[ use of rolling circle amplification for molecular identification of cladophialophora carrionii and cladophialophora yegresii]

Epidemiological studies indicate that not only the incidence of fungal infections is dramatically on the rise, especially in the immunocompromised hosts, but also the sensitivity of etiological agents to antifungal drugs shows a remarkable reduction. Therefore, early detection at the species level is critically important for proper clinical management. Because standard diagnostic procedures are time consuming, expensive, and less sensitive, PCR-based molecular techniques have been developed. In the present study, we aim to describe a rapid and sensitive technique based on the rolling circle PCR amplification for accurate and fast identification of Cladophialophora carrionii vs. C. yegresii. Specific padlock probes were designed based on a single nucleotide polymorphism [SNP] difference in the internal transcribed spacer [ITS] rDNA region of Cladophialophora strains to differentiate between C. carrionii and C. yegresii. The probe sequences are complementary to the target DNA leading to the linker position that after hybridization with the target DNA will be joined together by DNA ligase, form a closed molecule and hybridize to the target DNA for replication at single-temperature conditions. We successfully amplified the target fungi DNA at the species level without any false and negative cross reactivity. The RCA product was visualized on 1.5% agarose gel to clarify the specificity of the probe-DNA template binding. These results demonstrate that RCA is a powerful and accurate tool for discrimination and identification of pathogenic fungi

[Evaluation of the Immunogenic effect of lipopolysaccharide [LPS] and protein fractions of Legionella pneumophila in challenging with lethal dose of this bacterium in mice]

Legionella pneumophila is the cause of legionellosis disease that can be fatal. Yet, no vaccine has been available for this infection. Also antigens of these bacteria can stimulate the immune system. The purpose of this study was to compare the immunogenic effect of lipopolysaccharide fraction with that of protein fraction of Legionella pneumophila in challenging with lethal dose of these bacteria in mice. After preparation of bacterial biomass, LPS and protein fractions were separated by hot phenol method and precipitated by enzyme digestion. LPS and protein fraction electrophoresis on poly acrylamide gel was performed. For preparation of vaccines from LPS and protein fractions, 10 micro g of each antigen was solved in 0.5 ml of normal saline and used for injection. Six groups of female BALB/c mice [each group consisted of 15 mice] were selected. Four groups of mice were vaccinated by intraperitoneal [i.p] injections at fortnightly intervals for three times. The two control groups of mice received normal saline injections. Two weeks after the last immunization, two groups of vaccinated mice and one control group were challenged with LD100 of the virulent strain of L. pneumophila. Also six weeks after the last immunization the other three groups [2 vaccinated and one control groups] were challenged. Result: The results of the first challenge showed the immunogenic efficiency gains of 86.66% and 73.33% for protein fraction and LPS respectively, and after six weeks of the last immunization the immunogenic efficiency gains were 60% for LPS and 86.66% for protein fractions. This study showed that the protein fraction and LPS of L.pneumophila have high immunogenic activity and can be proper candidates for vaccine studies

[ effect of temporary middle cerebral artery occlusion on reduction of brain injuries in rat stroke model]

Recent studies suggest that sub-lethal ischemia protect the brain from subsequent ischemic injuries. This study was an effort to identify and shed light on the nature of changes in the blood brain barrier permeability and brain edema. Rats were divided into four main experimental groups, each of 21 animals. The first group acted as a model of ischemic preconditioning which was subjected to 10 minutes of temporary middle cerebral artery occlusion in the first day [tMCAO] and in the second day, was subjected to 60 min middle cerebral artery occlusion [MCAO]. The second group acted as a control group and did not receive any surgery except 60 min middle cerebral artery occlusion in the second day. The third group served as a sham group, and was subjected to surgery with 10 min of temporary middle cerebral artery occlusion [tMCAO] in the first day. The fourth group remained intact and was not subjected to any surgery. Each main group subdivided into three subgroups [n=7] for infarct volume [n=21], blood brain barrier permeability, and brain edema. After 24 hours, each main group was subjected to 60min of right MCAO occlusion. Then, neurologic deficit score [NDS], infarct volume, blood brain barrier permeability, and brain edema were assessed in the subgroups. Preconditioning with tMCAO decreased NDS and infarct volume, brain barrier permeability, and brain edema. Temporary middle cerebral artery occlusion [tMCAO] is associated with neurologic deficit scores, infarct, blood brain barrier permeability, and brain edema consistent with an active role in the genesis of ischemic protection

