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Ragweed pollen allergy is the most common seasonal allergy in western Romania. Prolonged exposure to ragweed pollen may induce sensitization to pan-allergens such as calcium-binding proteins (polcalcins) and progression to more severe symptoms. We aimed to detect IgE sensitization to recombinant Amb a 9 and Amb a 10 in a Romanian population, to assess their potential clinical relevance and cross-reactivity, as well as to investigate the relation with clinical symptoms. rAmb a 9 and rAmb a 10 produced in Escherichia coli were used to detect specific IgE in sera from 87 clinically characterized ragweed-allergic patients in ELISA, for basophil activation experiments and rabbit immunization. Rabbit rAmb a 9- and rAmb a 10-specific sera were used to detect possible cross-reactivity with rArt v 5 and reactivity towards ragweed and mugwort pollen extracts. The results showed an IgE reactivity of 25% to rAmb a 9 and 35% to rAmb a 10. rAmb a 10 induced basophil degranulation in three out of four patients tested. Moreover, polcalcin-negative patients reported significantly more skin symptoms, whereas polcalcin-positive patients tended to report more respiratory symptoms. Furthermore, both rabbit antisera showed low reactivity towards extracts and showed high reactivity to rArt v 5, suggesting strong cross-reactivity. Our study indicated that recombinant ragweed polcalcins might be considered for molecular diagnosis.

Aristolochia sp. plants are used in traditional medicine because of their immunostimulatory and anticarcinogenic properties, despite their content of aristolochic acids (AAs), carcinogenic and nephrotoxic agents. Therefore, ethanolic extracts of Aristolochia clematitis leaves, a specie growing in Western Romania, were obtained to study antioxidant and cytotoxic effects. The antioxidant capacity of the extract was evaluated by five in vitro chemical-based assays, proving that ABTS assay was a better method for this type of evaluation showing an IC50 of 160.89 ± 0.21 µg/mL. Furthermore, the cytotoxic effects of the extract were established by an IC50 of 216 µg/mL for 24 h by MTT assay, followed by a cell-based assay on Caco-2 cells by the ABTS method. The antioxidant effects of the A. clematitis extract demonstrate potential therapeutic applications in complementary medicine.

Currently, allergen-specific immunotherapy (AIT) for ragweed allergy is still based on natural allergen extracts. This study aimed to analyse the ability of four commercially available AIT vaccines (CLUSTOID, TYRO-SIT, POLLINEX Quattro Plus and Diater Depot) regarding their ability to induce IgG antibodies against ragweed pollen allergens in rabbits. Accordingly, the IgG reactivity of AIT-induced rabbit sera was tested for ten different ragweed pollen allergens (Amb a 1, 3, 4, 5, 6, 8, 9, 10, 11 and 12) by an ELISA. Furthermore, the ability of rabbit AIT-specific sera to block allergic patients’ IgE binding to relevant ragweed allergens (Amb a 1, 4, 6, 8 and 11) and to inhibit allergen-induced basophil activation was evaluated by an IgE inhibition ELISA and a mediator release assay. Only two AIT vaccines (Diater Depot > CLUSTOID) induced relevant IgG antibody levels to the major ragweed allergen Amb a 1. The IgG responses induced by the AIT vaccines against the other ragweed allergens were low and highly heterogeneous. Interestingly, the kinetics of IgG responses were different among the AIT vaccines and even within one AIT vaccine (Diater Depot) for Amb a 1 (long-lasting) versus Amb a 8 and Amb a 11 (short-lived). This could be due to variations in allergen contents, the immunogenicity of the allergens, and different immunization protocols. The IgE inhibition experiments showed that rabbit AIT-specific sera containing high allergen-specific IgG levels were able to inhibit patients’ IgE binding and prevent the mediator release with Diater Depot. The high levels of allergen-specific IgG levels were associated with their ability to prevent the recognition of allergens by patients’ IgE and allergen-induced basophil activation, indicating that the measurement of allergen-induced IgG could be a useful surrogate marker for the immunological efficacy of vaccines. Accordingly, the results of our study may be helpful for the selection of personalized AIT vaccination strategies for ragweed-allergic patients.

