. The currently used treatment methods include mesenchymal stem cells, exosomes, gene therapies, and blood-derived products. In addition, exogenous growth factors, platelet-rich plasma (PRP), platelet lysate, and autologous conditioned serum (ACS) are discussed with the application of tissue engineering techniques and biomaterials.

Effects of Topical Treatment of Foot Rot in Sheep Using Ozonated Olive Ointment.

INTRODUCTION: Foot rot in small ruminants is highly contagious, causes severe lameness, and impairs fertility and wool and meat production. It is usually treated with parenteral antibiotics, with attendant antibiotic resistance risk, and with bactericidal footbaths, potentially harmful to humans and the environment. An alternative treatment in sheep is proposed based on repeated topical ozonated ointment application. Its effectiveness and safety were evaluated by estimation of acute-phase response, biochemical indicators of organic damage, and antioxidant/oxidant balance (AOB). MATERIAL AND METHODS: The study was conducted on ten sheep with Egerton scale 2-3 lesions. Ozone application was repeated every day for seven days. Blood was drawn first (T0) after foot cleaning and before ozonation, then (T1) seven days after the first ozone application, and finally (T2) four days after the last application. RESULTS: High clinical effectiveness was observed, with total recovery by 28 days from the start of treatment. A significant increase in antiradical activity was noted on the basis of a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay from 1.16 ± 0.04 µmolTe/mL at T0 to 1.23 ± 0.03 µmolTe/mL at T1, with a slight decrease in oxidative stress. Calculated on the basis of antiradical capacity, AOB was higher at T1 (130 ± 19%) and decreased to 110 ± 16% at T2. Calculated on the basis of reducing power, it was 169 ± 22% at T1 and 131 ± 17% at T2. CONCLUSION: These results indicated that the AOB is efficient enough to prevent oxidative organ injury and the applied doses of ozone are safe for animals.

The Effect of Neutrophil-Derived Products on the Function of Leukocytes Obtained after Titanium Implantation in the Ovine Model.

Titanium (Ti) is currently the most common biomaterial used for orthopedic implants; however, these implants may cause deleterious immune response. To investigate the possible mechanisms involved in excessive inflammation, we assessed the activity of neutrophils and monocyte-derived macrophages (MDMs) during the insertion of the Ti implant in a sheep model. The study was conducted on 12 sheep, 4 of which were control animals and 8 were in the experimental group with inserted Ti implant. Neutrophil secretory response was estimated at two time points T0 before surgery and T1 1 h after implantation and was based on the release of enzymes from neutrophil granules and reactive oxygen and nitrogen species (RONS) generation. MDM function was evaluated 5 months after implantation, on the basis of RONS generation arginase activity and morphological changes. Moreover, the influence of some autologous neutrophil derived products, namely, antimicrobial neutrophil extract (ANE) and neutrophil degranulation products (DGP) on leukocytes was estimated. Our study revealed that Ti implant insertion did not cause any adverse effects up to 5 months after surgical procedure. Stimulation of neutrophil cultures with ANE decreased the enzyme release as well as superoxide generation. Treatment of MDM with ANE diminished superoxide and NO generation and increased arginase activity. On the other hand, MDM stimulated with DGP showed elevated superoxide and NO generation as well as decreased arginase activity. To summarize, ANE exerted an anti-inflammatory and pro-resolving effect on studied leukocytes, whereas DGP acted as pro-inflammatory.

Long-term Interactions of Circulating Neutrophils with Titanium Implants, the Role of Platelets in Regulation of Leukocyte Function.

