[Induction of allogeneic subcutaneous glioma tumor with GL 26 cell line in balb/c mice]

Glioma is the most common primary brain tumor. Despite many advances in treatment, all patients die within 6 to 18 months after diagnosis. In the cases of glioma, the immune system is suppressed in a local fashion. Therefore, unveiling the cellular and molecular mechanisms involved, with the aim of obtaining an appropriate new treatment is a priority. Designing an appropriate animal model is necessary before any clinical trials. In this study, we prepared fifteen 6-8 week-old female mice [Balb/C] from the Pasteur institute, Tehran, and also selected the mouse glioma cell line GL26 to induce a allogeneic subcutaneous tumor. After culturing the cell and anesthetization of the mice, we injected different cell doses into distinct groups of mice. Sterile PBS was injected into the control group. Animal behavior and clinical symptoms were regularly followed and recorded, and after tumor induction, it was surgically removed and evaluated in terms of macroscopic and microscopic characteristics. The tumor was induced more quickly with higher number of GL26 cells in mice. Atrophy and weakness was observed in the affected animals. In macroscopic examination, the tumor was relatively large, thick and full of blood. Moreover, in microscopic examination, cell proliferation, mitosis, abundant vessels, and tumor necrosis were observed. Regarding the limitations of a glioma syngeneic animal model, establishment of an allogeneic subcutaneous model, allows an easy evaluation of the size and volume of the tumor, without a requirement for sacrificing the animal. This model has the potential to provide opportunities for research on some immunological parameters, the testing of new therapeutic agents, and new discoveries in basic research, concerning glioma, for the first time in Iran

Production of human papilloma virus type 16 E6 oncoprotein as a recombinant protein in eukaryotic cells

Cervical cancer is one of the most important and widespread cancer which affects women. There are several causes of cervical cancer; among them HPV types 16 and 18 are the most prominent ones which are recurrent and persistent infections. These genotypes are currently about 70% of cervical cancer causes in developing countries. Due to the importance of these viruses in cervical cancer, we pioneered the production of Human Papilloma Virus type16 E6 oncoprotein as a recombinant protein in order to develop a vaccine. Two HPV oncoproteins, E6 and E7, are consistently expressed in HPV-associated cancer cells and are responsible for malignant transformation. These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm. In the present study, the cloned E6-oncoprotein of HPV16 in pTZ57R/TE6 vector was used to produce professional expression vector. The target gene was subcloned in a eukaryotic expression vector. The pcDNA3-E6 vector was propagated in E.coli strain DH5 alpha and transfected into CHO cells 72 hours posttransfection. The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody. HPV16-E6 target protein recognized by specific antibody could be an appropriate form of protein, which can be used for further studies. Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies

[ evaluation of probiotic effect of L.acidophilus on the immune responses in BALB/C mice against transplanted tumor derived from breast tissue]

Antitumor effect of lactic acid bacteria have been shown in many studies, this effect maybe due to the immunomodulatory properties of these bacteria. In present work we have studied the effect of Lactobacillus [L] acidophilus on the immune responses of BALB/c mice against transplanted tumor derived from breast tissue. 6-8 week-old in-bred BALB/c mice, each weighing 25-30 g, were used. The mice were divided into two groups each consisted of 9 mice as test and control groups. The L.acidophilus ATCC4356 strain was used in this study. It was inoculated in MRS agar and cultivated overnight under anaerobic conditions then collected and resuspended in PBS. After preparation of proper amount of this suspension it was orally [2.7 x 10[8] CFU/ml] administered to the mice with a gastric feeding 2 weeks before tumor transplantation and 3 weeks after that, with 3 days break and 7 days administration. The control mice received an equal volume of PBS during the study. Results of the present work showed that L. acidophilus can increase the production of immunomodulatory cytokine IL-12 and decrease the TGF-alpha which can suppress immune response. Moreover, the growth rate of tumor in group which received L. acidophilus were decreased and the results of delayed type hypersensitivity [DTH] of this group in 48h were better than control group. The results of our study suggest that daily use of L. acidophilus can regulate immune response with Th1 dominance and may be helpful for cancer immunotherapy, but further studies are needed to investigate the other mechanisms of this effect