Background and Aim: Human papillomavirus types 16 and 18 cause most cervical cancers worldwide. Existing VLP-based vaccines are unable to eliminate ongoing highly oncogenic infections. This study suggests an SLP-based vaccine for already infected patients, even with precancerous lesions. Methods: HPV16/18 E6 and E7 proteins' potent HLA class I and class II-restricted epitopes were extracted from IEDB or predicted by ANNs (IC50 < 50 nM, percentile rank < 2%). Population coverage was assessed via HLA-SLP interactions. SLP constructs, containing specific epitopes linked by cleavable peptides (LMRK, LLSVGG), underwent rigorous screening for allergenicity, toxicity, physicochemical parameters, antigenicity, and diversity. 3D structure prediction employed homology and ab initio modeling and was validated via QMEAN4 score and Ramachandran plots. Molecular docking (HADDOCK-2.4) evaluated SLP-TLR2 interactions. MD studies (GROMACS-2023.1) investigated SLP behavior in an electroneutral aqueous environment (300 K, 1 atm, 100 ns) via RMSD, RMSF, SASA, and Rgyr dynamics. Results: 28 class I and 16 class II-restricted epitopes had an IC50 < 50 nM and provided population coverages of 98.18% (class I) and 99.81% (class II). The selected 25 SLPs expressed a mean 1.5×10−2 toxicity score, no allergenic properties, a mean instability index of 37.61, and a mean VaxiJen score of 0.97. The QMEAN4 score ([0.665;0.813]), with >90% of the residues in the most favorable regions reflect good quality 3D structures. Molecular docking studies provided a ΔG of −13.20±1.42 kcal/mol, and an average Kd of 3.5×10−9 M. MD simulations reflect favorable thermodynamic properties, based on mild RMSD ([0.1;0.87] nm), RMSF, SASA (49.37±1.54 nm2) and Rgyr ([1.15;1.34]) fluctuations over the 100-ns simulations. Conclusions: 25 in silico-designed SLPs stimulate both innate and adaptive immunity, with little to no toxic or allergenic effects. The presented vaccination platform could exert both a therapeutic (by tumor cell clearance) and a prophylactic effect (by eradicating E6 and E7+ premalignant cells).

Three-dimensional (3D) bioprinting is the use of computer-controlled transfer processes for assembling bioinks (cell clusters or materials loaded with cells) into structures of prescribed 3D organization. The correct bioprinting parameters ensure a fast and accurate bioink deposition without exposing the cells to harsh conditions. This study seeks to optimize pneumatic extrusion-based bioprinting based on hydrogel flow rate and extrusion speed measurements. We measured the rate of the hydrogel flow through a cylindrical nozzle and used non-Newtonian hydrodynamics to fit the results. From the videos of free-hanging hydrogel strands delivered from a stationary print head, we inferred the extrusion speed, defined as the speed of advancement of newly formed strands. Then, we relied on volume conservation to evaluate the extrudate swell ratio. The theoretical analysis enabled us to compute the extrusion speed for pressures not tested experimentally as well as the printing speed needed to deposit hydrogel filaments of a given diameter. Finally, the proposed methodology was tested experimentally by analyzing the morphology of triple-layered square-grid hydrogel constructs printed at various applied pressures while the printing speeds matched the corresponding extrusion speeds. Taken together, the results of this study suggest that preliminary measurements and theoretical analyses can simplify the search for the optimal bioprinting parameters.

