Pathobiology Research
Year:2007 | Volume:10 | Issue:1|Papers Count:12

Objectives: Malaria is the most important tropical disease in terms of morbidity and mortality. The diagnosis of malaria can be conveniently subdivided into clinical, parasitological, biochemical, serological and molecular biological detection. The objective of the present work was to compare two techniques, blood smear and PCR-RFLP, for detection of Plasmodium species.Materials & Methods: Totally, 46 positive blood samples of malaria were examined by these two methods. In parasitological detection, direct observation of Plasmodium in Geimsa-stained thick and thin blood smears were carried out. In polymerase chain reaction (PCR) method, the target DNA was a segment of 18S rRNA gene. In RFLP technique three enzymes, Hinf I, Hae III and Tsp45 I, were used.Results: The results indicated that, by direct observation of thin smear, 35 and I leases were identified as Plasmodium vivax and Plasmodium falciparum respectively. But molecular analysis showed that 2 of 11 cases of Plasmodium falciparum were Plasmodium vivax whereas 3 of 35 cases of Plasmodium vivax were Plasmodium falciparum.Conclusion: In diagnosis of human Plasmodium species, the PCR-RFLP technique was found more appropriate and sensitive than blood smear technique.

Objectives: Human Immunodeficiency Virus type I is the causative agent of Acquired Immunodeficiency syndrome "AIDS" in human and demonstration of RN-I genome in samples is accepted as evidence of infection. Transmission of Infection during window period in blood transfusion settings is a worldwide concern. Also there is a need for a rapid, sensitive and accurate technique to detect RN -I infection prior to antibody appearance in patients and new borns.Material and Method: A rapid Visual DNA Chip based on RT-Nested PCR and Enzyme-Substrate detection system was developed. At first a specific RT-Nested PCR was developed and the products were confirmed in gel electrophoresis and the products were labeled with DIG (Digoxigenin).The labeled products were then hybridized with the pre-prepared chip with an anchored specific probe. After the washing procedure an antibody against DIG conjugated with alkaline phosphates enzyme was used. After the second washing procedure the BCIP/NBT substrate was used and development of color was interpreted as positive while the negative samples developed no color.Results: 35 sera samples from different stages of HIV infection (AIDS, Asymptomatic and Symptomatic Infection) as well as 20 confirmed negative sera samples were collected and checked with the developed assay. All the positive samples developed reaction while the negative samples had no reaction.Conclusion: In the current study the developed assay showed high sensitivity and specificity to detect HIV-I infection. It seems that the viral genome could be detected prior to antibody appearance and hence the window period could be shortened. Also the assay could be used to detect infection in new borns from infected mothers, because the maternal antibody could pass the placenta and antibody based assay have false positive results.Because of the high sensitivity, the developed assay could also detect infection in very low viral load conditions.

Objective: The antigen binding fragments of camelids heavy chain antibodies are comprised in one single domain (VHH). The crystal structure of an isolated VHH indicated that it is a problate particle of 2.5 nm in diameter and <4 nm high, and has been referred to as a nanobody. The very close similarity of these molecules to human VH3 illustrates the potential application of these novel products as an immunodiagnostic immunotherapeutic reagent. One of the key components in production, characterization and application of nanobodies in detection is the anti-nanobody HRP conjugate.Materials and Methods: In this study, we reported high expression and purification of some nanobodies against tumor markers. The nanobodies genes were sub-cloned into a pSJF9 vector to over-express the protein coupled with fusion tags in E. coli TG 1. The expressed nanobodies were purified by immobilized metal affinity chromatography (IMAC). Described here is the preparation, purification and characterization of anti-nanobody antibody HRP conjugate for use in the various nanobody detection systems.Results: Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the purified nanobodies. Because of application of several biochemical modifications, the produced anti-nariobodies HRP conjugate have efficient sensitivity and specificity.Conclusion: By setting the temperature, time and inducer reagent the nanobodies were produced in optimum yield. We concluded that the HRP conjugated anti-nanobody can detect nanobodies in various detection procedures with great sensitivity and accuracy.

