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Immunological safety of nanofibers remains poorly reported within the scientific literature and lacks specific in vitro testing models distinct from those used to test nanoparticles. To address the challenges of currently used conventional setups being described in the literature, we developed a novel in vitro model for nanofiber mats immunogenicity testing, which enables standardization of tested surface area, excludes nanofiber mat edges, and ensures stable contacts of cells with nanofibers during the experiment. The effect of nanofibers was assessed on peripheral blood mononuclear cells (PBMCs) by measuring their metabolic activity using MTS cell proliferation assay, where key performance parameters, i.e. cell number, phytohemagglutinin-L (PHA-L) concentration, incubation time and cell lysis were optimized. Repeatability of results obtained with non-activated and PHA-L-activated PBMCs in contact with differently thick polycaprolactone nanofiber mats was compared using both models. Our model provided more reproducible results with lower variability, exhibiting its higher reliability and accuracy than the conventional one. Furthermore, results showed the presence of thicker mats resulted in reduced metabolic activity and PBMC proliferation without any observed cytotoxicity, providing additional insights into their non-immunogenic characteristics. The developed model enables more accurate biological assessment that can support new guidelines for in vitro nanofiber testing and formulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Conventional treatments for chronic wounds are often ineffective, thus new therapeutic approaches are needed, such as the delivery of immunomodulatory drugs that can reduce inflammation, restore immune cell function, and facilitate tissue regeneration. A potential drug for such an approach is simvastatin, which has major drawbacks including poor solubility and chemical instability. With the aim of developing a dressing for wound healing, simvastatin and an antioxidant were incorporated into alginate/poly(ethylene oxide) nanofibers by green electrospinning without the use of organic solvents, thanks to their prior encapsulation into liposomes. The composite liposome-nanofiber formulations exhibited fibrillar morphology (160-312 nm) and unprecedentedly high phospholipid and drug content (76%). Transmission electron microscopy revealed dried liposomes as bright ellipsoidal spots homogeneously distributed over the nanofibers. After nanofiber hydration, the liposomes reconstituted in two size populations (~140 and ~435 nm), as revealed by cutting-edge MADLS ® analysis. Lastly, in vitro assays demonstrated that composite liposome-nanofiber formulations are superior to liposomal formulations due to a better safety profile in keratinocytes and peripheral blood mononuclear cells. Furthermore, both formulations exhibited similarly advantageous immunomodulatory effects, measured as decreased inflammation in vitro. A synergistic combination of the two nanodelivery systems shows promise for the development of efficient dressings for chronic wound treatment.

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6 months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Human skeletal stem cells (hSSCs) were recently identified as podoplanin (PDPN)/CD73/CD164-positive and CD146-negative cells that decline with age, and play a role in the pathogenesis of osteoarthritis (OA). The aim of this study was to identify the hSSC-like properties of bone-derived mesenchymal stem/stromal cells (MSCs) of patients with late and early OA. Methods : First, we performed gene expression profiling for the hSSC markers in 32 patients with late and early OA, and donors without OA. Having identified the low expression of hSSC markers in late OA patients, we further performed trilineage differentiation and immunophenotyping for hSSC makers in the selected subsets from each donor group. Results : Our results show no differences in osteogenesis, chondrogenesis, and adipogenesis between the MSCs from the three groups. However, the immunophenotyping shows lower CD164 in MSCs from early OA patients in comparison with late and no OA subjects ( p = 0.002 and p = 0.017). Conclusions : Our study shows that the in vitro hSSC-like properties of bone-derived MSCs are similar in patients with early and late OA, and in donors without OA. However, the lower percentage of CD164-positive MSCs in early OA patients indicates the potential of CD164 as a marker of the onset of OA.

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). New in vitro testing systems are needed to evaluate efficacies of new anti-inflammatory biological drugs, ideally in a patient-specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti-inflammatory drugs, spheroids were exposed to TNF-α, IL-1β, or to supernatant containing secretome from activated macrophages (MCM). The anti-inflammatory efficacies of anti-TNF-α biologicals adalimumab, infliximab, and etanercept, and the anti-IL-1β agent anakinra were assessed in short-term microspheroid and long-term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF-α or IL-1β. The differences in potency of anti-TNF-α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short-term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti-TNF-α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045-1058, 2018., (© 2018 American Institute of Chemical Engineers.)

