Pathobiology Research
Year:2010 | Volume:12 | Issue:4|Papers Count:10

Objective: Pseudomonas aeruginosa is the major cause of septicemia and wound infection in burned patients. Immunotraphy is the best practical way for prevention and treatment of these infections. Flagella as one of the most important bacterial virulence factors has important role in attachment, motility, chemotaxis and TLR-5-dependent immune response so that it propounded as a vaccine candidate. Production of anti-flagellar antibodies and evaluation of its protective effects in burned induced infection of mice was the main aim of this study. Materials and Methods: In the first step, flagellar antigen prepared by ultra-centrifugation. Anti-flagellar antibodies produced in rabbit and its impurity separated by absorption technique. Specification of the obtained antibodies for flagellar antigen was investigated via agglutination test. After determination of LD50 in a known strain, different dilutions of anti-flagellar antibodies injected in burned mice for passive immunization. The rate of bacterial spread from burn site was determined by quantification assay of bacteria in skin and liver. In this study, clinical isolate and PA103 in addition to ATCC 27853 strain were used for agglutination test. Results: H-antiserum reduced mortality of burned mice challenged with ATCC 27853 strains about 80%. Counting of bacteria in the skin and liver showed that the number of bacteria in immunized mice, in contrast with control group, was significantly low. Conclusion: The results of this study showed that anti-flagellar antibodies of Pseudomonas can inhibit invasion of Pseudomonas and facilitate its opsonization, so these antibodies have protective effects in burned wound infections.

Objective: HLA-G is a nonclassical major histocompatibility complex antigen and is expressed as seven isoforms, including four membrane bound (HLA-G1 to –G4) and three soluble (HLA-G5 to–G7) forms. The pattern of selective expression of HLA-G transcripts in tissues shows the existence of a tight transcriptional control on the gene expression. It has been revealed that cytokines including interfrons and IL-10 could cause stimulation of the HLA-G transcription. The purpose of this study was to examine the effects of IFN-g on the expression of HLA-G transcripts in both PBMCs of normal and SLE patients. Materials and Methods: Whole blood of 20 female SLE patients and 15 healthy donor candidates for Bone Marrow Transplantation were used. PBMCs were isolated from the whole blood by Ficoll gradient centrifugation and cultured with or without IFN-g/LPS for 48 hours. Total RNA was extracted from the cells by trizol method. After reverse transcription of RNA to cDNA and the performance of a multiplex PCR for beta actin and HLA-G, the PCR products were analyzed using electrophoresis on a 2 % agarose gel and stained with ethidium bromide. Results: The results showed that the transcription of HLA-G was higher in SLE patients compared to normal controls. Addition of IFN-/LPS could influence the expression of this molecule by increasing the transcription of HLA-G in both normal and patient PBMCs (P£0.05). Conclusion: Transcription of HLA-G gene could be increased by the cytokine IFN-g. This observation is in accordance with previous reports. This effect could be assigned to both normal and lupus Patients. The effects of the cytokine IFN-g/LPS in the induction of HLA-G transcription were higher in normal than lupus patients. Nevertheless, the total expression of HLA-G was higher in lupus patients.

Objective: Endothelium is a selective and permeable membrane for transferring nutrients and vital components to arterial wall. Endothelial damage might lead to altered biological function of endothelium and clinical consequences such as atherosclerosis. Blood pressure pulse always exerts circumferential tension to the arterial wall. Hence, such tension together with other loads, play important role in functional properties of endothelial cells. Previous studies verify effects of cyclic loading on adaptation and remodeling of endothelium. This study investigates structural properties of cultured endothelial cells subjected to uni-axial cyclic loading. Materials and Methods: Human umbilical vein endothelial cells, prepared from national cell bank of Iran (NCBI-C554), were cultured on silicon membrane, and then subjected to cyclic tension with 10% amplitude and 1 Hz frequency, and 2, 4, 6, 8 hour durations utilizing a custom made tensile device. Viscoelastic properties of endothelial cells were examined by micropipette aspiration technique. Results: Results show increase in elastic modulus (E) of cells due to tensile cyclic loading which results in stiffening of cell body. Also results show primary increase then subsequent decrease in viscose modulus. Conclusion: Previous studies verify generation of stress fibers due to accumulation and increase in actin fibers in endothelial cells after tensile cyclic loading. Since mechanical and structural properties of endothelial cells depend on actin fibers, results of this study show tensile cyclic loading causes increase in stiffness of endothelial cells through generation of stress fibers.

