Triple tandem mimotope peptide of epidermal growth factor receptor displaying on the surface of M13 phage induces anti-tumor response in mice tumor model

Epidermal growth factor receptor [EGFR] has been shown to play a critical role in tumor cell growth and its over expression has been observed in many epithelial tumors. In the field of cancer vaccine research, displaying the peptide mimotope on the surface of phage particles has shown promising results. In this study using m13-PVIII phage display system, two constructions were prepared: triple tandem repeat of EGFR mimotpe displaying particles [3M] and single EGFR mimotope displaying phage particle [1M]. To investigate the anti-tumor properties of phage vaccine, C57BL/6 mice Lewis lung carcinoma xenograft model was established and treated with 3M phage vaccine, 1M phage vaccine and control agents. Immunization of mice with these phage-based vaccines showed strong immune response against phage-mimotope. 3M phage vaccine showed more potency against tumor in comparison with control groups. Also the survival time was extended in phage vaccine treated tumor-bearing mice compared with untreated mice. Our findings suggest that mimotope-displaying phage vaccine can induce specific antibodies with antitumoral activity, which its potential as a candidate vaccine for EGFR-specific cancer immunotherapy needs to be more investigated in future studies

Retroviral transduction of fluonanobody and the variable domain of camelid heavy-chain antibodies to chicken embryonic cells

Single domain antibodies from camel heavy chain antibodies [VHH or nanobody], are advantages due to higher solubility, stability, high homology with human antibody, lower immunogenicity and low molecular weight. These criteria make them candidates for production of engineered antibody fragments particularly in transgenic animals. To study the development of transgenic chicken using a recombinant retrovirus containing fluonanobody. The retrovirus constructs containing nanobody genes along with secretory signals and GFP gene were established and packed. The virus particle containing the obtained fusion gene was injected into the eggs in stage X. Molecular detection and protein analysis was done in the G0 chickens. The rate of hatched chicken after gene manipulation was estimated to be about 33%. Real-Time PCR assay showed that the nanobody along with GFP gene were integrated in cells of 1.2% of chickens. We conclude that although the rate of gene transfer by recombinant viruses in chickens is low, it would be possible to transfect the target camel immunoglobulin gene into chicken genome.

[Development of agglutination test for detection of isolated mannoprotein antigen from candida albicans]

Candida albicans is one of the most common causes of hospital infections. The aim of this study was to develop a rapid, easy and sensitive way to detect candida albicans. Polyclonal antibodies were prepared in rabbits. The antibodies were purified by salt concentration and Ion exchange chromatography. The purified antibody was coated onto the colloidal gold. Color change [red to blue] was observed when the purified antigens [mannoprotein of Candida albicans cell wall] were added to Polyclonal antibodies. The method was sensitive and easy. This study indicated that using colloidal gold particle agglutination method can be used for accurate and rapid candida detection Method.

[Radiosensitization effect of PEGylated gold nanoparticles in orthovoltage x-ray irradiation of the MCF-7 cancerous cell line]

Due to recent advances in nanotechnology it is now possible to accumulate high atomic-number nanomaterial such as gold nanoparticles [GNPs] in cancerous cells and take advantage of their absorbed dose enhancement property as radiosensitizing agents. This study aimed to investigate the absorbed dose enhancement factor due to the presence of PEGylated GNPs under the irradiation of an MCF-7 cancerous cell line using orthovoltage X-ray beams. We synthesized GNPs with an average diameter of 47 nm and joined them to polyethylene glycol. A total of 50 micro g/mL of the pegylated GNPs were incubated with MCF-7 cells for 1, 2, 6, 12, and 24 hours, after which we compared their cytotoxicities. Then, PEGylated GNPs [50micro g/mL] were incubated with MCF-7 cells for 12 and 24 hours and their radiosensitizing effect during 2Gy delivery of 120, 180 and 200 kVp X-ray beams were compared by the MTT assay. Cytotoxicity studies showed no significant effect of GNPs on cell viability. Significant differences in cell survival were observed between the groups irradiated with and without GNPs, which lead to an average absorbed dose enhancement factor of 1.22 +/- 0.06. According to the results, there was no radiosensitization difference due to the usage of 120, 180 and 200 kVp X-ray beams. However increased incubation time increased the dose enhancement factor. By using PEGylated GNPs we can decrease the prescribed X-ray dose, yet maintain the same level of cancerous cell killing

[Combined effects of Curcuma longa and exercise training on kidney and spleen tissue levels of glutathione peroxidase and protein carbonyl in rats exposed to lead]