Despite the fact that different biomaterials are widely used in many biomedical applications, they can still cause side effects. Therefore, our aim was to assess neutrophil activity during the inflammatory phase of the repair process and long-term interactions between circulating neutrophils and Titanium (Ti) implants. Additionally, neutrophil in vitro response after stimulation by the extract of antimicrobial peptides (AMP extract), pentoxifylline (PTX) and some platelet-rich (L-PRP and PURE PRP) and platelet-poor (PPP) concentrates were tested. The study was conducted on eight sheep after Ti implant insertion into the tibia and revealed that the Ti implant did not cause any side effects during the course of experiment. After addition of L-PRP into neutrophils, culture activity of these cells significantly increased (p < 0.01), whereas treatment with AMP extract, PURE PRP, PPP or PTX caused decrease in neutrophil enzymatic response (on the basis of elastase, myeloperoxidase and alkaline phosphatase release) and free radical generation. These effects were observed in neutrophils isolated during the inflammatory phase as well as 4 and 10 months after implantation. Obtained results will be useful in regulation of inflammatory response during implantation of biomaterial and create possibility to modulate the cells response towards pro- or anti-inflammatory to reduce host tissue damage.

Application of Natural Neutrophil Products for Stimulation of Monocyte-Derived Macrophages Obtained before and after Osteochondral or Bone Injury.

We evaluated the use of some neutrophil products, namely; autologous rabbit antimicrobial neutrophil extract (rANE), heterologous porcine antimicrobial neutrophil extract (pANE), neutrophil degranulation products (DGP) and neutrophil microvesicles (MVs) for stimulation of monocyte-derived macrophages (MDMs) to improve healing. Two animal models were evaluated; the rabbit model for autologous osteochondral transplantation (OT) with application of rabbit ANE, DGP or MVs for MDMs stimulation, and the ovine model of the insertion of a Ti implant with the use of porcine ANE, and ovine DGP or MVs for MDMs stimulation. Macrophage activity was assessed on the basis of free radical generation and arginase activity. We estimated that DGP acted in a pro-inflammatory way both on rabbit and ovine MDMs. On the other hand, MVs acted as anti-inflammatory stimulator on MDMs in both experiments. The response to ANE depended on origin of extract (autologous or heterologous). Macrophages from rabbits before and after OT stimulated with autologous extract generated lower amount of NO and superoxide, especially after transplantation. In the ovine model of Ti implant insertion, heterologous ANE evoked increased macrophage pro-inflammatory activity. Our study revealed that these neutrophil products could regulate activity of macrophages, polarizing them into pro-or anti-inflammatory phenotypes that could enhance bone and osteochondral tissue healing.

Prospects and Applications of Natural Blood-Derived Products in Regenerative Medicine.

Currently, there are a number of therapeutic schemes used for the treatment of various types of musculoskeletal disorders. However, despite the use of new treatment options, therapeutic failure remains common due to impaired and delayed healing, or implant rejection. Faced with this challenge, in recent years regenerative medicine started looking for alternative solutions that could additionally support tissue regeneration. This review aims to outline the functions and possible clinical applications of, and future hopes associated with, using autologous or heterologous products such as antimicrobial peptides (AMPs), microvesicles (MVs), and neutrophil degranulation products (DGP) obtained from circulating neutrophils. Moreover, different interactions between neutrophils and platelets are described. Certain products released from neutrophils are critical for interactions between different immune cells to ensure adequate tissue repair. By acting directly and indirectly on host cells, these neutrophil-derived products can modulate the body's inflammatory responses in various ways. The development of new formulations based on these products and their clinically proven success would give hope for significant progress in regenerative therapy in human and veterinary medicine.

Relationship Between Neutrophil Activity, Oxidative Stress, Acute Phase Response, and Lameness Grade in Naturally Occurring Acute and Chronic Joint Disorders in Horses.

