Search Results (286)

Extracellular vesicles (EVs) have emerged as promising acellular tools for modulating immune responses for tissue engineering applications. This study explores the potential of human fibroblast-derived EVs delivered within a three-dimensional (3D) injectable scaffold composed of polycaprolactone (PCL) nanofibers and collagen (PNCOL) to reprogram macrophage behavior and support scaffold integrity under inflammatory conditions. EVs were successfully isolated from human fibroblasts using ultracentrifugation and characterized for purity, size distribution and surface markers (CD63 and CD9). Macrophage-laden PNCOL scaffolds were prepared under three conditions: macrophage-only (MP), fibroblast co-encapsulated (F-MP), and EV-encapsulated (EV-MP) groups. Structural integrity was assessed via scanning electron microscopy and Masson’s trichrome staining, while immunomodulatory effects were evaluated through metabolic assays, gene expression profiling, and immunohistochemistry for macrophage polarization markers (CD80, CD206). When co-encapsulated with pro-inflammatory (M1) macrophages in PNCOL scaffolds, fibroblast-derived EVs preserved scaffold structure and significantly enhanced macrophage metabolic activity compared to the control (MP) and other experimental group (F-MP). The gene expression and immunohistochemistry data demonstrated substantial upregulation of anti-inflammatory markers (TGF-β, CD163, and CCL18) and surface protein CD206, indicating a phenotypic shift toward M2-like macrophages for EV-encapsulated scaffolds relative to the other groups. The findings of this study demonstrate that fibroblast-derived EVs integrated into injectable PCL–collagen scaffolds offer a viable, cell-free approach to modulate inflammation, preserve scaffold structure, and support regenerative healing. This strategy holds significant promise for advancing immuno-instructive platforms in regenerative medicine, particularly in settings where conventional cell therapies face limitations in survival, cost, or safety. Full article

Background: Measuring frequencies of antigen-specific memory B cells (B_mem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot^® assays can provide such information; however, like most cell-based tests, they require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion in clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2–3 million peripheral blood mononuclear cells (PBMCs) per antigen, obtainable from 2–3 mL of blood from healthy adult donors. Results: Except when B_mem frequencies were very low, we found that testing PBMCs in singlet wells, but in serial dilution, enables as reliable B_mem frequency assessments as when testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot^® assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality by cryopreserved PBMCs equivalent to freshly isolated material implies that fewer than the standard 10 million PBMCs per vial can be frozen. This would reduce the number of individuals who could not be tested for B_mem due to insufficient availability of PBMCs, a common problem with such assays. Conclusions: The predictable need for and recovery of cryopreserved PBMCs facilitates planning of and optimal cell utilization in B cell ImmunoSpot^® assays and increases the practical feasibility of extensive B_mem characterization in larger cohorts. Full article

Tick-Borne Encephalitis Virus (TBEV) is impacting public health in the Eurasian region, with increasing case numbers. There is, therefore, a need to expand research efforts and the corresponding infrastructure capacity. Since TBEV is classified as a risk group 3 organism in Switzerland, handling infectious material containing the virus is restricted to biosafety level 3 laboratories. In some instances, downstream analyses may need to be performed outside of the containment facility. It is, therefore, essential to validate effective inactivation protocols compatible with the safe and accurate processing of samples. This study evaluated UV irradiation, chemical treatment with detergents, and mechanical filtration as candidate methods to inactivate TBEV infectious samples, including culture supernatants and tissue homogenates, while preserving their compatibility for different assays. Among the methods tested, 45 s of UV irradiation or Triton-X100 at concentrations between 0.05% and 0.1% effectively inactivated TBEV while mostly preserving the integrity of the processed samples for immuno- or enzymatic assays. These findings establish safe and reliable procedures for advancing TBEV research beyond high-containment settings. Full article

Background/Objectives: Type 2 diabetes mellitus (T2DM) is a major global health challenge, primarily driven by insulin resistance and beta-cell dysfunction. This study investigated the roles of p21-activated kinase 1 (PAK1) and calcium/calmodulin-dependent protein kinase II (CAMKII) in insulin secretion, aiming to elucidate their involvement in this process and their implications in T2DM pathophysiology. Methods: Using the Beta-TC-6 insulinoma cell line, we assessed colocalization and interaction of PAK1 and CAMKII under glucose stimulation through indirect immuno-fluorescence (IFI) and proximity ligation assays (PLA). To examine their expression dynamics in a physiological context, we performed immunohistochemistry (IHC) on pancreatic sections from wild-type (WT), prediabetic, and T2DM murine models. Additionally, bioinformatic analysis of publicly available RNA sequencing (RNA-Seq) data from human islets of healthy donors, prediabetic individuals, and T2DM patients provided translational validation. Results: High glucose conditions significantly increased PAK1-CAMKII colocalization, correlating with enhanced insulin secretion. Pharmacological inhibition of these kinases reduced insulin release, confirming their regulatory roles. Murine and human islet analyses showed a progressive increase in kinase expression from prediabetes to T2DM, highlighting their relevance in disease progression. Conclusions: The coordinated function of PAK1 and CaMKII in insulin secretion suggests their potential as biomarkers and therapeutic targets in T2DM. Further studies are warranted to explore their mechanistic roles and therapeutic applications in preserving beta-cell function. Full article