Time-dependent changes of immunologic responses after burn injury and immunomodulation by cimetidine and pyrimethamine in an animal model

Severe suppression of the immune system is the major cause of infections following burn injury. The aim of this study was to investigate the time-related alterations of immune responses following thermal injury in an animal model and also to modulate immune responses by use of the immunomdulators cimetidine and pyrimethamine. Male Balb/c mice were anesthetized and given a 10% total body surface area full-thickness burn. The timedependent changes of delayed type hypersensitivity [DTH] and antibody responses to sheep red blood cell [SRBC] were assessed at post-burn days [PBD]. The effects of different doses of cimetidine and pyrimethamine on DTH response were also quantitated at 10 PBD. Marked suppression of DTH response occurred during 30 days after burn trauma, with maximal suppression occurring between 10 to 14 days after burn injury. Simultaneously the antibody response to SRBC was significantly increased after thermal trauma. Cimetidine [at doses of 10 and 15 mg/kg] and pyrimethamine [at doses of 5 and 10 mg/kg] significantly augmented DTH response after thermal injury. These results showed that the severe time-dependent alterations occurred in DTH and antibody responses following burn injury. Cimetidine and pyrimethamine also restore burn-induced suppression of DTH response following thermal trauma

[ probiotic effects of Lactobacillus casei in BALB/c mice on the Tumor growth rate bearing breast cancer]

Antitumor effect of lactic acid bacteria [LAB] have been shown in many studies, this effect maybe as a result of immunomodulatory properties of these bacteria. In present work, we have studied the effect of Lactobacillus casei on the tumour growth rate in BALB/c mices bearing breast cancer. 6-8 week-old In-bred BALB/c mice, each weighing 25-30 g, were used. There are two experimental group consisted of 9 mices that they were used as controls in each assay. The L.casei ATCCT 39392 strain used in this study was inoculated in MRS broth and cultivated for a day at 37 °C under anaerobic conditions, collected by centrifugation and resuspend in PBS. After preparation of proper amount of these suspension it was orally administered to the mice with a gastric feeding, Control mice received an equal volume of PBS in duration of study. Results of this study showed that oral administration of L.casei can inhibit the tumour growth and increased the local inflammation in DTH assay as a result of increase in immune responses efficiency. In conclusion oral administration of Lactobacillus casei may regulate immune responses skewed Th1 balance and maybe helpful for cancer immunotherapy, but further studies is needed to investigate the other mechanisms of this effect

Purification of immunomodulator fraction components of garlic [R10] by HPLC

It has been reported that garlic extract and its components show medicinal effects including immunomodulatory activities. We have isolated the immunomodulatory fraction [R10] previously In this study we have proposed to purify the components of R10 using HPLC. Crude garlic extract purchased from Hamadan, Iran. R10 fraction including 10-50 KD molecules have been isolated using ultrafiltration. Further purification has been made using Vaydac 208Tpv10, a semi-preparative HPLC reversphase choromatography column. Tricine SDS-PAGE has been used to determine molecular weight of the samples. 6 major peakes were obtained from HPLC of R10 at gradient of 0.25% of buffer B/min through 60 minute. The molecular weight of 3 peaks [samples 0, 1 and 3] was 12 KD with tricine-SDSPAGE. Using C8 HPLC reversphase choromatography seems to be appropriate tool for purification of R10 components and the purified components at peak 0, 1 and 3 seems to be isotypic molecules

Micro-pixe analysis in invasive ductal carcinoma tissues after treatment of astaxanthin