Objective: The LRR (leucine rich proteoglycans) is a molecular recognition motif found in proteins with some roles in cell adhesion, signal transduction, DNA repair and RNA processing. Opticin is a member of this family. Takanosu et al (2001) detected messenger RNA expression of mouse opticin in the eye, heart, brain, testis, thyroid and epididymis by dot blot hybridization. In this study, expression levels of mRNA and protein of opticin was investigated by two monoclonal antibodies which were raised against opticin peptides. By this method structure of opticin in human and mouse has been studied. Materials and Methods: Mouse tissues including, kidney, testis, liver, lung, heart, brain, muscle, spleen and eye were isolated. Opticin expression was identified at mRNA and protein levels by RT-PCR and Western blot. Results: PCR analysis revealed that opticin mRNA is expressed in all the tissues studied except for the lung. However, opticin protein was detected in all tissues analyzed. Conclusion: Expression of opticin in the adult murine tissues may suggest functions other than that of putative regulation of vitreous collagen fibrillogenesis for this molecule.

Objective: Human CMV is the most causative agent of a very common viral infection contacted by most adults that have no noticeable or with only mild uncharacteristic symptoms. However when a pregnant women is infected with CMV as a primary infection, there is a risk for transmission of virus to the fetus as well as reactivation of virus in rare case. HCMV antibodies were already described in spontaneous abortion and fetal abnormalities cases. Also antibodies against HCMV in fetal abnormalities as well as abortion had been reported by several studies in different part of the world. Due to lack of published data about CMV epidemiology in Ilam, the aim of current study was to determine the seroprevalence of specific viral IgM and IgG in spontaneous abortion cases as well as the age and socioeconomic status in the studied population in Ilam. Materials and Methods: Sera sample from 42 patients in abortion process as well as 30 healthy pregnant and 30 healthy women as negative control were collected and quantitative serological test to assess IgM/IgG against HCMV was performed using a commercial ELISA assay. SPSS software was used to analysis the results and demographic information. Results: Among 42 patients in abortion process, IgG was found in 6 (14.28%) patients and IgM in 12(28.58%) cases. Based on demographical information, it was showed that IgG seropositivity correlate with the increase of age, but there is no correlation between IgM and age of patients. Conclusion: The results showed that there is a high seroPrevalence of HCMV IgM than IgG among pregnant women in the process of abortion in Ilam; correlation between Age and IgG anti body seroPrevalence was same as other reported. Based on the current studies, it seems that more sensitive and specific method such as NAT method is needed for determination of CMV and abortion procces.

Objective: Profound consumption of medicinal plants products worldwide and public misconception of the products safety puts the urgent need forward as to evaluation of their safe and harmful aspects. In the present study the Lavandula angustifolia essential oil was studied with a view to the foregoing criteria. Materials and Methods: The antimicrobial, antioxidative, hematologic and cytotoxic properties of Lavandula angustifolia essential oil were studied. Results: The bacterial strains sensitive to Lavandula angustifolia oil were in the following order: S.aureus> E.coli> K. pneumonia> Streptococcus faecalis> P. aeruginosa. Antioxidative property of the oil was carried out using beta carotene bleaching test and the results were compared with the standard synthetic antioxidants. Lipid peroxidation inhibitions were lower than the synthetic antioxidant BHT and BHA. The oil concentration required for 50% (IC50) free radical scavenging of DPPH was 56 mg/ml with total phenol contents of 85.43 mg GAE/mg for L. angustifolia oil. Ferric-reducing antioxidant power (FRAP) in the blood sera of the rats gavaged with a daily dose of 100 ml oil increased by 167.57%. Adverse therapeutic effects were noted as a result of feeding the rats with the essential oil. The volatile oil displayed cytotoxic effects on the human tumor cell line (HeLa cells) and peripheral blood cells with the IC50 of 26 and 21mg/ml respectively. The mutagenic and antimutagenic properties of various concentrations of Lavandula angustifolia oil on TA98 and TA100 strains Salmonella typhimurium in the presence and absence of S9 fraction were determined. Conclusion: The results show that the Lavandula oil used in the present study may not be consumed without dose determination.