The musculoskeletal system includes tissues that have remarkable regenerative capabilities. Bone and muscle sustain micro-damage throughout the lifetime, yet they continue to provide the body with the support that is needed for everyday activities. Our current understanding is that the regenerative capacity of the musculoskeletal system can be attributed to the mesenchymal stem/ stromal cells (MSCs) that reside within its different anatomical compartments. These MSCs can replenish various tissues with progenitor cells to form functional cells, such as osteoblasts, chondrocytes, myocytes, and others. However, with aging and in certain disorders of the musculoskeletal system such as osteoarthritis or osteoporosis, this regenerative capacity of MSCs appears to be lost or diverted for the production of other non-functional cell types, such as adipocytes and fibroblasts. In this review, we shed light on the tissue sources and subpopulations of MSCs in the musculoskeletal system that have been identified in animal models, discuss the mechanisms of their anti-inflammatory action as a prerequisite for their tissue regeneration and their current applications in regenerative medicine. While providing up-to-date evidence of the role of MSCs in different musculoskeletal pathologies, in particular in osteoporosis and osteoarthritis, we share some thoughts on their potential as diagnostic markers in musculoskeletal health and disease.

In vitro cell-based models are important tools for assessing efficacies of new leads in early phases of drug development. Human osteoarthritic chondrocytes (OACs), obtained from biomedical waste material, represent a valuable, relatively accessible cellular source that could be used for this purpose. By employing reverse transcription-polymerase chain reaction (qRT-PCR) we compared gene expression profiles of key anabolic, catabolic and inflammatory genes of freshly isolated vs. monolayer cultured OACs (passages P0-P2) and non-stimulated vs. tumor necrosis factor alpha (TNF-α) stimulated P2 OACs. After expansion of OACs in monolayer cultures, the expression of almost all analyzed genes significantly decreased. The subsequent addition of TNF-α to OACs at P2 significantly increased expressions of all catabolic and inflammatory genes, leaving the anabolic profile almost unchanged. TNF-α-treated OACs were later utilized for efficacy testing of anti-TNF-α drugs infliximab and etanercept and both significantly reduced the expressions of all catabolic and inflammatory genes tested., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Bisphenols (BPs) are widely spread pollutants that act as estrogen-like endocrine disruptors and are potentially affecting human health on a long run. We explored the effects of BPA, BPF and BPAF, on in vitro differentiation and maturation of MDDCs. Monocytes were treated with 17β-estradiol (E2) and each BP at the beginning of their differentiation into iMDDCs. We found that 10 and 50 μM of BPA and BPF, 10 and 30μM of BPAF and 10 and 50 nM of E2 did not affect cell viability. However, 50 μM of BPA and BPF, as well as 10 and 30 μM of BPAF, significantly decreased the endocytotic capacity of iMDDCs. Both, BPA (50 μM) and BPAF (30 μM) decreased the expression of CD1a and increased the amount of DC-SIGN molecules on iMDDCs. The E2 pre-treatment moderately decreased expression of CD80, CD86 and CD83 co-stimulatory molecules while increasing the numbers of HLA-DR on mMDDCs. Only BPAF significantly influenced the expression of CD80 and CD86 (both decreased), as well as CD83 and HLA-DR molecules (both increased) on mMDDCs. In addition, BPAF modulated DC maturation signaling pathways by lowering the phosphorylation of p65 NF-κB (nuclear factor-kappaB) and ERK (extracellular signal regulated kinase) 1/2 proteins. Consequently, the in vitro proliferation of allogeneic T cells, stimulated with differently pre-treated iMDDCs and mMDDCs, was significantly reduced only in case of BPAF., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions., Experimental Procedures: MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits., Results: We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients' survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM., Conclusion: The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.