Trace elements play an important role in a number of biological processes. Astaxanthin [ASX], a carotoid pigment found in certain marine plant and animals, has shown anti cancer and anti free radical properties. This work intended to understand the effect of Astaxanthin in breast cancer [invasive ductal carcinoma, IDC] by using micro-pixe method. For this aim the concentration of trace elements were compared in healthy, cancerous and cancer treated with astaxanthin in the breast and liver tissues of breast cancer bearing mice, using proton induced X-ray emission [PIXE]. Proton induced X-ray emission [PIXE] was used in a study intending to compare the concentration of trace elements in breast and liver tissues of mice bearing tumor, three groups of mice: healthy, cancerous, and cancerous treated by astaxanthin, were considered. Astaxanthin was supplied from Research Institute of women, Alzahra University. Comparing the untreated tumor tissue, treatment with Astaxanthin significantly decreased the amount Fe, P, S, and Ca elements level in tumor tissue of the breast cancer. It is also found that the concentrations of those elements in liver of the untreated mice and the liver of treated mice with astaxanthin were fairly equal. Astaxanthin significantly decrease the accumulation of elements in the site of tumor, and caused the breast cancer cell membrane to lose their desire to collect the elements from healthy tissues. The micro-pixe technique could calculate elemental concentrations in tissues. Changes in metallic elements may affect microenvironment and cell functions, which might led lead to cell degeneration or death, the results shows that astaxanthin reduces vital element concentration in tumor site, thus it could be used as an anti tumor agent

Surveying the effect of 100 kD protein of shark cartilage on erythroleukemic K[562] cell line and normal.lymphocytes

Shark cartilage is used in complementary medicine for the removal of tumor in cancer patients. However, scanty reports have addressed the effect of 100 KDa fraction on immunity system. The aim of the present study was to evaluate the effect of this fraction in human erythroleukemic K[562] cell line and normal lymphocytes. Gel chromatography purification strategy was employed for purification of 100 KDa fraction from the shark cartilage. Erythroleukemic K[562] cell line and normal lymphocytes were cultured in RPMI 1640 [Sigma], supplemented with 10% fetal calf serum, peniciline- streptomycin, L-glutamine and incubated at 37 °C for 2 days. Normal mononuclear peripheral blood cells were collected by ficole. Cellular vital capacity was determined by MTT. During MTT, erythroleukemic K[562] cell line revealed to have a meaningful suppression at 15 micro g protein concentration when compared with controls [p<0.0l]. To our knowledge, it is the first study that demonstrates l00kD protein of shark cartilage to induce cell death in erythroleukemic K[562] cell line in vitro

Free hydroxyl radical dosimetry by using 1 MHz low level ultrasound waves

In order to quantify effects of ultrasound irradiation parameters under therapeutic condition, especially sonodynamic therapy, it is initially necessary to evaluate inertial cavitation activity in vitro conditions; therefore, in this study, the effect of 1 MHz low level ultrasound based on OH radicals generated by acoustic inertial cavitation in aqueous solution was monitored by their reaction with terephthalic acid [TA] to produce fluorescent 2-Hydroxyterephthalate acid [HTA] by spectrofluorometry method [Terephtalic acid dosimetry]. The study was designed to measure hydroxyl radicals in a field near to 1 MHz sonotherapy probe in progressive mode and low level intensity. The effect of ultrasound irradiation parameters [1MHz] containing duty factor, mode, intensity ultrasound and, time sonication in hydroxyl radical production have been considered. After preparation of solution of dosimetry and plotting calibration curve of spectrofluorescence, the effect of mode of sonication [continuous and pulsating], duty factor [20-80%], intensity [0-2 W/cm[2], with step of 0.5 W/cm[2]], and sonication time [0-60min with step time of 10min] without increasing temperature to more than 3°C to determine the effective exposure in low level ultrasound were evaluated. The fluorescence intensity of TA solution before and after irradiation, in all cases was measured, and the results were reported as Mean +/- Standard Deviation [SD]. The result of experiments related to sonication mode for 1MHz ultrasound irradiation [2 W/cm[2]] show that continuous mode of sonication is 29% higher fluorescence intensity than the pulse mode in 80% duty cycle for sonodynamic therapy. With compensation of irradiation time for 1MHz sonication in different duty cycles, fluorescence intensity in continuous mode is 22% higher than the pulse mode in average. The amount of hydroxyl radicals production versus ultrasound intensity, and sonication time show with increasing intensity or sonication time in continuous mode, the hydroxyl radical production is linearity increased [R=0.99]. The results show that the terephthalic acid dosimetry is suitable for detecting and quantifying free hydroxyl radical as a criterion of inertial cavitation production over a range of condition in medical ultrasound fields

[Hyperthermia can accelerate the healing process of 2[nd] degree burn wounds]