Cardiac pathological hypertrophy is the major risk factor that usually progresses to heart failure. We hypothesized that extracellular vesicles (EVs), known to act as important mediators in regulating physiological and pathological functions, could have the potential to reduce the cardiac hypertrophy and the ensuing cardiovascular diseases. Herein, the effects of mesenchymal stem cell-derived extracellular vesicles (EV-MSCs) on cardiac hypertrophy were investigated. EVs were isolated from the secretome of human adipose tissue-derived stem cells (EV-ADSCs) or bone marrow-derived stem cells (EV-BMMSCs). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were stimulated with AngII and TGF-β1, in absence or presence of EVs. The results showed that exposure of hiPSC-CMs to AngII and TGF-β1 generated in vitro model of hypertrophic cardiomyocytes characterized by increases in surface area, reactive oxygen species production, protein expression of cardiac-specific biomarkers atrial natriuretic factor, migration inhibitory factor, cTnI, COL1A1, Cx43, α-SMA and signalling molecules SMAD2 and NF-kBp50. The presence of EV-ADSCs or EV-BMMSCs in the hiPSC-CM culture along with hypertrophic stimuli reduced the protein expressions of hypertrophic specific markers (ANF, MIF, cTnI, COL1A1) and the gene expressions of IL-6 molecule involved in inflammatory process associated with cardiac hypertrophy and transcription factors SMAD2, SMAD3, cJUN, cFOS with role in cardiomyocyte hypertrophic response induced by AngII and TGF-β1. The EV-ADSCs were more effective in reducing the protein expressions of hypertrophic and inflammatory markers, while EV-BMMSCs in reducing the gene expressions of transcription factors. Notably, neither EV-ADSCs nor EV-BMMSCs induced significant changes in cardiac biomarkers Cx43, α-SMA and fibronectin. These different effects of stem cell-derived EVs could be attributed to their miRNA content: some miRNAs (miR-126-3p, miR-222-3p, miR-30e-5p, miR-181b-5p, miR-124-3p, miR-155-5p, miR-210-3p hsa-miR-221-3p) were expressed in both types of EVs and others only in EV-ADSCs (miR-181a-5p, miR-185-5p, miR-21-5p) or in EV-BMMSCs (miR-143-3p, miR-146a-5p, miR-93-5p), some of these attenuating the cardiac hypertrophy while others enhance it. In conclusion, in hiPSC-CMs the stem cell-derived EVs through their cargo reduced the expression of hypertrophic specific markers and molecules involved in inflammatory process associated with cardiac hypertrophy. The data suggest the EV potential to act as therapeutic mediators to reduce cardiac hypertrophy and possibly the subsequent cardiovascular events.

Abstract Background Ragweed (Ambrosia artemisiifolia) is one of the most important allergen sources, worldwide, causing severe respiratory allergic reactions in late summer and fall, in sensitized patients. Amb a 1 has been considered as the most important allergen in ragweed but 12 ragweed pollen allergens are known. The aim of our study was to investigate IgE reactivity profiles of ragweed allergic patients and to associate them with clinical symptoms. Methods IgE sensitization profiles from clinically well‐characterized ragweed allergic patients (n = 150) were analyzed using immunoblotted ragweed pollen extract. Immunoblot inhibition experiments were performed with two Amb a 1 isoforms and CCD markers and basophil activation experiments were performed with IgE serum before and after depletion of Amb a 1‐specific IgE. Results By IgE‐immunoblotting 19 different IgE reactivity patterns with and without Amb a 1‐sensitization were found. The majority of patients (>95%) suffered from rhino‐conjunctivitis, around 60% reported asthma‐like symptoms and about 25% had skin reactions. Patients with complex IgE sensitization profiles tended to have more clinical symptoms. Serum with and without Amb a 1‐specific IgE induced basophil activation. Conclusions Ragweed pollen allergic patients exhibit complex IgE reactivity profiles to ragweed allergens including Amb a 1 isoforms and cross‐reactive carbohydrates indicating the importance of Amb a 1 isoforms and additional allergens for diagnosis and allergen‐specific immunotherapy of ragweed allergy.

Human papillomavirus types 16 and 18 cause the majority of cervical cancers worldwide. Despite the availability of three prophylactic vaccines based on virus-like particles (VLP) of the major capsid protein (L1), these vaccines are unable to clear an existing infection. Such infected persons experience an increased risk of neoplastic transformation. To overcome this problem, this study proposes an alternative synthetic long peptide (SLP)-based vaccine for persons already infected, including those with precancerous lesions. This new vaccine was designed to stimulate both CD8+ and CD4+ T cells, providing a robust and long-lasting immune response. The SLP construct includes both HLA class I- and class II-restricted epitopes, identified from IEDB or predicted using NetMHCPan and NetMHCIIPan. None of the SLPs were allergenic nor toxic, based on in silico studies. Population coverage studies provided 98.18% coverage for class I epitopes and 99.81% coverage for class II peptides in the IEDB world population’s allele set. Three-dimensional structure ab initio prediction using Rosetta provided good quality models, which were assessed using PROCHECK and QMEAN4. Molecular docking with toll-like receptor 2 identified potential intrinsic TLR2 agonist activity, while molecular dynamics studies of SLPs in water suggested good stability, with favorable thermodynamic properties.