Objective: DNA markers are one of the most important indicators for estimating Molecular weight of DNA samples, although it used in widespread medical and research laboratories. These markers are very divers and have been prepared in different manners and from different sources of DNA. But unfortunately, DNA markers haven't been made in our country and all of the markers that we use are made in a foreign country.The aim of this research is settings a suitable technology to produce this product in the lab.Material and Methods: With this aim, we used two different strains of lambda: c1857sam7 and EMBL3A both of which are lytic phages as a DNA source. These were grown in the suitable host, after plaque appearance on the bacterial lawn, suitable titer for phage collecting was determined. We also optimized plasmid purification method for extraction of pBR322, pUC18 and recombinant YZV plasmid DNA and designed fragments in the markers have been constructed by digesting these DNAs with variant enzymes. Results: In this study, we made seven DNA markers out of which four of them were made for the first time in the world (l/Hind III/BamH1, lHind III/EcoR1, Sam2, Sam1) and although foreign models of three of them exist but they were made in our country for the first time (l/Pst I, lHind III, pBR332/MspI).Conclusion: The' other goal of this study was to determining the best conditions for maintaining and preserving these markers in the lab which was successfully performed.

Objectives: The idiopathic minimal change nephritic syndrome (nephrosis) is responsible in 80% of nephritic syndromes in children. It is a clinical entity characterizedby inter and outer renal parameters. Main factor in glomerolar damages is proteinuria and the role of IgE is possible. Aim: In this research, serum IgE concentrations were measured in children with nephrosis in three stages: Relapse, Remission in treatment with steroids, Remissions after treatment with steroids.Materials and methods: Study was done on children under 16 years old that suffered from nephrosis. They treated with Prednisolone. Serum IgE concentrations were measured by ELISA technique.Results: Average Concentration of serum IgE in the relapse phase was 274.8 Iu/ml (SD=14.6); It was in the remission with steroids treatments 179.59 Iu/ml (SD= 14.82) and it was 234. 9 Iu/ml (SD=14.58) in the remission phase after treatment with steroids.Conclusion: In some cases IgE is significantly reduced after steroid treatment therefore it seems possible that allergic agents can trigger or increase the disease. Serum IgE concentration was not a main factor in nephritic syndrome.

Objective: Current of antibiotic treatment for Hpylori infection is often associated with frequent adverse effect and resistant to antibiotic. Alternative treatment method to control Hpylori infection is needed. The aim of this study was to test in vitro anti Helicobacter activity of Lactobacilli acidophilus isolated from yogurt.Material and Methods: Forty Hpylori strains isolated from 145 human gastric biopsies. After the primary culture of biopsies in the brucella brucella  agar and identification of Hpylori by biochemical tests, susceptibility tests to antibiotics (metronidazole, clarithromycin, amoxicillin and tetracycline) were performed with MDDM (Modified Disk Diffusion Method) and E test. The isolates were cultured in brucella broth and the effect of Lactobacillus acidophilus isolated from yogurt (cultured in MRS broth) on growth of Hpylori was tested by a mixed culture technique (co-culture).Results: Twenty five (62.5%) of the 40 H.pylori isolates were resistant to antibiotics. Resistance rates to metronidazole, clarithromycin, amoxicillin and tetracycline were 62.5%, 22.5%, 2.5% and 0% respectively. Twenty two (56%) of the 40 H.pylori isolates were inhibited by L. acidophilus in mixed culture. This inhibition occurred only when the two organisms were incubated together for more than 24 h. Lactobacillus acidophilus was able to inhibit the growth of both antibiotic sensitive and resistant H.pylori strains. Fifteen (68.2%) of the inhibited isolates were susceptible to antibiotics and seven (31.8%) were resistant to antibiotics.Conclusion: We observed a significant suppression in the growth of Hpylori in the presence of L. acidophilus. This occurred only when the two organisms were incubated together for more than 24h. Based on our findings, L. acidophilus was found to be a potentially effective probiotics against Hpylori and could enhance antibiotic therapy fur H.pylori eradication in humans.

