[Chitosan nanopolymers effect in activating of mouse bone marrow derived dendritic cells]

Background and Aim: Regarding to various problems in the activation of dendritic cells and immune system's responses, finding of a safe, effective and applicable agent is highly desirable. Chitosan is an effective gene delivery agent and also a part of nanoscaffolds. In the present study, chitosan nanopolymers effect on dendritic immune cells were assessed

Materials and Methods: In this experimental-laboratory study, chitosan [150 KD] in acetic acid 1% solution was depolymerized to 10 KD oligomers using NaNO2. The oligomers particles were obtained by means of 2 normal NAOH molecules. Denderitic cells were derived from the rats' bone marrow using GM-SCF. On the treated denderitic cells CD40, CD86 and MHC-II maturation markers were evaluated by flowcytometery and TNF-alpha release was evaluated using ELISA method and T cell proliferation

Results: It was observed that dendrtic cells purity on the 8th day was more than 90%. Flowcytometery analysis showed an increase in all evaluated CD40, CD86 and MHC-II maturation markers [p<0.05]. TNF-alpha release and T cell proliferation significantly increased by chitosan treated denderitic cells compared to unstimulated or lipofectamin treated cells [P<0.05]

Conclusion: Results showed that chitosan nanopolymers significantly increased dendertic cell maturation phenotype, proinflamatory cytokine production, and induction of T cell proliferation. Therefore, chitosan nanocomplexes and scaffolds can induce and accelerate immune responses

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.

Identification of live toxoplasma gondii by the nasba method in rat

Toxoplasmosis is a worldwide disease, for which different detection methods have been used. The nucleic acid sequence-based amplification [NASBA] method is shown to be highly efficient for diagnosis of live microorganisms. The present research evaluates the molecular isothermal method of NASBA to identify live Toxoplasma gondii [T. gondii] in rat. Tachyzoites of T. gondii were inoculated in the peritoneal cavities of mice [Mus musculus] and their RNA was extracted. The NASBA method was then used to amplify the tachyzoite B1 rRNA gene. Next, we examined blood samples from 30 experimentally infected case and control rats [Rattus norvegicus] by NASBA. Finally, the resultant band was investigated on an agarose gel. The B1 genes extracted from both the tachyzoites and blood samples were successfully amplified by the NASBA method. This amplified gene yielded an amplicon of approximately 116 bp on gel agarose. NASBA is highly efficient for the identification of live T. gondii. This method can be applied for early diagnosis of active toxoplasmosis in both newborns and immunocompromised individuals

Evaluattion of giardia lamblia genetic differences in khorramabad city and surrounding villages by use of PCR and sequencing

The intestinal protozoa Giardia lamblia is a parasite frequently involved in human parasitic gastroenteritis throughout the world. Transmission of G. lamblia cysts to human occurs mainly via ingestion of contaminated food and water. The aim of this study was to evaluate Giardia lamblia genetic differences in the Khorramabad City and its surrounding villages by means of PCR and sequencing. In this study 30 fecal samples positive for Giardia lamblia were collected from the patients in Khorramabad city and its surrounding villages. The samples were fixed in dichromat 5% after filtration. Before DNA extraction, all samples were washed with PBS solution in order to remove dichromat. For determination of genetic differences sequencing on 5 samples was performed. After DNA extraction, amplification of GDH gene from 24 of 30 samples was performed by PCR, successfully. Alignment of the obtained GDH sequences with reference sequences [gene bank] indicated the presence of only one genotype of G. lamblia; 5 specimens were identified as G. lamblia assemblage A. Assemblage A was the dominant genotype in Khorramabad City and its surrounding villages. Because of limited number of samples in this study, further studies with higher number of samples are recommended

Production of Pentameric Cholera Toxin B Subunit in Escherichia coli

Cholera toxin B subunit [CTB] has been extensively studied as an immunogen, adjuvant, and inducer of oral tolerance in many investigations. Production of CTB has been carried out in the bacterial, plant, insect and yeast expression systems. In this study the expression of the CTB containing a 6XHis-tagged was performed by Escherichia coli [E.coli] M15. The yield of purified pentameric recombinant CTB was about 1 mg/l. Western blot analysis demonstrated that the recombinant CTB was antigenically active. In addition, GM1-ganglioside ELISA showed that recombinant CTB binds to GM1-gangelioside receptor, confirming disulfide bond formation and proper folding of the recombinant protein in E.coli. Overall, in regard to the vast applications of CTB in medicine, this bacterial expression system will be a fast, cost-effective and simple system for production of pentameric CTB and CTB conjugated proteins