Metabolomic analysis methods were employed to determine biomarkers for various chronic kidney diseases (CKDs). Modern analytical methods were developed and applied successfully to find a specific metabolomic profile in urine samples from CKD and Balkan endemic nephropathy (BEN) patients. The aim was to explore a specific metabolomic profile defined by feasible/easy-to-identify molecular markers. Urine samples were collected from patients with CKDs and BEN, and from healthy subjects from endemic and nonendemic areas in Romania. Metabolomic analysis of urine samples, extracted by the liquid-liquid extraction (LLE) method, was performed by gas chromatography-mass spectrometry (GC-MS). The statistical exploration of the results was performed through a principal component analysis (PCA) evaluation. Urine samples were statistically analyzed using a classification based on six types of metabolites. Most urinary metabolites are distributed in the center of a loading plot, meaning that these compounds do not represent significant markers for BEN. One of the most frequent and higher-concentration urinary metabolites in BEN patients was p-Cresol, a phenolic compound that implies a severe injury of the renal filtration function. The presence of p-Cresol was associated with protein-bound uremic toxins, which have specific functional groups such as indole and phenyl. In prospective studies for future investigation, prevention, and disease treatment, we suggest a larger sample size, sample extraction using other methods, and analysis using other chromatography techniques coupled with mass spectrometry, which can generate a more significant data set for statistical analysis.

Ragweed pollen is highly allergenic and elicits type I hypersensitivity reactions in the exposed populations. Amb a 11 is a recently discovered component of this pollen, and its biological role in allergy is still being researched. In our study, ragweed allergy patients were recruited prospectively over a three-year period; a comprehensive questionnaire was administered, and sera were collected and stored. The production of recombinant Amb a 11 was achieved in parallel with patients’ recruitment. The gene coding for mature protein was inserted in E. coli and in Sf9 Spodoptera frugiperda cells. The recombinant allergens (designated eAmb a 11 and iAmb a 11) were tested for His-tag presence in Western blot. IgE reactivity was evaluated in 150 patients’ sera for both recombinant allergen forms in ELISA, with 5 positive sera being tested further by hRBL (humanized rat basophilic leukemia) hexosaminidase release assay. Both allergen forms were proven to be IgE-reactive His-tagged proteins, with an extensive overlap of positive sera (92 toward the former recombinant allergen, 100 toward the latter) and an overall Amb a 11 sensitization prevalence estimated at 68.67%. The hRBL mediator release assay revealed a significant, slightly weaker effect of recombinant allergens when compared with nAmb a 1. Sensitization to this major allergen appears to be associated with more severe asthma symptoms (OR = 4.71, 95% CI = 1.81–12.21). In conclusion, recombinant Amb a 11 is a bona fide allergen, which is IgE-reactive and an inducer of hRBL degranulation. It is an important IgE-reactive component from ragweed pollen, with high IgE sensitization prevalence in the sample population and allergenicity of the recombinant allergen comparable to Amb a 1.

Cancer is an increasingly health problem worldwide, and new diagnostic methods are developing each year. Next generation sequencing (NGS) represents already a state-of-the-art method in this field, while mutation-based therapies are standard-of-care in many countries for cancer treatment. We present the methods and results that can be obtained using an NGS platform from IonTorrent, while employing a limited panel of genes (HotSpot Panel) and appropriate software for results analysis. This method is very fast (2-3 days) and requires low amout of DNA as input material. The analysis reports using either IonReporter or Oncomine Reporter software can give the clinicians information related to mutations localisation, type of mutations (synonymus, missense), mutation-targeted therapies and ongoing clinical trials, which could be of real bennefit for the oncologic patients. [ABSTRACT FROM AUTHOR]