We hypothesized that in horses with naturally occurring joint disorders, the neutrophil response, acute phase response (APR), and oxidative stress parameters elevated significantly and are markers of increased inflammatory response in these conditions. Therefore, the first aim of the study was to evaluate neutrophil response, oxidative status, and APR. The neutrophil activity was assessed on the basis of elastase, myeloperoxidase (MPO), and alkaline phosphatase release, whereas free radical generation was assessed on the basis of nitric oxide and superoxide production. Acute phase response was estimated on the basis of fibrinogen or haptoglobin plasma concentration and oxidative stress on the basis of malondialdehyde plasma concentration. Then, these parameters were compared with lameness grade, and correlation coefficients between them were calculated. The study was conducted on 43 horses divided on control group of healthy horses (n = 17), acute lameness (AL) group (11 horses), and chronic lameness (CL) group (15 horses). The neutrophil activity from horses of both groups of joint disorders (AL and CL) was significantly (P < .01) higher in comparison with healthy horses. Elastase release was 67.28 ± 1.89% of maximal activity in AL group in comparison with 51.72 ± 1.75% in healthy horses and 62.61 ± 1.54% in CL group. The highest values of other enzymes were also noted in AL group. Moreover, in AL group release of elastase and MPO positively correlated (P < .01) with grades of lameness. These findings revealed the mutual relation between studied parameters and obtained results may be useful in the development of new therapeutic strategies in the treatment of acute and chronic joint disorders in horses.

Changes in the Activity of Ovine Blood-derived Macrophages Stimulated with Antimicrobial Peptide Extract (AMP) or Platelet-rich Plasma (PRP).

INTRODUCTION: Antimicrobial peptides (AMP) are a large group of innate immune effectors, which apart from antimicrobial activity show immunomodulative properties. Platelet-rich plasma (PRP) is a source of autologous growth factors and is used for stimulation of bone and soft tissue healing. The purpose of this study was to assess the influence of PRP and AMP extract on ovine monocyte-derived macrophage cultures. MATERIAL AND METHODS: The study was conducted on ovine macrophages (Mfs) previously stimulated with LPS or dexamethasone and then with preparations of PRP or AMP. Following activation of the Mfs their morphological and functional features were assessed. RESULTS: The study revealed pro-inflammatory influence of both examined preparations on Mfs cultures on the basis of morphology, ROS generation and arginase activity. Both preparations enhanced the pro-inflammatory response of cultured Mfs. CONCLUSION: This activity may intensify the antimicrobial action of Mfs, however, in cases of excessive and prolonged inflammation the use of these preparations should be limited.

The Neutrophil Response to Rabbit Antimicrobial Extract After Implantation of Biomaterial into a Bone/Cartilage Defect.

BACKGROUND/AIM: In this study, the neutrophil response to an antimicrobial extract was evaluated as a potential marker of inflammatory process after implantation of alginate and carbon fiber biomaterials into a bone or cartilage defect in rabbits. MATERIALS AND METHODS: The response to biomaterials used was assessed based on enzyme release, generation of reactive oxygen species (ROS) and survival of neutrophils isolated from the rabbit's blood after implantation. RESULTS: The implantation of alginate biomaterial increased elastase and alkaline phosphatase release, whereas carbon fibers did not evoke increased elastase release. The implantation of both biomaterials resulted in a higher myeloperoxidase (MPO) release after 30-min incubation. Stimulation with different fractions of antimicrobial peptide (AMP) extract diminished MPO release and nitric oxide generation, as well as slightly reducing neutrophil survival. CONCLUSION: Our study permits the assessment of the neutrophil intravital response to an implant without the need for preparation of histological sections. Additionally, AMP extract restricted some manifestations of the pro-inflammatory response and may be considered a regulator of neutrophil activity in the early inflammatory phase, preventing rejection of the implant.

Application of Platelet-rich Plasma and Tricalcium Phosphate in the Treatment of Comminuted Fractures in Animals.

AIM: To assess the applicability of ß-tri-calcium phosphate (TCP) and platelet-rich plasma (PRP) in the treatment of comminuted fractures in small animals. MATERIAL AND METHODS: The experimental study was carried out on 16 New Zealand White rabbits. After creating the bone defect and performing tibial osteotomy, TCP implants containing activated PRP were introduced into the fracture and the defect. The fracture was stabilised using external fixators or intramedullary nails. After 12 weeks, the animals were euthanised, and radiological, histological, scanning electron microscopy and peripheral quantitative computed tomography examinations were performed. The analysis also covered the results of fracture treatment in 37 small animals (cats and dogs) in which treatment with TCP containing PRP was used as an alternative to cancellous bone implantation. RESULTS: Correct bone union was observed in the experimental groups, TCP remained visible at the site of the fracture after 12 weeks. In the clinical application in small animals, bone union was observed in over 91% of treated animals. CONCLUSION: ß-TCP and activated PRP may be an effective method of bone union enhancement in the treatment of comminuted fractures in small animals.