Functional concentrations of BMP4 on differentiation of mouse embryonic stem cells to primordial germ cells

Bone morphogenetic protein 4 [BMP4] has a significant role in primordial germ cells [PGCs] differentiation from mouse embryonic stem cell [mESC]. The aim of this study is to determine the best concentration of BMP4 at a time of two days on differentiation PGCs from mESC. To differentiate PGCs, embryoid bodies [EBs] from mESCs were cultured in concentrations of 0, 5 and 10 ng/ml BMP4 for two days. Germ cell markers Oct4 [Pou5f1], Stella [Dppa3] and Mvh [Ddx4] were analyzed by flow cytometry, immunocytochemistry and reverse transcriptase polymerase chain reaction [RT-PCR]. Flow cytometry data demonstrated most Mvh-positive cells were observed only in the treated groups. Immunocytochemistry of EBs in the treated groups identified cells positive for Mvh. PCR results showed expression of Oct4 in the control group and treated groups. Stella and Mvh were expressed only in the treated groups. Low concentrations of BMP4 during two days had an optimal effect on differentiation of PGCs from mESC

[TGF-bReceptor2 knocked down by SiRNA increases cord blood CD34+ HSCs self-renewal]

Nowadays, cord blood Hematopoietic stem cells [HSCs] are known as a valuable source for bone marrow transplantation but unfortunately their insufficient number is a limiting factor for using them in adult bone marrow transplantation. Cord blood HCSs expansion is an approach to overcome this problem, by inducing their self-renewal. TGF-b signaling pathway is a key inhibitory agent for HSCs self-renewal. In this study, we tried to enhance self-renewal of long term culture initiating cell by inhibiting TGFbR2 expression. CD34+ HSCs were isolated from cord blood units with MACS column. SiRNA against TGFbR2 was transfected by Lipofectamine [TM] RNAiMAX as transfection reagent. HSCs were cultured in IMDM medium containing 10% FBS and early acting cytokines [Flt3L, SCF, Tpo] for 8 days. Then we evaluated TGFbR2 expression by QRT-PCR. The CD34+ subpopulation of cultured cells were examined by flow cytometry on the 8th day. Finally the expanded cells were evaluated for the presence of early hematopoietic stem cells by LT-CIC and clonogenic assays. According to our results, TGFbR2 down regulation increases CD34+ subpopulation of HSCs. In addition, LT-CIC assay showed an enhancement in primitive hematopoietic stem cell capable of self-renewal. All in all, it seems that positive regulators have attracted more attention in the field of HSCs expansion while negative regulators have same importance in self-renewal process of HSCs and their inhibition can be a beneficial tool for enhancement of HSCs self-renewa

correlation between the endometrial integrins and osteopontin expression with pinopodes development in ovariectomized mice in response to exogenous steroids hormones

The ovariectomized animals are good models to evaluate the effect of different steroid hormone treatments on implantation events and the pattern of integrin expression. Therefore, this study was performed to compare the expression of integrins and osteopontin [OPN] in correlation with pinopode development in ovariectomized mice endometrium which was subjected to steroid hormones. Ovariectomized mice were subjected to estrogen, progesterone and estrogen-progesterone hormones. Their uterine horns were evaluated for integrin expression by immunohistochemistry and real-time RT-PCR and for pinopode development by transmission and scanning electron microscopic studies. No immunostaining for integrin and OPN molecules were detected in the endometrium of non-ovariectomized mice except in metestrus phase. The alpha4 and beta 1 integrin genes were expressed in all phases of estrous cycle. In ovariectomized mice, no reaction was detected in the endometrium of control, sham and estrogen-treated groups, but in both progesterone-treated groups, all examined genes were expressed. There was not any correlation between pinopodes and integrin expression in ovariectomized mice. The progesterone is more effective on endometrial integrin expression than estrogen and differences in the expression pattern of integrins reflect their important and different roles in embryo implantation. The pinopodes may have minor effect in mice implantation or have some delay in their expressions in ovariectomized mice which were subjected to exogenous hormones