The present study aimed at evaluating the effect of local hyperthermia on the healing of burn wounds. Right and Left flunks of 8 Balb-c mice [as treated and control wounds respectively] were burned. Local hyperthermia was applied only for the burn wounds of the right flunks [the treated wound]. Sampling was accomplished on the 6[th] day for half of the mice and on the 9[th] day for the other half. The treated wounds had significantly smaller sizes than control wounds on the 6[th] day [P=0.019] and the 9[th] day [P=0.007]. The number of hair follicles and sebaceous glands of the treated wounds were significantly more than those of control wounds both on the 6[th] day [P=0.025 and P=0.043, respectively] and on the 9[th] day [P=0.012 and P=0.033, respectively]. Regarding the neovascularization there was no significant difference between the treated and control wounds on the 6[th] day but on the 9[th] day the neovascularization of the treated wounds was significantly more comparing to the control wounds [P=0.025]. Acute inflammation of the treated wounds was significantly less than the control wounds [P=0.015]. Collagen formation and reepithelialization in the treated wounds were more than the control wounds both on the 6[th] and 9[th] days. It is concluded that local hyperthermia can accelerate the healing process of the second degree burn wounds

cytotoxicity pathway of natural killer cells in cord blood compared to peripheral blood

The cytotoxic activity of natural killer cells is usually tested by radioactive assay [51Cr release assay], which detects the release of cytoplasmic contents after plasma membrane disintegration of dying cells. In contrast to this indirect evaluation of cytotoxicity, the assessment of cell damage by flow cytometry aims to provide a more exact characterization of the death pathway via detection of the percentage of apoptosis and necrotic cells. Annexin V-FITC [Axv -FITC] can be used to label cells in the early apoptopic state, while propidium iodide [PI] indicates late apoptosis or necrosis. The NK cytotoxicity of cord blood [CB] and peripheral blood [PB] was determined after 4 hours of incubation in the absence of cytokines. After 4 hours in vitro incubation, co-staining with Annexin V-FITC [Axv-FITC] and propidium iodide [PI] permitted discrimination between viable, early apoptotic and necrotic cells. As we would expect, the cytotoxicity pathway in PB mononuclear cells [MNCs] consists of both apoptosis and necrosis pathways but in CB MNCs it almost consists of early apoptosis; and necrosis is negligible. With escalating E: T [effector: target] ratio changes in the percentage of apoptotic cells in PB samples were significantly higher than CB samples. The mechanism [s] of the low cytotoxicity of resting cord NK cells is not well understood. Complementary research in this field is recognized to elucidate the phenotypical and functional properties of CB cells and how they relate to maturational stages. CB studies are important for transplantation research and may provide insight to the suppressive mechanism by which the host-recipient could evade GVHD and rejection

Production of monoclonal antibody against human immunoglobulin E

Immunoglobulin E is one of the five classes of immonoglobulins that plays an important role in allergic diseases. Production of monoclonal antibodies by a single clonotype against different epitopes of immunoglobulin E has high priority in development of diagnostic kits. In this study, an attempt was made to produce monoclonal antibodies against human immunoglobulin E. Balb/c mice were immunized with semipurified immunoglobulin E and spleen cells fused with SP2.0 mouse myeloma cell line in the presence of polyethylene glycol. Supernatant of hybridoma cells was screened for detection of antibody by enzyme linked immonosorbent assay method. Cloning of selective high absorbance wells were done with limiting dilution method. The suitable clone [monoclone] was selected by enzyme linked immunosorbent assay and confirmed by immunoblot. The subclass of the chosen monoclonal antibodies was determined and the clones freezed and kept in liquid nitrogen. During this study three successful fusions were carried out, which resulted in development of 156 clones with high production of anti-IgE. Fourteen clones with the highest titres were selected for cloning. After limiting dilution more than 100 monoclonal antibodies were produced and the suitable one [G10F7] I.E.; the clone which displayed the high absorbance in reaction with purified immunoglobulin E and the lowest cross-reactivity with immunoglobulin M, immunoglobulin G and immoglobulin A was chosen. In immunoblotting, presence of high density band in reaction with immunoglobulin E was confirmed. The suitable mab was shown to be IGG1 subclass with kappa light chain. It seems that, this mab could be successfully used in diagnostic kits