Leech therapy is a biotherapeutic approach that has been traditionally used for centuries and is currently being re-evaluated in modern medicine. The efficacy of this treatment is attributed to various bioactive compounds found in leech saliva, which exhibit anticoagulant, anti-inflammatory, antioxidant, and anticancer properties. It has been demonstrated that leech saliva possesses the potential to modulate inflammatory processes and apoptotic mechanisms. In this study, the therapeutic potential of the saliva of Hirudo verbana was evaluated, and its biological and pharmacological effects were comprehensively investigated. The anticancer effects, antioxidant capacity, and anti-inflammatory activity of the crude leech saliva were assessed using human umbilical vein endothelial cells and epithelial ovarian cancer cells. The chemical composition of the saliva was analyzed using gas chromatography–mass spectrometry, while the protein content was determined by the Bradford assay. Antioxidant activity was measured using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, inflammatory effects were evaluated by Enzyme-Linked ImmunoSorbent Assay, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The findings revealed that crude leech saliva had a minimal effect on healthy cells but showed a selective effect on the viability of ovarian cancer cells. At low concentrations (3.13%), 99.16% of healthy cells remained viable, whereas this rate decreased to 89.25% in cancer cells; at high concentrations (50%), cell viability in cancer cells declined to 63.02%. Gas chromatography–mass spectrometry analysis identified compounds such as gibberellic acid and 6-[(4-methoxyphenyl)methoxy]-4,4,5,7,8-pentamethyl-3H-chromen-2-one, which demonstrated high affinity for the antiapoptotic proteins Bcl-2 and Survivin in molecular docking analyses. In conclusion, the crude leech saliva was confirmed to possess anti-inflammatory, antioxidant, and anticancer properties. However, further biochemical and clinical research is needed to elucidate the underlying mechanisms of these biological effects in greater detail. Full article

Reservoirs and downstream rivers draining Oregon’s Cascade Range provide critical water supplies for over 1.5 million residents in dozens of communities. These waters also support planktonic and benthic cyanobacteria that produce cyanotoxins that may degrade water quality for drinking, recreation, aquatic life, and other beneficial uses. This 2016–2020 survey examined the sources and transport of four cyanotoxins—microcystins, cylindrospermopsins, anatoxins, and saxitoxins—in six river systems feeding 18 drinking water treatment plants (DWTPs) in northwestern Oregon. Benthic cyanobacteria, plankton net tows, and (or) Solid-Phase Adsorption Toxin Tracking (SPATT) samples were collected from 65 sites, including tributaries, reservoirs, main stems, and sites at or upstream from DWTPs. Concentrated extracts (320 samples) were analyzed with enzyme-linked immuno-sorbent assays (ELISA), resulting in >90% detection. Benthic cyanobacteria (n = 80) mostly Nostoc, Phormidium, Microcoleus, and Oscillatoria, yielded microcystins (76% detection), cylindrospermopsins (41%), anatoxins (45%), and saxitoxins (39%). Plankton net tow samples from tributaries and main stems (n = 94) contained saxitoxins (84%), microcystins (77%), anatoxins (25%), and cylindrospermopsins (22%), revealing their transport in seston. SPATT sampler extracts (n = 146) yielded anatoxins (81%), microcystins (66%), saxitoxins (37%), and cylindrospermopsins (32%), indicating their presence dissolved in the water. Reservoir plankton net tow samples (n = 15), most often containing Dolichospermum, yielded microcystins (87%), cylindrospermopsins (73%), and anatoxins (47%), but no saxitoxins. The high detection frequencies of cyanotoxins at sites upstream from DWTP intakes, and at sites popular for recreation, where salmon and steelhead continue to exist, highlight the need for additional study on these cyanobacteria and the factors that promote production of cyanotoxins to minimize effects on humans, aquatic ecosystems, and economies. Full article