Objective: Nowadays, as the field of neural tissue engineering advances, the fabrication and application of combined structures open a new window of research for the regeneration of nervous system injuries. In this study, chitosan/poly (vinyl alcohol)-carbon nanotube nanocomposites has been exploited as scaffolds. Materials and Methods: Electrospinning was used to fabricate chitosan/poly (vinyl alcohol)-carbon nanotube scaffolds. Raman spectroscopy and scanning electron microscopy (SEM) was used to evaluate the chemical and physical structure of the electrospun scaffolds. Then, the biocompatibility of the scaffolds was evaluated using MTT assay and Neutral red assay. Results: The results showed that the chitosan/poly (vinyl alcohol)-carbon nanotube nanocomposites have suitable structural and morphological aspects for human brain-derived cells growth and proliferation. Therefore, the cells could maintain their usual morphology while adhering to the surface of the nanocomposites due to an appropriate biocompatibility of the scaffolds. Conclusion: Chitosan/poly (vinyl alcohol)-carbon nanotube nanocomposites could enhance the proliferation of human brain-derived cells due to their proper structure and biocompatibility.

Objective: The secreted aspartic proteinases (Sap2) of Candida albicans has prominent role on Candida adherence, invasion, and pathogenicity. The aim of this study was cloning, expression and characterizing of Sap2 enzyme. Also in this study for the first time, the expression system P. pasturis was used for expressing the recombinant protein. Materials and Methods: C. albicans Sap2 gene was amplified by PCR with sticky ends, EcoR1 and SacII, and it was subcloned into the T/A vector. The sequencing of this gene was done with universal primers and then the Sap2 gene was cloned into pGAPZaA expression vector. The construct was transformed into P. pasturis yeast; the Sap2 gene integration into the yeast genome was accomplished by the homologous recombination. The expressed protein was confirmed by western blotting using monoclonal antibody against Sap2 protein. Finally, the recombinant protein was purified by Ni-NTA chromatography column, and the activity of the enzyme was confirmed. Results: In this study, we successfully amplified C.albicans Sap2 gene and subsequently integrated into the yeast pichia pasturis genome by homologous recombination. Moreover, we were able to identify a yeast clone secreting the recombinant protein. The optimum over expression of sap2 protein was obtained after 96 h, at 30oC. Conclusion: Expression of Sap2 gene in P. pasturis, in comparison to bacterial expression system, leads to a high-level expression, and also need for post translation modifications, that might be required for the activity of enzyme, is obviated in the yeast system. Based on our results, the purified acid aspartyl proteinase purified from P. pasturis was capable of degrading BSA as a substrate in-vitro. The recombinant Sap2 protein had maximum activity in an acidic pH.

Objective: Today, AIDS is considered as a global problem and many efforts to generate an effective vaccine against this disease have been made, but remain inconclusive. DNA vaccines are a member of the new generation of vaccines that can efficiently stimulate the immune system. However, recent findings indicate low immunogenicity for these vaccines and it is believed that these types of vaccines require strategies that could infer more immunogenicity. The employment of adjuvants could be considered as one of the most important methods involved. In this study, a DNA vaccine candidate for HIV P24-Nef is constructed and then using genetic adjuvants IL-15 and GM-CSF, cellular immune responses have been studied. Materials and Methods: In this study the gene structure of HIV P24-Nef in eukaryotic expression vector was constructed and expression vectors of IL-15 and GM-CSF were used as adjuvants. After inoculation of the candidate vaccine to BALB/c mice, cytokine patterns, lymphocytes proliferation and cytotoxicity were analyzed. Results: Our findings indicate that candidate vaccine significantly stimulated cellular immune responses. The usage of IL-15 and GM-CSF as DNA adjuvants together and separately with candidate vaccine has strengthened cellular immune responses significantly. Co-administration of DNA adjuvants significantly increased cellular immune responses when the ratio of the vaccine dose was more than the adjuvants. Conclusion: The sequences that we selected as candidate vaccine demonstrated good immunogenicity in mouse model and co-administration of IL-15 and GM-CSF DNA adjuvants increased cellular immune response to DNA vaccine construct.