Effects of different doses of bone morphogenetic protein 4 on viability and proliferation rates of mouse embryonic stem cells

In this study, we examined the effect of different doses of bone morphogenetic protein 4 [BMP4] on CCE mouse embryonic stem cells [ESCs] viability and proliferation rates in order to improve the outcome of induction processes and make a system with highest viability and proliferation rates for further studies on BMP4 roles in multiple developmental stages. Expression of Oct-4 was studied and confirmed in this cell line immunocytochemically. Also, in order to evaluate the proliferation and viability rates in BMP4-treated cells, ESCs were cultured in Dulbecco's Modified Eagle Medium [DMEM] containing different doses of BMP4 [0, 0.01, 0.1, 1, 5, 25, 50 and 100ng/ml]. The mean number of whole cells and living cells were considered as proliferation and survival rates respectively. Data analysis was done with ANOVA test. The results showed that there were significant differences between the mean percent of viability between 1ng/ml and 0 ng/ml [control] and 50 and 100 ng/ml BMP4 [p

[Isolation and identification of listeria monocytogenes in vaginal samples by PCR]

Listeria monocytogenes is a facultative, gram-positive bacterium which is found in soil, water, decaying vegetables, raw milk, and contaminated dairies. Listeria monocytogenes causes listeriosis. Listeriosis is a zoonosis disease, which transfers from animals to humans by animal feces and contaminated dairies. Listeriosis causes the flu like disease or self-limited enteritis, but it leads to serious disease in elderlies, new-borns, pregnant women and immunocompromised persons. If pregnant women are infected by Listeria monocytogenes, the newborn probably will be miscarried, prematured or stillborn. For the importance of the bacteria on pregnant women's and newborn's health, there are so much concentration and studies on it. Culturing of the bacteria is so difficult and time-consuming and it needs at least 5 days to confirm. Our goal in this survey was to develop an PCR based molecular method for fast detection of the bacteria from the vaginal samples. In this survey 100 vaginal samples were examined. All of the samples were cultured and assayed with PCR method. Among them, 7 samples for culture and 36 samples by PCR were positive for Listeria monocytogenes. In this study we showed that the PCR is a faster, more accurate and sensitive than culture method for the detection of Listeria monocytogenes in vaginal samples

Designing and development of a silencer vector based on second generation of RNA interference [RNAi] technique to study downregulation of steroid receptor RNA activator [SRA]

RNA interference [RNAi] is the most potent technique for gene silencing in eukaryotic cellular system at transcriptomic level. Genetic disorders and cancers are important targets for therapeutic development of this technique. In order to bypass the temporary dpwnregulation by siRNA, a new generation of shRNA named shRNAmir has developed. Silencing construct with structure similar to microRNA [shRNAmir], mimics a natural microRNA pathway inside the cell. Steroid receptor RNA activator [SRA] is one of the regulators of steroid receptor like ER. Prostate, uterus and breast tissue express a low level of SRA, there is an increase of expression during their tumorgenesis. So SRA may participate in tumorgenesis or proliferation of tumors. We used RNAi technique to silence expression of SRA. The SRA silencer was designed and constructed by Soe-PCR, then cloned into an expression vector pEGFPCl. Human breast cancer [MCF7] cells were transfected with silencer plasmid then the changes in the SRA expression estimated by Real-Time PCR at 24, 72 hours and after l0days. The results showed about 60% decrease in relative expression of SRA gene, after 72 hours and 10 days, which shows that shRNAmir-SRA could successfully knockdown the expression of target gene. It seems that the designed shRNAmir may be a suitable tool for a variety of applications because it could stably knockdown the expression of target gene

Transplantation and homing of mouse embryonic stem cells treated with erythropoietin in spleen and liver of irradiated mice