The aim of our work was to produce a modern nanomaterial with incorporated blood-derived growth factors, produced by electrospinning, applicable in treatment of chronic wounds. Platelet-rich plasma was chosen as a natural source of growth factors. Results showed that platelet-rich plasma stimulates keratinocyte and fibroblast cell growth in vitro. Its optimal concentration in growth medium was 2% (v/v) for both types of skin cells, while higher concentrations caused alterations in cell morphology, with reduced cell mobility and proliferation. In the next step hydrophilic nanofibers loaded with platelet-rich plasma were produced from chitosan and poly(ethylene oxide), using electrospinning. The morphology of nanofibers was stable in aqueous conditions for 72 h. It was shown that electrospinning does not adversely affect the biological activity of platelet-rich plasma. The effects of nanofibers with incorporated platelet-rich plasma on cell proliferation, survival, morphology and mobility were examined. Nanofibers limited cell mobility, changed morphology and stimulated cell proliferation. Despite of the small amount of blood-derived growth factors introduced in cell culture via platelet-rich plasma-loaded nanofibers, such nanofibrillar support significantly induced cell proliferation, indicating synergistic effect of nanotopography and incorporated growth factors. The overall results confirm favorable in vitro properties of produced nanofibers, indicating their high potential as a nanomaterial suitable for delivery of platelet-rich plasma in wound healing applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) can express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT) astrocytes and in astrocytes devoid of IFs., Methods: The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software., Results: Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent., Conclusions: Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.

A molecule with a relatively simple chemical structure, resveratrol has been found to interact with multiple molecular targets, many of them associated with inflammation and immunity. Indeed, it has been shown to act directly on central players of both innate and adaptive immunity, such as macrophages, lymphocytes, and dendritic cells. In addition, there is very little evidence suggesting significant deleterious side effects of resveratrol, further highlighting its potential future use as a therapeutic agent. This review provides an up-to-date discussion on recent advances regarding anti-inflammatory effects of resveratrol, mechanisms of action, and its potential for therapeutic use.

Background: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable., Methodology/principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex "crossomics" analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4+T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset., Conclusions/significance: Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.

A novel lectin, isolated from the basidiomycete mushroom Clitocybe nebularis and termed C. nebularis lectin (CNL), exhibits an immunostimulatory effect on the most potent antigen-presenting cells, the dendritic cells (DCs). Treatment of human monocyte-derived DCs with CNL in doses from 1 to 10 μg/ml resulted in a dose-dependent induction of overall DC maturation characteristics. Exposure of DCs to CNL for 48 hr resulted in extensive up-regulation of co-stimulatory molecules CD80 and CD86, as well as of the maturation marker CD83 and HLA-DR molecules. Such CNL-matured DCs (CNL-DCs) were capable of inducing a T helper type 1-polarized response in naive CD4+  CD45RA+ T cells in 5-day allogeneic co-cultures. The allostimulatory potential of CNL-DCs was significantly increased relative to untreated controls, as was their capacity to produce several pro-inflammatory cytokines such as interleukin-6, interleukin-8 and tumour necrosis factor-α. By using a specific Toll-like receptor 4 (TLR4) signalling inhibitor, CLI-095, as well as Myd88 inhibitory peptide, we have shown that DC activation by CNL is completely dependent on the TLR4 activation pathway. Furthermore, activation of TLR4 by CNL was confirmed via TLR4 reporter assay. Measurement of p65 nuclear factor-κB and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CNL stimulation of DCs revealed primarily an increase in nuclear factor-κB activity, with less effect on the induction of p38 MAPK signalling than of lipopolysaccharide-matured DCs. The CNL had the ability to activate human DCs in such a way as to subsequently direct T helper type 1 T-cell responses. Our results encourage the use of mushroom-derived lectins for use in therapeutic strategies with aims such as to strengthen anti-tumour immune responses., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

