Pathobiology Research
Year:2008 | Volume:11 | Issue:1-2|Papers Count:12

Objective: Helicobacter pylori is a spiral, microaerophilic gram negative bacterium, that multiplies and causes infection in human gastric mucosal layer. H.pylori infection, followed by destruction of gastric epithelial tissue, leads to gastric chronic inflammation, which can cause gastric and peptic ulcers. New approaches have focused on using specific treatments, such as immunotherapy, to eradicate this infection. Urease, as one of the most important virulent and antigenic factors of the bacterium, is a suitable target for this purpose. This study is aimed at production of specific IgY against urease UreC subunit.Materials and Methods: In this study, initially for preparing recombinant UreC, after purification of the genomic DNA, ureC gene was amplified by polymerase chain reaction (PCR). The PCR product was ligated to pET28a. The recombinant protein was expressed followed by transformation of recombinant construct into E. coli BL21DE3. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to hens. IgY recovered from egg yolk, was purified by PEG precipitation at >70% purity. The purified IgY was analyzed by ELISA and SDS-PAGE.Results: SDS-PAGE analysis revealed a good expression and >70% purification of the recombinant protein. ELISA observation demonstrated high immunogenicity of the recombinant protein.Conclusion: With a view to higher potential of IgY-HpUc in recognition of UreC subunit, the results are in favour of the oral administration of the IgY obtained from hens immunized by H.pylori may provide a novel approach to the management of H.pylori infections.

Objective: DNA vaccines have been widely used to develop immunity against various pathogens including parasites and viruses. The potential of DNA vaccine to induce an effective immune response is related to the expression levels of the encoded protein in eukaryotic cells. Therefore, optimization of plasmid DNA delivery system is a major concern in protein expression in order to make an efficient DNA vaccination. Non-viral vectors such as polymers and cationic peptides have been recently known as efficient gene delivery systems into eukaryotic cells. In this study, transfection efficiency of HPV16E7 gene was evaluated by two non-viral delivery systems in vitro.Materials and Methods: DNA construct encoding HPV16E7 (pEGFP-E7) was prepared in large scale with high purity. Then, two delivery systems including polymer PEI 25 kDa and polymer-peptide hybrid as PEI600-Tat conjugate were used to compare their efficiency for HPV16E7 DNA transfection in vitro.Results: Our data demonstrated that both delivery systems including PEI 25 kDa and PEI600-Tat are efficient tools for E7 gene transfection. Although the level of transfected COS-7 cells is higher using PEI 25 kDa in comparison with PEI600-Tat. Conclusion: Our study indicated that PEI potency for E7 gene transfection was higher than PEI600-Tat in vitro, but its toxicity was obstacle in vivo. Therefore, with regard to low toxicity of PEI600-Tat delivery system and its potent plasmid DNA delivery, it is critical issue to study its potency as new delivery system in vivo

Objective: 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.Materials and Methods: During this study specimen biopsies were collected from 180 patients who underwent routine gastrointestinal endoscopies to the internal medicine ward, Hazrat Rasoul Akram Hospital, IUMS, 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.Result: In this study out of 180 samples of 92 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 s1/m2 genotype. 57 (62%) 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.Conclusion: 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.

Objective: Ischemic preconditioning (IPC) is an endogenous phenomenon that can induce ischemic tolerance (IT) in variety of organs such as brain. In this study, we examined the intermittent and prolonged normobaric hyperoxia (HO) on neurologic deficit scores, infarct volume, and superoxide dismutase activity.Materials and Methods: The rats were divided to four main groups. First two main groups were exposed with HO in prolonged (24 h; PrHO) and intermittent (4 h×6 days; InHO) groups and second two main group acted as controls, and were exposed to 21% oxygen in the same chamber (room air, RA) continuously (24 h; PrRA) and discontinuously (4 h×6 days; InRA). Each group subdivided to three subgroups. After 24 h, first subgroup were subjected to 60 minutes MCAO followed by 24 h of reperfusion. Then, IT induced by InHO and PrHO were measured by neurologic deficit scores and infarct volume. Second and third subgroups were called sham-operated and intact subgroups for assessment of the effect of HO on superoxide dismutase activity.Results: Our findings indicate that InHO and PrHO are involved in the induction of IT. Pretreatment with InHO and PrHO reduced neurologic deficit scores and infarct volume significantly. InHO and PrHO increase superoxide dismutase activity significantly.Conclusion: Although further studies are needed to clarify the mechanisms of ischemic tolerance, InHO and PrHO seem to partly exert their effects via increase superoxide dismutase activity.