The present study was designed to evaluate the homing potential of mouse embryonic stem cells [ESC] treated with erythropoietin [EPO] in hematopoietic organs such as spleen and liver after transplantation using morphological and immuno-histochemical techniques. Day-four embryoid body [EB]-derived cells were dissociated and re-plated in medium in the presence and absence of EPO for three days. The EPO- and untreated differentiated cells were labeled with 5-bromo-2 deoxyuridine [BrdU] before transplantation and analyzed using flow cytometry and reverse transcription-PCR methods. BrdU-labeled cells were injected via the tail vein into irradiated adult mice in both groups. The spleen colony-forming unit assay [CFU-S] was performed 12 days after transplantation. Immuno-histochemistry was also carried out to trace transplanted cells. The percentage of CD34 positive cells was 5.51 +/- 1.06% in the EPO-treated group and 1.63 +/- 0.225% in untreated group. The RT-PCR analysis showed that the EPO-treated cells expressed epsilon globin, beta H1 globin, RUNX1 and EPO receptor genes, but the beta-major globin gene was not expressed. The number of colonies formed in the spleens of treated group [17.33 +/- 4.726] was significantly different from the control group [6 +/- 1]. The population of BrdU positive cells in spleen of EPO-treated cell-transplanted group was higher than that of the control group. Also, BrdU positive cells were observed in the central vein of the liver sections of EPO-treated and control groups but were not observed in the liver parenchyma. There were not BrdU positive cells in the spleen and liver sections of the sham group. Our results confirm that ESC have the ability to home and form colonies in spleen after transplantation and EPO-treated EB-derived cells caused an increase in the number of colonies in spleen after CFU-S

Studying of the helicobacter pylori vacA cytotoxin gene prevalence by PCR technique among the Iranian patients with gastroduodenal disorders

Helicobacter pylori is a major etiological agent in gastro-duodenal disorders, and has spread in the world. The prevalence of infection with H. pylori is more than 80% in some populations, but only 10% to 20% of them are infected with this organism. Also infection with this pathogen is associated with peptic ulcer disease [PUD], Gastritis [G], Duodenitis [Du], and non-ulcer dyspepsia [NUD]. The development of diseases depends on the virulence of the infecting H. pylori strain and the susceptibility of the host. The vacuolating cytotoxin and the cytotoxin associated protein, encoded by vacA and cagA genes are important virulence determinants of H. pylori which are divided into different pathogenic types, to cause varities of infections. This may be used as a marker of infection and could be used to distinguish between pathogenic and nonpathogenic strains of H. pylori in Iran. The aim of this study was to assess the prevalence of vacA genotype of H. pylori and its development of PUD, G, Du, and NUD from Iranian patients who were admitted to Hazrat Rasoul Akram [peace upon him] as an educational and research society, affiliated to Iran University of Medical Sciences and Health Services [IUMS] in Tehran, Iran. During this study specimen biopsies were collected from 180 patients who underwent routine gastrointestinal endoscopies to the internal medicine ward, Hazrat Rasoul Akram Hospita, LIUMS, Tehran, Iran. Positive H. pylori strains were identified by cultured isolates, standard biochemical methods, and molecular typing was performed by PCR technique to detect vacA gene and its alleles. In this study out of 180 samples of 93 H. pylori strains were isolated and identified by biochemical tests, the PUD, G, Du and NUD were 79%, 60%, 90% and 30% respectively. 87 [94%] strains contained vacA gene and the predominant genotypes are in the vacA gene. Also 59 [64%] strains had displayed the sl/m2 genotype. 57 [625] strains were classified as type 1, 31[33%] strains were type IV, 3 [3.26%] strains were type II, and 2[2.17% strains were type III. The significant difference [P<0.01] between type I and type IV isolated strains from PUD [62% type I] showed that type I strains are more pathogenic than type IV strains

Application of PCR-ELISA method based on RE domain for diagnosis of toxoplasmosis in experimentally infected rat [Rattus norvegicus]

Toxoplasmosis may cause significant damage to the developing fetus and is as life-threatening opportunistic infection in immunocompromised persons. Molecular methods are known to be more sensitive and more specific than serological assays for diagnosis of toxoplasmosis. Application of quantitative PCR has evolved sensitive, specific, and rapid method for the detection of RH strain of Toxoplasma gondii DNA. In the present study, quantitative PCR-ELISA [Polymerase Chain Reaction- enzyme linked immunosorbent assay] was used for quantization of Taxoplasma gondii in the blood of 15 rats [Rattus norvegicus] infected experimentally with the parasite. In this regard Polymerase PCR - ELISA was developed for rapid detection of Toxoplasma gondii. DIG-labeled [digoxigenin-labeled] amplicons were hybridized with a specific biotinylated oligonucleotide probe in solution phase and subsequently transferred to streptavidin coated plates. The captured DNA-DNA hybrids were colorimetrically detected by the addition of anti-digoxigenin antibody peroxidase conjugate and substrate. DNA of Toxoplasma gondii were efficiently detected within 4 hours and no interference was encountered in the amplification and detection of the parasite. Efficiency of PCR-ELISA system was evaluated we found with several advantages in terms of sensitivity, rapidity and simplicity in this system

