Design of an Epitope-Based Peptide Vaccine Against the Major Allergen Amb a 11 Using Immunoinformatic Approaches.

Allergic diseases are a socially significant problem of global importance. The number of people suffering from pollen allergies has increased dramatically in recent decades. Pollen allergies affect up to 30% of the world population. Pollen of the common ragweed (Ambrosia artemisiifolia L.) is one of the most aggressive allergens in the world. We have used a series of immunoinformatics approaches to design an effective epitope-based vaccine, which might induce a competent immunity against a major allergen Amb a 11. CD8+ and CD4+ T-cell epitopes and their corresponding MHC restricted alleles were identified by prediction tools provided by immune epitope database (IEDB). Among T-cell epitopes, MHC class I peptide (GLMEPAFTYV) and MHC class II peptide (LVCFSFSLVLILGLV) were identified as most suitable. From all predicted B-cell epitopes, only one epitope (GKLVKFSEQQLVDC) containing sequence from the conserved region was chosen for next processing. Selected epitopes have been validated by molecular docking analysis. These epitopes showed a very strong binding affinity to MHC I molecule and MHC II molecule with binding energy scores - 729.3 and - 725.0 kcal/mole respectively. Performed experimental validation showed that only the MHC class II peptide (LVCFSFSLVLILGLV) can stimulate T cells from ragweed allergic patients and IgE antibodies specific to the ragweed pollen do not recognize this epitope. Therefore, this peptide could be potentially used as a vaccine against the major allergen Amb a 11. The B-cell epitope GKLVKFSEQQLVDC forms a stable complex with the IgE molecule (energy weighted score - 695,0 kcal/mole). Tested sera from patients with ragweed allergy showed that the ragweed specific IgE antibodies can bind to the identified B-cell epitope. Population coverage analysis was performed for CD8+ and CD4+ T-cell epitopes. It was predicted that CD4+ T-cell epitope (LVCFSFSLVLILGLV) covers 90.56% of the population of Europe and 99.36% of the world population. CD8+ T-cell epitope (GLMEPAFTYV) has a population coverage of 77.37% for Europe and 71.35% for all the world.

Disease-Modifying Potential of Metformin and Alendronate in an Experimental Mouse Model of Osteoarthritis.

Osteoarthritis (OA) is the most common degenerative joint disease causing progressive damages of the cartilage and subchondral bone, synovial inflammation, and severe pain. Despite the complex pathomorphological changes that occur in OA, the approach to different forms of OA is standardized. The global results from pharmacological treatment are not satisfactory. Hence, this study aimed to explore the effects of metformin, alendronate, and their combination on OA development and progression in mice with collagenase-induced osteoarthritis (CIOA). Female ICR (CD-2) mice were randomized to five groups: control group, CIOA untreated, CIOA + metformin, CIOA + alendronate, and CIOA + metformin + alendronate. OA was induced by the intra-articular (i.a.) injection of collagenase. OA phenotype was analyzed by flow cytometry (bone marrow cell differentiation), ELISA (serum levels of the adipokines leptin and resistin), and histology (pathological changes of the knee joint). Treatment with metformin, alendronate, or their combination inhibited the expression of RANK and RANKL on osteoblasts and osteoclasts obtained by ex vivo cultivation of bone marrow cells in mineralization or osteoclastogenic media. In addition, metformin treatment was effective for the attenuation of fibroblast differentiation, but not of mesenchymal stem cells (MSCs), while alendronate had an opposite effect. The combination of metformin and alendronate had a suppressive effect on both MSCs and fibroblasts differentiation. Treatment with metformin, alendronate, and their combination decreased serum concentrations of leptin and resistin in the chronic phase of arthritis. The histopathological examination showed that compared with the untreated CIOA group (OA score 9), the groups treated with metformin (OA score 4) or alendronate (OA score 6) had lower scores for cartilage changes. Metformin combined with alendronate significantly decreased the degree of cartilage degeneration (OA score 2), suggesting that this combination might be a useful approach for the treatment of OA patients.

Serum Leptin and Resistin Levels in Knee Osteoarthritis-Clinical and Radiologic Links: Towards Precise Definition of Metabolic Type Knee Osteoarthritis.