Objectives: Salmonella typhimurium is important food-borne pathogen responsible for gastroenteritis. In this work a polymerase chain reaction based enzyme linked immunosorbent assay (pCR-ELISA) was developed to identify Salmonella typhimurium.Materials and Methods: The rfb gene which is responsible for biosynthesis of the Salmonella O-antigenic lip polysaccharide was selected as the target sequence. The selected primers amplified fragment size of 882bp of S. typhimurium. Food samples contaminated with Salmonella typhimurium as well as clinical and standard samples were used in this investigation. The PCR products randomly labeled with Dig-II-dUTP were transformed to a plate coated with streptoavidin and tested with anti digoxigenin. An internal biotin labeled probe was used to confirm the amplified PCR product.Results: The specificity of the assay toward S.typhimurium samples was confirmed by testing 20 Salmonella and 6 non Salmonella strains. ELISA increased the sensitivity of the conventional PCR method by approximately 1000 fold.Conclusion: The method presented here is a rapid and specific alternative for the traditional time consuming culture methods for the detection of S. typhimurium in food and clinical samples. Due to its high specificity and sensitivity, our method finds its place as an alternative to PCR in large scale screenings, for detection of S. typhimurium.

Objectives: Tissue homeostasis is the result of strict regulatory mechanisms, which control self-renewal, differentiation, prevention of premature Senescence and apoptosis of stem cells. Bmi-1, a Polycomb group represses or protein, represses genes that induce cellular senescence and cell death, and can contribute to cancer when improperly expressed.Material and methods: Bladder tumoral and nontumoral samples were collected from Labbafi-Nejad hospital.RNA was extracted from each sample, reverse transcribed and amplified by RT-PCR technique, using specific primers for Bmi-1 and b2-microglobolin, as an internal control. The production and distribution of Bmi-1 protein was also examined by western blotting and immunohistochemistry technique. Results: To clarify the role of Bmi-1 in bladder tumors, we examined the expression of Bmi-1 in tumoral and nontumoral samples. RT-PCR generated a 683 bp product, corresponding to the expected size of the Bmi-1 amplified region. The identity of the amplified fragment was then confirmed by direct DNA sequencing. The mean of expression of the Bmi-1 detected in tumoral tissues was significantly higher than the non-tumoral tissues and there is also a significant correlation between the mean of gene expression with stage of malignancy (p< 0.05).The expression of Bmi-1 at protein level was further confined by western blotting and imrinmohistochemistty.Conclusion: The tumor suppressor locus Cdkn2a (Ink4a/Arflocus) codes for two proteins, p16ink4a and p14 arf. Ink4a and Arf are playing important roles in the retinoblastoma (pRB) and p53 pathways, respectively. Bmi-1 is a potent repressor of both pathways and hence elucidating its role in tumorigenisis is very important. Here, for the first time we are reporting the expression of Bmi-1 and its correlation with malignancy in bladder tumors.

Objectives: Familial Adenomatous Polyposis (FAP) is an autosomal dominant predisposition to colon cancer.This hereditary genetic disease is characterized by more than 100 adenomatous polyps in colon and rectum.Additional features may include desmoids tumors, polyps in the upper gastrointestinal tract, osteomas and Congenital Hypertrophy of the Retinal Pigment Epithelium (CHRPE). A mutation in APC (Adenomatous Polyposis Coli) is found in the majority of cases. Mutation detection and genetic analysis of APC in this syndrome is highly recommended as the penetrance is almost 100% by 40 years of age. The APC gene expanding on 5q21- q22 has 15exons and has an ORF with 8538 nucleotides which codes a protein with 2843 amino acids.Materials and Methods: 5 families among 150 families were selected according to accepted diagnosis criteria of FAP. CSGE (Conformation Sensitive Gel Electrophoresis) technique for the first time was set up to screen mutations in all 15 exons of APC gene by this technique. Direct sequencing was used as gold standard to confirm CSGE results.Results: CSGE analysis showed electrophoresis migration anomalies and heteroduplex formation in exon 15 of APC in all patients. Further analysis by direct sequencing characterized these heteroduplexes as deleterious mutations. These mutations can be classified as non sense, frame shift and deletion.Conclusion: For the first time, CSGE technique was set up for mutation screening of coding and some parts of non coding regions of APC. In comparison with other screening methods, this technique has many advantages so it can be used in routine clinical laboratories. As mutation detection in APC is laborious, needs high tech technology and is expensive, finding sensitive and cost effective mutation screening technique would have direct positive effect on clinical management of families with familial susceptibility to colon cancer.