Pathobiology Research
Year:2016 | Volume:18 | Issue:4|Papers Count:8

In recent years, electrospinning that has the capability to form polymeric nano-/microfibers has gained substantial attention for fabrication of tissue engineering scaffolds. The morphological resemblance to native extracellular matrix (ECM), high surface to volume ratio, high porosity, and pore interconnectivity are amongst the brilliant features of electrospun structures. The high surface area to volume ratio and interconnected pores of these fibrous meshes confer desirable cell attachment and growth. However, due to small pore sizes and high packing density of electrospun nanofibers, cell penetration into a conventional electrospun mat is completely restrained. Scarce cell infiltration in turn prohibit cell migration into internal parts of the scaffold, cause inhomogeneous cell distribution throughout the structure, limit vascularization, and impede tissue ingrowth. In fact, traditional electrospun nanofibrous scaffolds in practice act as two-dimensional (2D) surfaces rather than three-dimensional (3D) microenvironments. Thus far, a number of approaches have been employed to solve this problem, which range from simple variations in electrospinning parameters to intricate post-processing modifications. Some efforts directly manipulate the electrospun mat characteristics to enhance cell penetration, while others combine cells with scaffolds or encourage cells to migrate into internal parts with different stimuli. In the present study, we have attempted to provide an overview of different approaches offered for improving cell infiltration in electrospun scaffolds.

Objective: Cholera is an endemic disease in Iran. Early detection, especially in times of disease outbreaks, is of vital importance. The antibody against the lipopolysaccharide (LPS) is an important method for bacterial detection. This study intends to extract and purify the LPS of Vibrio cholerae and evaluate the cholera antibody for detection purposes.Methods: Vibrio cholerae was cultured in tryptone extract medium. LPS was extracted by the hot phenol water method, purified, and dialyzed. We measured the LPS protein and sugar content, purity, and biological activity. Antibodies were produced by injection of the killed bacteria with Freund's complete adjuvant into rabbits and then the LPS was injected three times with Freund's incomplete adjuvant. After the last booster, blood samples were taken. We used ELISA to determine the antibody titers against the Inaba and Ogawa serotypes, the LPS of these serotypes, and several other similar bacteria.Results: The amount of protein in the purified LPS was approximately zero and sugar was 0.5 mg/ml. The LPS had a titer activity of 1024, and consisted of three bands (5.2, 4, and 5.14 KD). Antibodies produced by the rabbits identified the bacterial Inaba and Ogawa serotypes, and the purified LPS. Ogawa and Inaba serotypes cross-react with each other but not with other species of Vibrio and other bacteria. The LPS antibody titer against the Ogawa serotype was 1: 32000, whereas for Inaba it was 1: 16000.Conclusion: Due to the low cost of production, high sensitivity, and importance of cholera diagnosis in Iran, the antiserum produced in this study can be used as a tool for early screening of cholera and discrimination of O1 strains from non-O1 strains in immunologic tests.

Objective: This study attempted to generate monospecific antibodies through immunization with recombinant proteins and subsequent purification by synthetic peptides (the PrIPeP model).Methods: The SRY gene was cloned on a pet-28a vector and the recombinant protein was expressed in the Escherichia coli (E.coli) BL21 strain. The purified antigen was emulsified in Freund’s adjuvant and injected into rabbits according to a standard time table. Then, a specific peptide was designed, synthesized, and conjugated to sepharose 4B to generate an affinity purification column. As a control, the peptide was conjugated to KLH and used for immunization, as above. Antisera against the conjugated peptide (Pepantisera) and SRY recombinant protein (Pro-antisera) were evaluated by ELISA and subsequently subjected to the affinity purification column. Sensitivity and specificity of the purified antibodies against SRY recombinant protein as well as negative controls (recombinant HSFY, RBMY, and RPSFY) were assessed by Western blot analysis.Results: Titration by ELISA confirmed proper immunization and specificity of both antigens. Western blot analysis validated the specificity and sensitivity of the IgG class purified antibodies.Conclusion: By applying the PrIPeP model, it is possible to develop antibodies against the native structure of a protein whilst avoiding challenges of peptide-carrier protein conjugation.