The measurement of serum antibodies that specifically recognize self-antigens is a critical diagnostic in autoimmunity. A limitation of such an approach is sensitivity to detect the antibody, particularly when abundant self-antigens in the body may bind and sequester circulating specific antibodies. The presence of specific memory B cells (B_mem) may provide a more sensitive and robust indicator of an autoimmune response, as is suggested for certain anti-viral responses. B cell enzyme-linked ImmunoSpot (ELISPOT) is capable of detecting antigen-specific B_mem cells in blood at the single cell level, following stimulation of peripheral blood mononuclear cells (PBMCs) to expand and differentiate the B_mem cells into functional antibody-secreting cells (ASCs). While this assay has been widely utilized in infectious diseases and vaccination, detection is more difficult for autoantigens due to self-tolerance and specific tissue compartmentalization of immune responses, making autoantigen-specific B cells rare in the circulation. The cycles of re-activation of B_mem cells to become ASCs, that may reflect disease flare-ups in autoimmunity, are not well defined. For several autoimmune diseases (ADs), the targeting of B cells via depleting monoclonal antibodies has proven to be an effective treatment, where B_mem cells are likely being targeted. The measurement of autoantigen-reactive B_mem cells may aid in diagnosis and staging of clinical severity, or be a metric for efficacious treatments, thus providing an additional informative biomarker of ADs. How B cell ELISPOT has been utilized to characterize B_mem cells in human ADs is described here, including the advantages and disadvantages of the assay. Full article

Background: Allergic rhinitis (AR) is a common disease that requires more convenient, safe, and effective therapy. This study aimed to investigate the therapeutic effect of Forkhead box protein3 (Foxp3) introduced with poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (Foxp3 NPs) in an AR mouse model. Methods: A murine model of allergic rhinitis was established using BALB/c mice through initial sensitization by intraperitoneal administration of ovalbumin (OVA), followed by repeated intranasal OVA challenges. Foxp3 plasmid-loaded PLGA nanoparticles were subsequently administered via either the intranasal or intraperitoneal route to evaluate therapeutic efficacy. Episodes of sneezing and nose rubbing were counted. The serum total IgE, OVA-specific IgE, and cytokine levels in nasal lavage fluid (NALF) were determined by ELISA (Enzyme-Linked ImmunoSorbent Assay). Nasal mucosa from each group were analyzed using protein, reverse transcriptase–polymerase chain reaction (RT-PCR), and histological analyses. Result: Rubbing and sneezing symptoms improved in the Foxp3 NPs intranasal administration group. Foxp3 NPs intranasal administration markedly ameliorated OVA-induced nasal allergic inflammation. The total IgE and OVA-specific IgE serum level and IL-4, IL-13 expression levels of NALF were significantly decreased in the treated Foxp3 NPs group. The histopathological results of nasal mucosa were also normal, with no cellular infiltration and no inflammation in the Foxp3 NPs group. Conclusions: These results suggest that Foxp3 NPs alleviate nasal allergic inflammation and may have therapeutic value in the treatment of AR. Full article

The purpose of this study was to evaluate in vitro whether the type of tooth preservation before treatment with the Tooth Transformer^® (TT) device affects the osteoinductive characteristics of the extracted tooth. Forty extracted teeth from healthy non-smoking patients were selected. All teeth were cleaned of caries, tartar, and filling material and then roughly sectioned and divided into four experimental groups according to storage type: room-temperature (RT) tooth samples, frozen tooth samples, RIPA tooth samples, and fresh tooth samples. Each sample was minced, demineralized, and disinfected using the TT device. The Enzyme-Linked ImmunoSorbent Assay (ELISA) test revealed the presence of bone morphogenetic protein-2 (BMP-2) and collagen type-I (COL-I) in all of the samples, demonstrating that the fresh teeth retained the most significant amount of osteoinductive protein. In contrast, the tooth samples stored at room temperature (RT) showed the most important loss of BMP-2 and COL-I. A Western Blot analysis demonstrated the presence of the Mineralization Protein LIM-1 (LMP-1) and Transforming Growth Factor-β (TGF-β) in all of the dental samples analyzed. The fresh and frozen dental samples showed significantly higher levels of LMP-1 than those in the other samples. In contrast, the levels of TGF-β were similar in all of the dental samples examined, regardless of the type of storage. These experimental results suggest that an extracted tooth should be treated with the TT device as soon as possible to maximize its osteoinductive potential in surgical bone preservation and regeneration procedures. Full article