Monocyte-derived dendritic cells (DCs) are considered an indispensible and one of primary tools for in vitro DC-based studies. For majority of in vitro DC-based studies the medium of choice is supplemented RPMI, with certain variable ingredients such as HEPES buffer or Phenol Red (PHR). In effort to identify potential obstruction of DC differentiation process due to presence of mentioned additives, we differentiated DCs using RPMI either with or without HEPES or PHR. Although PHR caused a certain down-regulation of immature DCs (iDCs) differentiation markers and lower expression of co-stimulatory molecules on mature DCs, these changes were not significant. In contrast, use of RPMI also containing HEPES resulted in significantly lower CD1a and DC-SIGN expression on iDCs and extensively lowered co-stimulatory molecule expression after DC activation (HEPES-DCs). Furthermore, DCs differentiated in HEPES-free RPMI possessed more genuine immature/mature DC characteristics in context of Th1 polarization. Additionally, during classical differentiation procedure, fewer DCs remained adherent and possessed better overall morphology in HEPES-free medium. In summary our study clarifies a seemingly minor, but a very important issue, that will most likely facilitate lab work for many scientists dealing with monocyte-derived DCs.

Dendritic cells (DCs) constitute a unique set of antigen-presenting cells (APCs), equipped with the potential to initiate strong immune responses as well as to critically regulate immunity. Tolerogenic or "alternatively activated" DCs show remarkable properties in regulating immune responses both in vitro and in vivo. Furthermore, tolerogenic DCs are now beginning to be tested in clinical studies. Use of pharmacological agents to induce maturation-resistant tolerogenic DCs is a popular approach. In this review, we will discuss already recognized, as well as recently discovered, potential pharmacological agents, their mechanism of action, and the way in which they induce tolerance in DCs.

Basic functional test for evaluation of in vitro cultured human dendritic cells (DC) is primary allogeneic one-way mixed lymphocyte reaction (MLR). In this way, one can evaluate stimulating capacity, which is a basic characteristic of DC. The proliferation of cells is measured through incorporation of 3 H-thymidine. Normally proliferation is measured at days 5-7. We studied kinetics of proliferative responses initiated with different stimulating cell suspensions to evaluate differences and possibly reduce time needed to perform this test.Gradual increase in response from days 1 to 7 and a significant difference from controls (peripheral blood mononuclear cells) seen from day 4 was noted if macrophages were used as stimulators. A consistently higher proliferation, compared to controls, was always found already on day 2 when mature DC were used as stimulators. The reaction peaked 2 to 3 days earlier and was also more than two times more intense.This maximal and significantly higher response, consistently seen already after 48 hours, allows us to confirm the presence of mature DC in stimulating suspensions much earlier than previously.

Chondrocytes present in articular cartilage survive as a resident cell population throughout the lifespan of the individual organism. However, articular chondrocytes as other cells also undergo apoptosis and there is an ever increasing list of diverse stimuli that can induce this phenomenon in vitro. Our main interest was to investigate potential cytotoxic effects of vitamin C (L-ascorbic acid) on human articular chondrocytes. The present study suggests that vitamin C can induce apoptosis in a cell culture of chondrocytes after 18 h of cultivation. Apoptosis-inducing activity of L-ascorbic acid is dose dependent and significantly affected by the presence of serum. The increased number of vitamin C induced apoptotic cells was associated with DNA fragmentation and morphological changes of the cells.

The variability in the immune response modulated by HLA alleles may be an important factor for the induction of the protective effect of HBsAg vaccines. We present here the analysis of HLA-DRB1, DQB1 and DQA1 alleles and their combinations in the group of 36 individuals with poor humoral immmune response to HBsAg vaccination. Comparison with the control group, consisted of 60 randomly choosen healthy subjects, revealed that the DRB1*1601, DQB1*0502, DQA1*0102 haplotype is overrepresented in the group of hyporesponders and may therefore be regarded as a factor influencing poor antibody responsiveness. We observed that after revaccination two of three individuals who failed to develop anti-HBs antibodies carry the same phenotype DRB1*0101,DRB1*0301;DQB1*0501, DQB1*0201;DQA1*0101,DQA1*0501, which supports the conjecture that immunogenicity of the HBsAg vaccine depends on specific combination of HLA DR and DQ molecules on antigen presenting cells.