[ changes of alpha v, beta 3, alpha 4, beta 1 integrins expression and osteopontin their ligands in murine estrous cycle]

Considering the importance of integrin molecules in the implantation and lack of sufficient information in the expression pattern of these molecules in various phases of estrous cycle. It seemed to be necessary to investigate these molecules in mouse endometrial during the various phases of oestrous cycle. Female NMRI mice [n=15] aged 6-8 weeks were studied. Various phases of estrous cycle including: proestrus, estrus, metestrus and diestrus were determined by vaginal smear. The mice were sacrificed [at least 3 per each phase] by cervical dislocation and the tissues were obtained from the middle 1/3 part of their uterine horns at each phase then the cryosections at thicknesses between 8-10 micro were obtained. Then the immunohistochemistry were done for integrins of alpha4, beta1, alphav, beta3 and their ligand osteopontine. The integrins were expressed only in the metestrous phase of oestrous cycle in the different locations of mouse endometrium. The positive reactions were observed for alphav, alpha4 and beta3 in the apical and basal membrane of glandular epithelium. Also the positive reaction for beta1 was found in surface and glandular epithelium as well as stroma. The osteopontin expression was seen in the apical membranes of surface and glandular epithelium and was not seen in other locations. It seems that expression of integrins in endometrium is based on their role in the implantation, therefore the molecules alpha4, beta1 and OPN that are expressed on the surface epithelial may be involve in the adhesion of cell to cell and integrins of alphav, beta3 that are expressed in the glandular

study of glutamate transporter 3 [EAAT3] expression, serum TNF-alpha level, and NF-KB activity in ischemic tolerance induced by prolonged and intermittent normobaric hyperoxia

Recent studies suggest that intermittent and prolonged normobaric hyperoxia [HO] results in ischemic tolerance to preventing ischemia brain injury. In this research attempts were made to see the changes in excitatory amino-acid transporter 3 [EAAT3], TNF- alpha levels, and NF- kappa B activity following prolonged and intermittent NBHO preconditioning. Rats were divided into four experimental groups, each with 21 animals. The first two groups were exposed to 95% inspired HO for 4h/day for 6 consecutive days [intermittent HO; InHO] or for 24 continuous hours [prolonged HO; PrHO]. The second two groups acted as controls, and were exposed to 21% oxygen in the same chamber [nomobaric normoxia, RA; room air] continuously for six days [intermittent RA, InRA] or for 24 hours [prolonged RA; PrRA]. Each main group was subdivided to MCAO- operated [middle cerebral artery occlusion], sham-operated [without MCAO], and intact [without any surgery] subgroups. After 24 h, MCAO-operated subgroups were subjected to 60min of right MCAO. After 24h reperfusion, neurologic deficit score [NDS] were assessed in MCAO-operated subgroups. Immediately and 48 h after pretreatment, blood sampling for assessment of serum TNF- alpha levels were performed. Then, the effect of InHO and PrHO on serum TNF - alpha levels, NF - kappa B activity and EAAT3 expression were measured. Reconditioning with InHO and PrHO decreased NDS and upregulate EAAT3 and increase serum TNF- alpha level and NF- kappa B activity significantly. Although further studies are needed to clarify the mechanisms of ischemic tolerance, InHO and PrHo seems to partly exert their effects via increase in serum TNF- alpha levels, NF- kappa B activity and upregulation of glutamate transporters

Application of SYBR-Green Real Time RT-PCR for quantification of human immunodeficiency virus type-1 [HIV-1] and comparison with COBAS AMPLICOR test

In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26_seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIB-1 Monitor test. The results demonstrated that this technique could detect up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers [5x10[2]- 5x10[9]]. Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results [R[2] =0.95]. On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test [5x10[9] versus 7.5x10[5]]. This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis, Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than 3% and 4.5% accordingly. The above results, indicates that the new developed test can be a used in substitution of the commercial assay for quantitative analysis of HIV-1