[Isolation of legionella pneumophila from environment and water system samples and evaluation of immuno-protective efficiency of its whole killed cell in mice model]

Legionella pneumophila is a cause of pneumonia in human beings. The purpose of this study was to separate L.pneumophila from stagnant and waste water, city squares, coolers and faucets and evaluation of the immunoprotective efficiency of its whole killed cells in mice model. Water samples were prepared, concentrated and then cultured on selective [GVPC] media. After identification of L.pneomophila the biomass of the organism was fixed with 0.5% formalin in sterile saline at 37§C for 24 hours in order to prepare whole killed cells. Four groups of female BALB/c mices [each group consisted of 15 mices] were selected. Two groups of mice were immunized by three intraperitoneal administrations of prepared antigen in a dose of 4x108 CFU from whole killed cells at two week intervals and control groups received only sterile saline injections. Two weeks after the last injection, one group of immunized mice and one of the control groups were challenged with the lethal dose of virulent strain of L.pneuophila and also the two other groups of mice were challenged six weeks after the last immunization. From 120 water samples 27 samples were contaminated with L.pneumophila. Challenge results showed that the immune efficiency of whole killed cell was 93.33% after two weeks of the last immunization, and 86.66% after six weeks of the last immunization. This study showed that, stagnant water had the highest rate of contamination with L.pneumophila and the whole killed cell of L.pneumophila is a proper candidate for L.pneumophila vaccine studies

[ effect of increasing blood flow rate on complications and dialysis adequacy in hemodialysis patients with low KT/V]

Insufficient dialysis, increasing complication, duration of hospitalized and expenditures in end stage renal disease patients. Several factors, including increased blood flow rate in the absence of complications can improve dialysis adequacy. So the aim of this study is to investigate the Effected of increasing blood flow rate on complications and dialysis adequacy in hemodialysis patients with Low KT/V. In this quasi-experimental study ,one group [pre-post test], all 45 hemodialysis patients, at 6 month bygone had 3 times in week, each time 3 to 4 hours of hemodialysis and KT/V was less than 1.2, in two sessions, that in first session adjustment blood flow rate on 230 ml/min and other increased 15% of blood flow rate in patients<65 Kg of body weight and 20% in patients>65 Kg in 2009 By the questionnaire and Check list were studied, and than we control KT/V and complications and finally by the statistical tests such Paird-Samples /-test and McNamara were compared. Mean systolic and diastolic blood pressure, incidence of hypotension, nausea, vomiting, muscle cramp did not have a significant difference within the tow status and the mean KT/V with the before and after increased blood flow rate ,was respectively 0.99 +/- 0.09 and 1.3 +/- 0.12 and results showed significant difference [P=0.001] comments from dialysis adequacy. Although the Complications during hemodialysis, while the two study conditions did not have a significant difference and also increase blood flow rate caused increase dialysis adequacy can be concluded that increased blood flow rate is effective on dialysis adequacy

[Investigation of TNF-alpha converting enzyme and activity of nf-kb in tolerance to ischemia induced by intermittent normobaric hyperoxia in rat model]

Recent studies suggest that intermittent and prolonged normobaric hyperoxia [HO] results in ischemic tolerance to reduce brain injury. In this research attempts were made to see the changes in TNF-alpha converting enzyme [TACE] and NF-kB activity following intermittent HO and ischemia preconditioning. The rats were divided into two experimental groups, each consisted of 20 animals. The first group was exposed to 95% inspired HO for 4h/day for 6 consecutive days [intermittent HO; InHO]. The second group acting as the control was exposed to 21% oxygen in the same chamber [normobaric normoxia or room air; RA] continuously for six days [intermittent RA; InRA]. Each main group was subdivided to MCAO-operated [middle cerebral artery occlusion], sham-operated [without MCAO], and intact [without any surgery] subgroups. After 24hr, MCAO-operated subgroups were subjected to 60 min of right MCAO. After 24 h reperfusion, neurologic deficit score [NDS] and infarct volume were assessed in MCAO-operated subgroups. Immediately and 48 h after pretreatment, blood sampling for assessment of serum TNF-alpha levels were subjected. Then, the effect of intermittent HO and ischemia on NF-kB activity and TACE expression were measured. Preconditioning with intermittent HO and ischemia decreased NDS and infarct volume. Moreover InHO and MCAO-InHO upregulate TACE and increase NF-kB activity significantly. Although further studies are needed to clarify the mechanisms of ischemic tolerance, InHO and ischemia seem to partly exert their effects via increase upregulation of TACE and NF-kB activity