Biomolecular detection plays essential and irreplaceable roles in safeguarding human health, impeding the transmission of diseases, and augmenting the efficacy of treatments. The precise and specific identification of biomarkers holds profound significance for the early diagnosis, real-time surveillance, and targeted treatment of various diseases. In the initial phases of numerous diseases, the absence of distinct biomarkers in the bloodstream often leads to weak detection signals when using traditional immune detection methods such as enzyme-linked immunosorbent assays (ELISAs), chemiluminescence, and fluorescence chromatography. With the surge in research on surface plasmons, innovative approaches have recently emerged that combine surface plasmon resonance (SPR) with immunological detection techniques, reducing the detection sensitivity to 283 ag/mL, shrinking the sensor size to 2.228 µm², and shortening the detection time to 5.5 min. This review provides an overview of the theoretical foundations of surface plasmon resonance and immunoassays and then delves into the latest advancements in biosensors based on these principles, categorizing them according to their detection mechanisms and methodologies. Finally, we discuss future research directions, opportunities, and the challenges hindering the development of highly sensitive immuno-biochips. Full article

Background/Objectives: Feline coronavirus (FCoV) belongs to the family Coronaviridae and includes two pathotypes, the less virulent feline enteric coronavirus (FECV), which replicates in the enteric epithelial cells, and feline infectious peritonitis virus (FIPV), which is more virulent, replicates efficiently within monocytes/macrophages with systemic involvement and may cause feline infectious peritonitis (FIP), a progressive and often fatal disease. The diagnosis of FIP is complex and requires different examinations. Among serological tests, the indirect immunofluorescent antibody test (IFAT), considered the gold standard, and the enzyme-linked immunosorbent assay (ELISA) are the most widely used to detect FCoV antibodies. The aim of this work was the development of FCoVCHECK Ab ELISA, a new rapid indirect test for the detection of FCoV antibodies in feline serum/plasma samples. Methods: FCoVCHECK Ab ELISA was developed after a meticulous set-up and cut-off analysis through several methods, including the Youden’s index and ROC curve, to achieve the best test performance. It was validated by testing 110 feline sera (62 positives and 48 negatives) against the reference IFAT and compared with two other rapid ELISA tests, INgezim Corona Felino (Gold Standard Diagnostics) and ImmunoComb Feline Coronavirus (FCoV) [FIP] Antibody Test Kit (Biogal). Conclusions: FCoVCHECK Ab ELISA agreed with IFAT at 96.4% (93.5% sensitivity, 95% confidence interval (CI): 83.5–97.9%; 100% specificity, 95% CI: 90.8–100%), with ImmunoComb FCoV at 93.6% and with INgezim Corona Felino at 82.7%. Intra- and inter-assay accuracy and precision gave coefficients of variation lower than 20%. Compared to IFAT, the new assay correctly identifies positive and negative samples with a good correlation, and, in addition, it is simpler, faster and provides a less subjective reading of the results. Full article

Mesenchymal stem/stromal cells (MSCs) have been exploited as an experimental cell therapy in a broad array of clinical applications but have underperformed based on results from pre-clinical studies due to gaps in translating pre-clinical findings to human patients. Herein, we isolated mouse MSCs from pelvic bone marrow (BM^P), a preferred source for human MSCs, and compared their growth, differentiation, and immuno-modulatory activity to those derived from long bone marrow (BM^L), the traditional source of mouse MSCs. We report that BM^P-MSCs exhibit significantly enhanced growth kinetics in 5% and 21% oxygen saturation and superior bi-lineage differentiation and hematopoiesis-supporting activity as compared to BM^L-MSCs. Additionally, we show that TNF upregulates inducible nitric oxide synthase (NOS2) in BM^L- and BM^P- MSCs and augments their immune suppressive activity in cell-based assays, while interferon-gamma (INFG) upregulates indoleamine, 2-3, dioxygenase (IDO1) and enhances the immune suppressive activity of only BM^P-MSCs. These results indicate that mouse MSCs sourced from different bone compartments exhibit measurable differences in critical quality attributes, and these differences are comparable to those observed across species. Based on these differences, BM^P- MSCs represent a useful resource to model the behavior of human BM-derived MSCs. Full article