Objective: One of the most significant damages to the genetic material is oxidative deamination of DNA and free nucleotides in the cell pool. Incorporation of deaminated purine nucleotides such as inosine triphosphate (ITP, dITP) into the DNA can increase the frequency of base substitution mutation. it has been suggested that presence and accumulation of these rough nucleotides can lead to genetic instability which is the perquisite of different types of diseases or cancers. Inosine triphosphate pyrophosphates (ITPase) encoded by ITPA gene, is responsible for protecting the cells by omitting deaminated purines from the free nucleotide pool. The objective of this study was to examine the possible dysfunction of ITPA gene activity as an important factor in genetic background predisposing to chromosomal disorders and malignancies such as CML. Materials and Methods: ITPA gene expression study performed on 23 CML patients and 21 controls using semi-quantitative RT-PCR technique and the expression level of GAPDH gene was used as an internal control. Unusual variants obtained from cDNA amplification of ITPA gene were cloned.Results: Our results showed a significant reduction of ITPA gene expression in CML patients in comparison to the controls. Two types of transcripts were produced in addition of expected transcript in some samples. One of them had a 123 nucleotide and the other had 77 nucleotide deletions in their open reading frame.Conclusion: Due to decreased expression of ITPA gene; it seems that the function of ITPase is not normal in CML patients. Therefore the altered expression of this gene can be considered as an additive factor for genetic instability in these patients

Objective: Despite sensitive antibody based blood donor screening, infection can be transmitted during window period. Therefore sensitive methods based on nucleic acid tests (NATs) have been considered. The aim of this project was to design a more sensitive method for detection of PCR products and diagnosis of HIV-1/HCV co-infection accurately.Materials and Methods: After designing specific primers and probes, the Multiplex RT-PCR method was optimized and the PCR products were labeled with Digoxigenin. The PCR product was denatured under alkaline condition and was hybridized with the specific probe that had a biotin at 5' end, and then was added to streptavidin coated wells. After washing an antibody against DIG, conjugated with alkaline phosphates enzyme was used, following second washing, the substrate (ABTS) solution was added to each reaction well. Development of green color shows the positive where as no color shows negative results. Results: 35 samples were tested with the developed method including 27 positive samples, 8 confirmed negative and 4 standard panels. False negative or positive reactions were not observed. Conclusion: This method had acceptable sensitivity and specificity for detecting HCV and HIV-1 infections during window period, also the method can be quantified which can be used for the flow-up and treatment of patients. In addition to the very high sensitivity of the test, it is cost effective and takes less time to performe

Objective: Sarcocystosis is caused by species of Sarcocystis, an intracellular protozoan parasite in the phylum Apicomplexa. The intermediate hosts are the herbivores eating infected food or water containing sporocysts excreted by the carnivorous final host, and that result in tissue cysts in corpses. The present study was carried out to identify Sarcocystis species of sheep using PCR-RFLP.Materials and Methods: In the present study 60 specimens from the diaphragm, heart and esophagus muscles of sheep slaughtered in Qazvin Ziaran slaughterhouse were collected. 40 specimens contained macroscopic Sarcocystis cysts and the next 20 contained microscopic Sarcocystis cysts. They were examined by Dob Smear and digestion method. DNA extraction was carried out by a kit. PCR conditions were optimized for 18S rRNA amplification. Meanwhile, a comparative study was done to investigate the specific primers of the Toxoplasma and Neospora along with the Sarcocystis cases. According to the position of restriction sites, restricted enzymes were selected.Results: Results indicated that the primers were completely unique and specific for detecting Sarcocystis. RELP-PCR analysis showed that macroscopic cysts and microscopic cysts belonged to Sarcocystis gigantea and Sarcocystis arieticanis, respectively.Conclusion: Sarcocystis species of sheep can be recognized through PCR-RFLP technique using the designed specific primers. By this technique applying TaqI enzyme for microscopic cysts as well as TaqI and HincII enzymes for macroscopic cysts were found more efficient than the others.

