Characterization of M1A2 monoclonal antibody and in vitro cytotoxicity assessment

Despite all the big achievements in diagnosis and clinical advances, cancer as a life threatening illness, remains a problematic issue. Clinical successes of monoclonal antibodies confirm the capacity of immunotherapeutics to amend the demands in cancer treatment. To find a potential therapeutic and diagnostic product, several hybridoma clones were established earlier by fusion of lymphocytes from BALB/c mice sensitized with MCF7 breast carcinoma cell line and Sp20/0-Ag 14 myeloma cells. M1A2 is one of the stable hybridoma clones producing an IgM monoclonal antibody with < light chain. In this study, the M1A2 hybridoma clone was recloned by limiting dilution technique and the monoclonal antibody reactivity was screened against MCF7 and HT29 cell lines using cell-ELISA. High producer hybridoma clone was selected and the monoclonal antibody was produced in culture media (in vitro) and in ascitic fluid of peritoneal cavity of pristine primed BALB/c mice. Then the antibody was purified by an affinity chromatography on an F PLC system. The purified monoclonal antibody was characterized by immunological experiments such as immunofluorescence assay and immunohistochemistry. Then the target antigen was identified using protein techniques such as gel electrophoresis, immunoblotting and MALDI TOF/TOF mass spectrometry. Finally, the cytotoxicity potential of antibody was examined by MTT assay and complement-dependent cytotoxicity as well as antibody-dependent cellular cytotoxicity experiments. The results displayed the reactivity of M1A2 monoclonal antibody against tested human, mouse and rabbit cell lines in cell-ELISA technique. The reactivity results were further confirmed by immunofluorescence assay, which illustrated FITClabelling in cells’ cytoplasm. Immunohistochemical studies also revealed the positive staining of both human normal and cancerous tissues with M1A2 mAb with positive nuclear staining in less-differentiated carcinomas and cytoplasmic in welldifferentiated cancerous as well as normal breast, colon and ovary tissues. The flow cytometry analysis also verified the reactivity of M1A2 mAb toward both normal and cancerous cell lines. The HT29 cell line showed the highest percentage of stained cells with 90.07±1.15% followed by PBMC, HeLa and MCF7 with 88.7±0.35%, 77.3±2.66% and 52±0.76%, positive staining respectively measured by flow cytometry. Additional experiments were performed to identify M1A2 mAb target protein. A 65 kDa protein band was recognized on PVDF membrane in western blot experiment and a protein with a same molecular weight was immunoprecipitated from HT29 whole cell lysate by the M1A2 mAb. Then the m target protein was identified using MALDI TOF/TOF mass spectrometry and data mining suggested this protein belongs to heat shock protein family named Hsp60. The antibody also displays in vitro cytotoxicity toward MCF7 and PBMC with an approximate IC50 value of 403 μg/ mL. These findings support the introduction of M1A2 mAb as a new monoclonal antibody that recognize Hsp60 protein. With regard to the importance of heat shock proteins especially Hsp60 in new cancer research studies, the M1A2 mAb has the potential to develop as diagnostic and or therapeutic monoclonal antibody in future works

Modulation of Immune Function in Rats Using Oligosaccharides Extracted from Palm Kernel Cake

To investigate the prebiotic and immunomodulatory effects of PKC extract (OligoPKC) a total of 24 male rats were randomly assigned to three treatment groups receiving basal diet (control), basal diet containing 0.5% OligoPKC, or basal diet containing 1% OligoPKC for four weeks. We found that OligoPKC had no significant effect on the tested growth parameters. However, it increased the size of the total and beneficial bacterial populations while reducing pathogen populations. OligoPKC increased the concentration of immunoglobulins in the serum and cecal contents of rats. It also enhanced the antioxidant capacity of the liver while reducing lipid peroxidation in liver tissue. OligoPKC affected the expression of genes involved in immune system function in the intestine. Therefore, OligoPKC could be considered a potential mannan-based prebiotic for humans and animals due to its beneficial effects on the health and well-being of the model rats

Antiproliferation effects and antioxidant activity of two new Lactobacillus strains

<div><p>Abstract The microorganisms most commonly used as probiotics are lactic acid bacteria, especially those of the genus Lactobacillus. In the present study, two Lactobacillus strains, L. pentosus ITA23 and L. acidipiscis ITA44, previously isolated from mulberry silage, were characterized for their antiproliferative and antioxidant activities. The antiproliferative effects of the strains were investigated using the MTT assay with breast cancer (MDA-MB-231), liver cancer (HepG2) and normal liver (Chang) cell lines. The strains were tested for their antioxidant activity using the FRAP and ABTS methods. The results showed that the two Lactobacillus strains had good antiproliferative effects against both cancer cell lines tested, while their effects on the normal cells were weak. Based on the results of the antioxidant tests, the intact cells and cell-free extracts of the two Lactobacillus strains showed more than 135 and less than 50 µg trolox/ml of antioxidant activity, respectively. Lactobacillus pentosus ITA23 and L. acidipiscis ITA44 can be considered as potential probiotic candidates for humans because of their antioxidant activity and antiproliferation effects against cancer cells.</p></div

In vitro assessment of Pediococcus acidilactici Kp10 for its potential use in the food industry

Background: Selection of a microbial strain for the incorporation into food products requires in vitro and in vivo evaluations. A bacteriocin-producing lactic acid bacterium (LAB), Pediococcus acidilactici Kp10, isolated from a traditional dried curd was assessed in vitro for its beneficial properties as a potential probiotic and starter culture. The inhibitory spectra of the bacterial strain against different gram-positive and gram-negative bacteria, its cell surface hydrophobicity and resistance to phenol, its haemolytic, amylolytic and proteolytic activities, ability to produce acid and coagulate milk together with its enzymatic characteristics and adhesion property were all evaluated in vitro. Results: P. acidilactici Kp10 was moderately tolerant to phenol and adhere to mammalian epithelial cells (Vero cells and ileal mucosal epithelium). The bacterium also exhibited antimicrobial activity against several gram-positive and gram-negative food-spoilage and food-borne pathogens such as Listeria monocytgenes ATCC 15313, Salmonella enterica ATCC 13311, Shigella sonnei ATCC 9290, Klebsiella oxytoca ATCC 13182, Enterobacter cloaca ATCC 35030 and Streptococcus pyogenes ATCC 12378. The absence of haemolytic activity and proteinase (trypsin) and the presence of a strong peptidase (leucine-arylamidase) and esterase-lipase (C4 and C8) were observed in this LAB strain. P. acidilactici Kp10 also produced acid, coagulated milk and has demonstrated proteolytic and amylolactic activities. Conclusion: The properties exhibited by P. acidilactici Kp10 suggested its potential application as probiotic and starter culture in the food industry