Environmental pollution is of major concern today and lead is considered to be one of the most important environmental pollutants. Long-term contact with lead causes harmful effects to humans. This study seeks to determine the effects of Curcuma longa [turmeric extract] consumption and exercise training on glutathione peroxidase and protein carbonyl in kidney and spleen tissues from rats exposed to lead. We randomly classified 60 male rats into the following six groups of 10 rats per group: 1] control; 2] sham [turmeric extract solvent]; 3] lead; 4] training + lead; 5] turmeric extract + lead; and 6] training + lead + turmeric extract. The training program for groups 3 and 6 consisted of running on a level treadmill for 40 sessions [eight weeks at five sessions per week] at a speed of 22 to 15 m/min for 26 to 64 minutes. Turmeric extract [30 mg/kg] was injected three times per week for eight weeks. Amounts of glutathione peroxidase and protein carbonyl were measured by ELISA. The amount of protein carbonyl in the kidney and spleen tissues of the lead group increased compared to the sham, training, combined and extract groups. Rats in the combined, extract and practice groups [F=4.787; P=0.002] had lower levels of protein carbonyl in their kidney and spleen tissues compared to the sham group [F=6.970, P=0.000]. Glutathione peroxidase levels in the kidney and spleen tissues were less in the lead group compared to the sham group. However these levels in the training, extract, and combined groups increased compared with the sham group [respectively, in kidney and spleen P=0.051, F=2.466 and P=0/086, F=2.11]. Intake of turmeric extract and exercise alone did not cause complete inhibition of the oxidative effects in kidney and spleen tissues. However, exercise and consumption of turmeric extract can be effective in reducing the harmful effects of lead

new monoclonal antibody radiopharmaceutical for radioimmunoscintigraphy of breast cancer: direct labeling of antibody and its quality control

Radioimmunoscintigraphy [RIS] has found widespread clinical application in tumor diagnosis. The antibody [Ab] PR81 is a new murine anti-MUCl monoclonal antibody [MAb] against human breast carcinoma. In this study a very simple, rapid and efficient method for labeling of this MAb with [99m] Tc, particularly suitable for development of a [kit] is described. The reduction of Ab was performed with 2-mercaptoethanol [2-ME] at a molar ratio of 2000:1 [2-ME:MAb] and the reduced Ab was labeled with [99m] Tc via methylene diphosphonate [MDP] as a transchelator. The labeling efficiency which was determined by instant thin layer chromatography [ITLC] was 94.2% +/- 2.3. Radiocolloides measured by cellulose nitrate electrophoresis were 2.5% +/- 1.7. In vitro stability of the labeled product in human serum which was measured by gel filtration chromatography [FPLC] was 70% +/- 5.7 over 24 hr. The integrity of labeled MAb was checked by means of SDS-PAGE and no significant fragmentation was observed. The results of the cell-binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF 7 cells. Biodistribution studies were performed in normal BALB/c mice at 4 and 24 hrs post-injection and no important accumulation was observed in vital organs. These results show that the new radiopharmaceutical may be considered as a promising candidate for imaging of breast cancer

Cloning and expression of recombinant camelid single-domain antibody in tobacco

Antibodies provide a suitable tool in fundamental research and their high affinity and specificity make them invaluable for diagnostic and therapeutic applications. A promising alternative to conventional antibodies are the heavy chain antibodies [VHH] of Camelidae having short length, high solubility and stability are preferred to other antibody derivatives. In this study, our goal was production of recombinant VHH antibody fragments [against cancer associated mucin, MUC1] in tobacco plants. The VHH gene cDNA was cloned in TA vector and then subcloned into a plant expression binary vector pBI 121. The VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. The presence of VHH gene in transformed plants was confirmed by PCR. Western blot analysis showed that the recombinant VHH protein was expressed in tobacco plant. ELISA results with MUC1 antigen confirmed that the biological activity and antigen-specific responses of the plant derived VHH protein compare favorably with that of the parent recombinant antibodies. This is the first report of production of camelied VHH antibody against tumor specific antigen from two-humped camel [Camelus bactrianus] in plants

Extraction and purification of anti-proteinase 3 [PR3] antibodies from egg yolk

Recently it has been reported, that immunoglobulin Y [IgY] can be used instead of polyclonal antibodies extracted from mammals [IgG] for the purpose of diagnosis and therapy. These antibodies are found to have better properties in terms of specificity and ease of large-scale production. In addition, IgY binds neither to mammalian complement or Fc-receptors nor does it interfere with rheumatoid factors [RF], which has proven to be advantageous in many immunological tests. Proteinase 3 [PR3], a constituent of azurophil granules of neutrophils, is the target antigen for most anti-neutrophil cytoplasmic antibodies [c-ANCA] in Wegener granulomatosis [WG]. Capture ELISA was found to be the method of choice in case of c-ANCA determination for the diagnosis and management of WG. However, in this method, the reaction of RF with the Fc portion of IgG in capture ELISA leads to false positive in the assay of c-ANCA and is found to be the most important short-comings of available diagnostic immunochemical tests using mammalian antibodies. To avoid such unwanted interactions, laying hens were inoculated with PR3, and IgY was purified from egg yolk by acidic extraction with chloroform. The aqueous phase was treated with sodium sulphate and the precipitate collected, was dissolved in buffer and was purified using a T-gel chromatography method. The prepared IgY-anti-PR3 was used to set up a capture ELISA. Our results showed that the prepared IgY-anti-PR3 had good titer [lu g/ml in a coating system] and specificity. Hence, IgY based immunoassay would be a useful alternative to mammalian IgG antibody used in PR3 immunoassays