Objective: Bacterial meningitis is a dangerous and sometimes fatal infection that affects the central nervous system. Because some antibiotics can prevent some types of these Bacteria and supress them from spreading and infecting, therefore it is important to know what type of virus or bacterium is causing meningitis. Haemophilus influenzae and Neisseria meningitides are the two main pathogens causinig acute bacterial meningitis. Different methods are used for the detection of H. influenzae and N. meningitidis but they are of low sensitivity, taking long time and difficult to perform. Therefore, complementary methods are necessary for more sensitive detection of these agents.Materials and Methods: In this study, a multiplex polymerase chain reaction (mPCR) assay was developed for detection of H. influenzae and N. meningitidis. These strains were confirmed by biochemical methods. Two specific primer pairs were designed for lic-1 and opa genes of H. influenzae and N. meningitidis respectively.Results: DNA amplification product fragments were 150bp and 320bp for H. influenzae and N. meningitidis, respectively. Streptococcus pneumoniae used as a negative control and did not yield a PCR product.Conclusion: The results of this study indicated that PCR is a useful complementary diagnostic technique, especially when Gram stain, culture, or antigenic detection is negative or inconclusive.

Objective: Molecular epidemiology of Visceral Leishmaniasis (VL) is currently used widely for different objectives such as vector incrimination studies.Materials and Methods: In this study three different loci including kinetoplast DNA (kDNA), ribosomal DNA (rDNA), and Cystein protease B (CPB) of Leishmania parasite genome were used for detection and identification of natural infection of sand flies of Germi district of Ardebil province, the most important VL or Kala-azar foci in Iran.Results: The results showed that the three loci of kDNA, rDNA and CPBs are respectively more appropriate for leptomonad infection/initial screening, identification of the L.donovani complex, and discrimination of the species complex. It was also verified that both members of the complex, L. donovani and L. infantum, are present in the study area and are transmitted to the hosts by Phlebotomus perfiliewi trenscaucasicus sand flies.Conclusion: This is the first report on natural infection of sand flies to L. donovani in the country and since the ecology and biology of L. donovani differs extensively from L. infantum, it is necessary to perform further studies to highlight the role of L. donovani in epidemiology of VL in the region and country.

Objective: Ajowan is an annual herbaceous essential oil of Carum copticom. The main components of the oil are Tymol, b-pinene, g- terpinene and Sabinene. The fruit oil of Carum copticum has been reported to have several therapeutic effects including anti fungal, anti bacterial and anti viral, ... Candida albicans is an opportunistic fungus and transforms into pathogenic form in favorable conditions, causing fungal diseases.Materials and Methods: In this study, essential oil and alcoholic extract of Carum copticum were gained and Microdilution Broth method were used for detection of minimum inhibition concentration (MIC) and minimum fungicide concentration (MFC) of 11 clinical isolates of Candida albicans and Standard strain (PTCC50-27).Results: Results show that MIC for essential oil is 0.43mg/ml, 0.87mg/ml and for alcoholic extract is 3.51mg/ml, 7.03mg/ml, 1/75mg/ml. Thus, it seems that Carum copticum could inhibit Candida albicans growth by a similar mechanism which occurs by Fluconazole (FLZ). In general, the results obtained in this study indicate that Carum copticum has potential values for growth inhibition of Candida albicans in vitro.Conclusion: In recent years, systemic fungal infections due to Candida species have been received major consideration about inducing mortality in nosocomial patients because of increasing in immunocompromised disorders such as AIDS and hematological disorders as well as long term use of broad spectrum antibiotics and corticosteroids. The present study was done with the aim of evaluating antifungal effects of essential oil and alcoholic extract from Carum copticum against Fluconazole (FLZ) susceptible and Fluconazole resistance Candida albicans strains isolated from different types of Candidiasis. Standard drug susceptibility tests with broth dilution technique were used to measure the in vitro antifungal activity of essential oil and alcoholic extract from Carum copticum. According to our results, it seems that Carum copticum could inhibit Candida albicans growth by a similar mechanism which occurs by FLZ and could be used as a potential antifungal agent especially with FLZ.

Objective: Toxoplasmosis may cause significant damage to the developing fetus and is a 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.Materials and Methods: In the present study, quantitative PCR-ELISA (Polymerase Chain Reaction-enzyme linked immunosorbent assay) was used for quantization of Toxoplasma 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. Specific primers targeting RE gene were selected and used for the amplification of toxoplasmic DNA. 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.Results: DNA of Toxoplasma gondii were efficiently detected within 4 hours and no interference was encountered in the amplification and detection of the parasite.Conclusion: Efficienty of the PCR-ELISA system was evaluated we found with several advantages in terms of sensitivity, rapidity and simplicity in this system.