Different activation of monocyte-derived macrophages by antimicrobial peptides at a titanium tibial implantation in rabbits.

The aim of our study was to assess the functional and morphological features of monocyte derived Mfs from rabbits with titanium (Ti) tibial implants, their ability to polarize towards M1 or M2 and their reactivity after stimulation with neutrophil antimicrobial peptides extract (AMP). The study was conducted on six White New Zealand rabbits with tibial implants of pure titanium Grade 2. Blood was taken before implantation and 14days after biomaterial implantation. Then, blood-derived Mfs were cultured and their function was assessed on the basis of morphological changes, generation of nitric oxide (NO), superoxide, and proteases release after treatment with LPS or dexamethasone (Dex). As a result of polarization we specified two subpopulations of Mfs with features characteristic of each subtype. M1 stimulated with LPS exhibited an increased NO and superoxide level, and M2 stimulated with Dex which showed higher arginase and lower free radical generation. Addition of AMP evoked further changes in Mfs morphology and function. After 24h stimulation with AMP an increase of NO was observed in all cultures, whereas after 48h it decreased. Production of superoxide lowered, especially after 48h, when M1 generated 6.00±0.2nM and M1/AMP culture generated 5.2±0.1nM of superoxide. Our study revealed that activated Mfs stimulated with AMP demonstrated both pro- and anti-inflammatory features. Moreover, we did not detect significant differences between the response of Mfs cultured from blood derived monocytes before and after implantation of Ti implants.

Evaluation of Platelet-Rich Plasma and Neutrophil Antimicrobial Extract as Two Autologous Blood-Derived Agents.

The platelet-rich plasma (PRP) and antimicrobial peptides neutrophil extract (AMP extract) were prepared from rabbit neutrophils as two autologous blood-derived preparations, which could be applied locally to enhance healing process of tissues. Both preparations were analyzed using the MALDI TOF method for accurate qualitative assay. Growth factors (PDGF and VEGF) and microbicidal protein were reported in PRP. In AMP extract α-defensins, namely; NP-1, -2, -3a, -3b, -4, and -5 and cathelicidins represented among other by 15-kDa antibacterial protein (p15s) were detected. In the second part of our study the influence of antimicrobial extract on macrophages in vitro was tested. Then, degranulation of neutrophils in vitro and generation of reactive intermediates by these cells under the influence of AMP extract were assessed. As estimated, the addition of AMP extract into cultures of macrophages decreased superoxide anion generation after 5 days of incubation. Furthermore, AMP extract inhibited degranulation and respiratory burst in neutrophils, therefore in this regard it suppress proinflammatory effect of two studied populations of leukocytes.

In Vitro Proinflammatory Polarization of Macrophages Isolated from Hepatocarcinogenic Stage in Humans and Rats.

Macrophages have multiple functions in both inhibiting and promoting hepatocarcinogenesis, which are dependent on their phenotypes. Thus, we were interested in clarifying the 'training' proinflammatory effects exerted by barley ß-glucan (BBG) on monocyte-derived macrophages from patients with hepatocellular carcinoma (HCC-Mfs). After isolation and differentiation HCC-Mfs were treated with different concentrations of BBG and functional assays were then conducted after 24 h, and 3 and 5 days of incubation. The release of reactive oxygen species, arginase concentration and cell morphology were analyzed. Under the influence of BBG neoplastic cells slightly elongated and dendric-like filopodia were observed. In HCC-Mfs, the significant generation of NO and O2- was seen on days 3 and 5 of culture, concomitantly with significant depletion (p<0.05) of arginase activity. In summary, we showed that HCC-Mfs, 'trained' in a BBG microenvironment keep a highly dynamic plasticity, together with their proinflammatory polarization, expressed by reactive oxygen species and reactive nitrogen intermediates (RNI) augmentation.