Obesity is considered a major risk factor for the development and progression of knee osteoarthritis (OA). Apart from the mechanical effect of obesity via increase in mechanical overload of weight-bearing joints, an association with hand OA has been observed. There has been increasing interest in the role of adipokines in the pathogenesis of OA in the recent years. It has been suggested that their systemic effects link obesity and OA. In this regard, the aim of the current study was measurement and analysis of serum levels of leptin and resistin in patients with knee OA with different body mass index (BMI). Seventy-three patients with primary symptomatic knee OA at the age between 35 and 87 years (mean age 66 years) were included in the study (67 women and 6 men). The patients were from 2nd to 4th radiographic stage according to Kellgren-Lawrence scale. 43 patients were with concomitant obesity (BMI ≥ 30 kg/m2, mean values 38.34 ± 8.20) and 30 patients with BMI < 30 kg/m2 (mean values 25.07 ± 2.95). Eleven individuals with different BMIs, including cases with obesity but without radiographic knee OA, were examined as a control group. Serum levels of leptin and resistin were measured via ELISA method. In patients with knee OA and BMI ≥ 30 kg/m2, serum levels of leptin (39.546 ± 12.918 ng/mL) were significantly higher as compared with healthy individuals (15.832 ± 16.531 ng/mL, p < 0.05) and the patients with low BMI (p < 0.05). In patients with BMI < 30 kg/m2 the levels of leptin (13.010 ± 10.94 ng/mL) did not differ significantly from the respective values in the control group (p = 0.48). Serum levels of resistin were also higher in knee OA patients in comparison with healthy controls, but the difference was statistically significant only for patients with high BMI (2.452 ± 1.002 ng/mL in the group with BMI ≥ 30 kg/m2; 2.401 ± 1.441 ng/mL in patients with BMI < 30 kg/m2; 1.610 ± 1.001 ng/mL in the control group, p < 0.05). A correlation was found between the serum levels of leptin and radiographic stage of OA, i.e., higher leptin levels were present in the more advanced 3rd and 4th radiographic stage, while for resistin a correlation was observed in the patient subgroup with BMI < 30 kg/m2. Serum leptin and resistin levels and clinical characteristics were analyzed in patients with different clinical forms of OA. Novel clinical correlations have been found in the current study in patients with isolated knee OA vs. cases with presence of other disease localizations. It has been observed that patients with isolated knee OA were significantly younger and had higher BMI as compared with cases in whom OA is combined with other localizations i.e., spondyloarthritis ± presence of hip OA and with generalized OA. This supports the hypothesis that presence of obesity promotes earlier development of knee OA as an isolated localization of the disease in younger patients before appearance of osteoarthritic changes at other sites. The levels of leptin and resistin in isolated knee OA were also higher. Serum levels of leptin and resistin in combination with patients' clinical characteristics suggest existence of different clinical and laboratory profile through which more precise definition of metabolic phenotype of knee OA would be possible. Considering the fact that obesity is a modifiable risk factor that has an impact on progression of knee OA, different approaches to influence obesity may offer potential for future disease-modifying therapeutic interventions.

Selective Anticancer Properties, Proapoptotic and Antibacterial Potential of Three Asplenium Species.

The ferns Asplenium ceterach L., Asplenium scolopendrium L. and Asplenium trichomanes L. have wide application in traditional medicine worldwide. However, the scientific research on their anticancer and antibacterial properties is insufficient. The present article aims to provide more information on this topic. Extracts derived from the aerial parts of A. ceterach, A. scolopendrium and A. trichomanes were examined using a panel of in vitro assays with different bacterial and mammalian cells. The cytotoxicity and anticancer activity of the samples were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays with three human (A549, FL, HeLa) and three murine (3T3, TIB-71, LS48) cell lines. Inhibitory effects on the growth of Gram-positive (Bacillus cereus) and Gram-negative (Pseudomonas aeruginosa) bacteria were determined by the agar diffusion assay. Apoptosis-inducing properties of the extracts were analyzed by flow cytometry. Superoxide dismutase (SOD) activity in extract-treated cells was investigated by ELISA. The obtained results demonstrate selective anticancer activity of all three Asplenium species. The extract from A. ceterach displayed the strongest inhibitory properties against human cervical cancer cells and bacterial cells. It induced a lower level of cytotoxicity against mouse cell lines, indicating a species-specific effect. The extract from A. trichomanes demonstrated better anticancer and antibacterial properties than the sample from A. scolopendrium. Further experiments linked the mechanism of action of A. ceterach extract with oxidative stress-inducing potential and strong proapoptotic potential against the cervical cancer cell line HeLa. A. trichomanes and A. scolopendrium extracts appeared to be potent inducers of necrotic cell death.

Tight Junction Protein Claudin-12 Is Involved in Cell Migration during Metastasis.

Claudins are important components of the tight junctions determining barrier properties, cell polarity, and paracellular permeability. Although many functions of claudins in cancer cells have not been elucidated, recent studies have shown that claudins play an important role in cell migration and metastasis. Loss of epithelial/endothelial integrity, disruption of tight junctions, and increased paracellular leakage are often observed during metastasis. The aim of our study was to investigate the involvement of claudin-12 in the process of cell migration as well as to evaluate the possibility of using this protein as a specific target for the regulation of tumorigenesis. We have performed immunocytochemistry assays to detect the expression of claudin-12 in different epithelial/endothelial human cell lines, and selected three (A549, LS180, and HeLa) for further experiments. Using transwell chamber migration assays, we found that anti-claudin-12 antibodies inhibited both the migration and proliferation of claudin-12 expressing cells (A549 and LS180), inducing apoptosis, as well as the migration capacity of Jurkat cells through the monolayers formed from A549 or LS180 cells. In addition, co-cultures of Jurkat cells on monolayers from A549 or LS180 cells, in the presence of synthetic claudin-12 peptides representing the extracellular domains of the claudin-12 protein, also reduced the number of migrated Jurkat cells. Two of the tested peptides (p5 and p6) almost completely blocked the migration of Jurkat cells. All migrated Jurkat cells expressed LFA-1 and CD62L, but not CD44. Thus, claudin-12 is a suitable biomarker for tumor progression and metastasis and an attractive target for antitumor therapy. Anti-claudin-12 antibodies and competitive inhibitory peptides could be useful in the therapeutic approach applied to cancer metastasis in tissues expressing claudin-12.