Methods: In this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA.
Results: In silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls.
Discussion: In silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.
METHODS: C. nutans leaves were subjected to methanol extraction and divided into two different concentrations, 200 mg/kg (low-dose) and 1000 mg/kg (high-dose). The antitumor effects of C. nutans extracts were assessed using bone marrow smearing, clonogenic, and splenocyte immunotype analyses. In addition, hematoxylin and eosin, tumor weight and tumor volume profiles also used to indicate apoptosis appearance. Serum cytokine levels were examined using ELISA assay. In addition, nitric oxide assay reflecting antioxidant activity was performed.
RESULTS: From the results obtained, the methanol extract of C. nutans leaves at 200 mg/kg (P C. nutans extract (1000 mg/kg) also showed a significant decrease in the number of mitotic cells, tumor weight, and tumor volume. No inflammatory and adverse reactions related to splenocytes activities were found in all treated groups of mice. Despite its promising results, the concentration of both C. nutans extracts have also reduced the number of colonies formed in the liver and lungs.
CONCLUSION: In conclusion, C. nutans extracts exert antitumor and antioxidant activities against 4 T1 mouse breast model with no adverse effect and inflammatory response at high dose of 1000 mg/kg, indicating an effective and complementary approach for cancer prevention and treatment.
METHODS: In this study, the anti-inflammatory effect of the NESTE aqueous extract and raw soybean aqueous extract (SBE) were evaluated by quantifying the inhibition of IL-1β, TNF-α and nitric oxide (NO) secretion in LPS treated RAW 264.7 cell in vitro. On the other hand, in vivo oral acute toxicity effect of the extract was tested on mice at the dose of 5000 mg/kg body weight. In vivo oral analgesic effect of both aqueous extracts at 200 and 1000 mg/kg body weight was evaluated by the hot plate test.
RESULTS: In the in vitro anti-inflammatory study, 5 mg/mL NESTE was able to inhibit 25.50 ± 2.20%, 35.88 ± 3.20% and 28.50 ± 3.50% of NO, IL-1β and TNF-α production in LPS treated RAW 264.7 cells without inducing cytotoxic effect on the cells. However, this effect was lower than 4 μg/mL of curcumin, which inhibited NO, IL-1β and TNF-α production by 89.50 ± 5.00%, 78.80 ± 6.20% and 87.30 ± 4.00%, respectively. In addition, 1.5 to 2.5-fold increase of latency period up to 120 min for mice in the hot plate test was achieved by 1000 mg/kg NESTE. The analgesic effect of NESTE was better than 400 mg/kg of acetyl salicylic acid, which only increased ~ 1.7-fold of latency period up to 90 min. Moreover, NESTE did not show acute toxicity (no LD50) up to 5000 mg/kg body weight.
CONCLUSION: NESTE is a nutritious food ingredient with potential anti-inflammatory and analgesic effects.
METHODS: Two hybridoma clones (Aa3c10, b10c1) were used for the production of ascites in BALB/c mice. Purification of monoclonal antibodies from the ascites was carried out using affinity columns. The thermal stability study of monoclonal antibodies was done by storing it at 37°C and 45°C for thirty days. The stored antibodies were analyzed using SDS-PAGE and flow-through device where the antigenantibody interaction was visualized by Protein A colloidal gold solution. Sensitivity was determined by endpoint dilution ELISA and the dissociation constant by competitive ELISA. Sensitive pair optimization was done by sandwich ELISA using biotinylated antibodies. Prototype preparation for lateral flow assay had a colloidal gold-based detection system.
RESULTS: Thermal stability experiments showed that both mAbs (Aa3c10; b10c1) are stable up to thirty days at 45°C while the commercially available mAbs were stable up to fifteen days only. Compared to commercial antibodies, the mAb Aa3c10, showed the highest sensitivity in end-point titre. In sensitive pair optimization, it was observed that the mAb, b10c1, as a detector and the mAb, Aa3c10, as a capture antibody showed the highest absorbance to detect 50pg/ml PfHRP2 antigen. The prototype formulation of lateral flow assay using the mAbs (Aa3c10; b10c1) showed good reactivity with WHO panel and no false-positive results were observed with twenty clinically negative samples and five P. vivax positive samples.
CONCLUSIONS: The novel monoclonal antibodies (Aa3c10, b10c1) against truncated PfHRP2, could be a strong potential candidates that can be included in making RDTs with better sensitivity and stability.