Objective: Enterohemorrhagic Escherichia coli (EHEC) which produces shiga-like toxin type 2 (Stx2) is a major cause of bloody diarrhea. This pathogen can lead to hemolytic uremic syndrome (HUS) and renal failure with a high mortality rate. Stx2 is the major virulence factor of EHEC. Neutralization of toxin by specific antibodies is known to be the best way to prevent and cure HUS. In this study, we describe the cloning, expression, purification, and immunization of the Stx2B subunit which is responsible for toxin binding to the target cell surface.Methods: The Stx2B gene was amplified by PCR and subcloned into a pET28a expression vector and transformed into E. coli BL21-DE3. We evaluated recombinant protein expression and rSTX2B was purified by the Ni-NTA column. The purified rSTX2B was administered subcutaneously to BALB/c mice in three separate doses as an immunogenic candidate. The raising of anti-rSTX2B antibodies in immunized mice sera was evaluated by Elisa assay. The neutralizing immune response was verified by an in vitro assay on HeLa cells and an in vivo assay on mice by challenging them with a lethal dose of Stx2.Results: The IgG titration verified the induction of a humeral response in immunized mice. The HeLa cell assay indicated that the Stx2 toxin was neutralized by immune mice sera. In the challenge assay, 70% of immunized mice survived.Conclusion: Recombinant rSTX2B can induce a neutralizing immune response in mice. It can be used as a major component in development of EHEC vaccines.

Objective: Vitrification is a convenient, effective method for freezing and storing embryos. Under certain situations, such as an unsuitable endometrial environment, extra embryos can be re-vitrified for future use. There is inadequate data on the effects of revitrification on embryos, so we have evaluated the effects of re-vitrification on the development rate and expression of apoptotic and implantation genes.Methods: Female NMRI mice, ages six-eight weeks were super-ovulated with 7.5 IU PMSG and 7.5 IU hCG. Females were mated with males from the same strain and inspected for the presence of vaginal plugs the following morning. Females with the presence of vaginal plugs were considered to be pregnant and killed 62 h post hCG injection. Eight-cell embryos were flushed from their oviducts and subsequently divided into three experimental groups: fresh, vitrified-warmed 8-cell embryos, and re-vitrified warmed blastocyst embryos. RNA was extracted and we used real-time PCR to evaluate expressions of Bax, Bcl-2, and ErbB4. Data was analyzed by the chi-square and ANOVA tests.Results: A significant difference existed in blastocyst formation rate, degeneration rate, and expressions of Bax, Bcl-2, and ErbB4 in re-vitrified embryos compared to fresh embryos.Conclusion: The vitrification and warming process did not affect the developmental rate and expressions of Bax, Bcl-2, and ErbB4 in the eight-cell stage embryos. However, we observed a change in development rate and expression rates of Bax, Bcl-2, and ErbB4 after re-vitrification in the early blastocyst stage.

Objective: Hepatitis C virus (HCV) is considered to be a worldwide health problem. In most cases, HCV infection becomes chronic and may proceed to fibrosis, cirrhosis, and hepatocellular carcinoma. Many pathological effects in cells may occur by viral proteins. The purpose of this study is to evaluate the effect of the HCV core protein on cells to induction of the fibrogenesis process.Methods: We use the LX-2 cell line that originated from hepatic stellate cells. Plasmid which expressed HCV core protein was transfected to the cells. After 72 h, RNA was extracted and treated with DNase, followed by synthesis of cDNA. Positive control cells were treated with the leptin fibrotic hormone. We used real-time PCR to measure and statistically analyze a-SMA gene expression.Results: The HCV core protein significantly increased α-SMA gene expression (p<0.05). There was more a-SMA gene expression in cells treated with leptin compared to cells treated with the HCV core protein.Conclusion: HCV infection is an impressive factor in the development of chronic hepatitis to hepatic fibrogenesis. The HCV core protein can induce a fibrogenesis process in HCV infection.

Objective: Cerebrospinal fluid (CSF) has a broad range of molecules and neurotrophic factors essential for neurogenesis. Bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells that can differentiate into the cells with neural-like phenotype under the induction of appropriate growth factors. According to the significant role of retinoic acid (RA) in neurogenesis, this study aims to differentiate BMSCs into neuronlike cells using CSF, RA, and the combination of CSF and RA.Methods: Rat BMSCs were isolated and characterized. The CSF was prepared from the cisterna magna of 19-day-old Wistar rat embryos. The BMSCs were induced by either 5% CSF (CSF group), 10-6 mM RA (RA group), or CSF plus RA (CSR group) for 12 days. Morphology of differentiated cells was examined by inverted microscope and axonal outgrowth measured using Image J software. In addition, the expression of neural-specific markers (Nestin and MAP-2) was examined by immunocytochemistry.Results: We observed specific-neuronal morphology in the differentiated cells. The maximum axon length was seen in the CSR group on the 12th day of induction. Immunocytochemistry results showed that the neural progenitor marker (Nestin) was expressed in all treated groups. However, MAP-2, as a mature neural marker, was only expressed in the CSR group.Conclusion: The findings suggest that CSF accompanied RA lead to differentiation of cells with neuronal and glial phenotypes from BMSCs in vitro.