Determining an individual’s humoral immune reactivity to a pathogen, autoantigen, or environmental agent is traditionally accomplished through the assessment of specific antibody levels in blood. However, in many instances, titers of specific antibodies decline over time and thus do not faithfully reveal prior antigen exposure or establishment of immunological memory. To estimate an individual’s humoral immune competence, it is therefore necessary to assess functional B cell memory. Here, we describe novel B cell ELISPOT and FluoroSpot assays (collectively referred to as ImmunoSpot) that can be rapidly developed and validated to characterize the memory B cell (B_mem) repertoire specific for any desired antigen ex vivo and at single-cell resolution. Moreover, multiplexed variants of the B cell FluoroSpot assay enable high-throughput testing of antigen-specific B cells secreting distinct antibody classes and/or IgG subclasses, with minimal cell material requirements. B cell ImmunoSpot assays also enable measurement of affinity distributions within the antigen-specific B_mem compartment and permit cross-reactivity measurements that can provide insights into B_mem established against future pathogen variants. Collectively, the ImmunoSpot^® system presented here is highly reproducible, and can be readily validated for regulated tests. The newly gained ability to monitor the antigen-specific B_mem compartment should catalyze a more comprehensive understanding of humoral immunity in health and disease. Full article

Fumonisins, a class of mycotoxins predominantly produced by Fusarium species, represent a major threat to food safety and public health due to their widespread occurrence in staple crops including peanuts, wine, rice, sorghum, and mainly in maize and maize-based food and feed products. Although fumonisins occur in different groups, the fumonisin B series, particularly fumonisin B1 (FB1) and fumonisin B2 (FB2), are the most prevalent and toxic in this group of mycotoxins and are of public health significance due to the many debilitating human and animal diseases and mycotoxicosis they cause and their classification as by the International Agency for Research on Cancer (IARC) as a class 2B carcinogen (probable human carcinogen). This has made them one of the most regulated mycotoxins, with stringent regulatory limits on their levels in food and feeds destined for human and animal consumption, especially maize and maize-based products. Numerous countries have regulations on levels of fumonisins in foods and feeds that are intended to protect human and animal health. However, there are still gaps in knowledge, especially with regards to the molecular mechanisms underlying fumonisin-induced toxicity and their full impact on human health. Detection of fumonisins has been advanced through various methods, with immunological approaches such as Enzyme-Linked Immuno-Sorbent Assay (ELISA) and lateral flow immunoassays being widely used for their simplicity and adaptability. However, these methods face challenges such as cross-reactivity and matrix interference, necessitating the need for continued development of more sensitive and specific detection techniques. Chromatographic methods, including HPLC-FLD, are also employed in fumonisin analysis but require meticulous sample preparation and derivitization due to the low UV absorbance of fumonisins. This review provides a comprehensive overview of the fumonisin family, focusing on their biosynthesis, occurrence, toxicological effects, and levels of contamination found in foods and the factors affecting their presence. It also critically evaluates the current methods for fumonisin detection and quantification, including chromatographic techniques and immunological approaches such as ELISA and lateral flow immunoassays, highlighting the challenges associated with fumonisin detection in complex food matrices and emphasizing the need for more sensitive, rapid, and cost-effective detection methods. Full article

Background: A limited number of studies have investigated the role of environmental chemicals in the etiology of mild cognitive impairment (MCI). We performed a cross-sectional study of the association between exposure to selected trace elements and the biomarkers of cognitive decline. Methods: During 2019–2021, we recruited 128 newly diagnosed patients with MCI from two Neurology Clinics in Northern Italy, i.e., Modena and Reggio Emilia. At baseline, we measured serum and cerebrospinal fluid (CSF) concentrations of cadmium, copper, iron, manganese, and zinc using inductively coupled plasma mass spectrometry. With immuno-enzymatic assays, we estimated concentrations of β-amyloid 1-40, β-amyloid 1-42, Total Tau and phosphorylated Tau181 proteins, neurofilament light chain (NfL), and the mini-mental state examination (MMSE) to assess cognitive status. We used spline regression to explore the shape of the association between exposure and each endpoint, adjusted for age at diagnosis, educational attainment, MMSE, and sex. Results: In analyses between the serum and CSF concentrations of trace metals, we found monotonic positive correlations between copper and zinc, while an inverse association was observed for cadmium. Serum cadmium concentrations were inversely associated with amyloid ratio and positively associated with Tau proteins. Serum iron concentrations showed the opposite trend, while copper, manganese, and zinc displayed heterogeneous non-linear associations with amyloid ratio and Tau biomarkers. Regarding CSF exposure biomarkers, only cadmium consistently showed an inverse association with amyloid ratio, while iron was positively associated with Tau. Cadmium concentrations in CSF were not appreciably associated with serum NfL levels, while we observed an inverted U-shaped association with CSF NfL, similar to that observed for copper. In CSF, zinc was the only trace element positively associated with NfL at high concentrations. Conclusions: In this cross-sectional study, high serum cadmium concentrations were associated with selected biomarkers of cognitive impairment. Findings for the other trace elements were difficult to interpret, showing complex and inconsistent associations with the neurodegenerative endpoints examined. Full article