Tehran University Medical Journal
Year:2015 | Volume:73 | Issue:3|Papers Count:12

In situ hybridization (ISH) is a method that uses labeled complementary single strand DNA or RNA to localize specific DNA or RNA sequences in an intact cell or in a fixed tissue section. The main steps of ISH consist of: probe selection, tissue or sample preparation, pre-hybridization treatment, hybridization and washing, detection and control procedure. Probe selection is one of the important aspects of successful hybridization. ISH sensitivity and specificity can be influenced by: probe construct, efficiency of labeling, percentage of GC, probe length and signal detection systems. Different methods such as nick translation, random priming, end tailing and T4 DNA polymerase replacement are used for probe generation. Both radioactive and non-radioactive labels can be used in order to probe labeling. Nucleic acid maintenance in samples, prevention of morphological changes of samples and probe penetration into tissue section are the main aims of sample preparation step. Then, a small amount of solution containing probe, is added on slides containing tissue sections for hybridization process, then slides are incubated overnight. Next day, washes are carried out to remove the probes which are not bound to target DNA or RNA. Finally, in order to be sure that the observed labeling is specific to the target sequence, using several control procedures is very important. Various techniques based on ISH consist of: Fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), genomic in situ hybridization (GISH), comparative genomic hybridization (CGH), spectral karyotyping (SKY) and multiplex fluorescence in situ hybridization (MFISH). One of the most common techniques of ISH is fluorescence in situ hybridization. FISH can be used to: 1) detect small deletions and duplications that are not visible using microscope analysis, 2) detect how many chromosomes of a certain type are present in each cell and 3) confirm rearrangements that are suspected after microscope analysis. In this technique different fluorescent labels are attached to the probes. In this review article ISH, its different types, their application, advantages and disadvantages have been considered.

Background: Stem cells represent an ideal cell source for application in tissue engineering and regenerative medicine due to their ability to proliferate and differentiate to a wide variety of cell lineages. With recent development of medical sciences and tissue engineering, usage of adipose-derived mesenchymal stem cells, their culture and differentiation on suitable scaffolds are considered as a successful clinical and research strategy. One of the most crucial factors in a successful tissue engineering technique is to choose an appropriate scaffold which allow cell migration transferring of bioactive factors as well as providing optimal growth environment for stem cells. In this study, the ability of two scaffolds is investigated as a suitable environment for the proliferation and differentiation of adipose-derived mesenchymal stem cells.Methods: This is an in vitro study that was performed in Laboratory of Stem Cell in Academic Center for Education, Culture and Research, Qom province from April 2013 to February 2014. In this study, two scaffolds including fibrin glue and alginate were prepared as two separate groups and after isolating mesenchymal stem cells from adipose tissue and adequate proliferation, they were seeded into each scaffold in chondrogenic medium. After 14 days, the evaluation of viability and gene expression of collagen II and I, SOX9 and aggrecan were done by MTT (3-{4,5-dimethylthiazol-2yl}-2,5- diphenyl-2H tetrazolium bromide) assay and real-time PCR technique respectivel. Also, cartilaginous tissue formation on scaffolds was evaluated by histological analysis.Results: According to the obtained results, the fibrin glue scaffold showed significant difference in terms of viability in comparison to alginate scaffold in chondrocyte differentiating medium (P<0.05). Also the results of real-time PCR analysis showed that the fibrin glue scaffold express cartilage specific genes at a higher level than alginate scaffold.Conclusion: The use of natural fibrin glue scaffold can be considered as a suitable environment for proliferation and differentiation of adipose-derived mesenchymal stem cells in cartilage tissue engineering.

Background: Environmental signals have an essential role in the maturation of neural circuits during critical period of brain development. It has been shown that, change in visual signals during critical period of brain development changes structure and function of glutamate receptors in the visual cortex. After processing in visual cortex, part of visual signals goes to the hippocampus and makes memories. The aim of this study was evaluating effects of visual deprivation during critical period of brain development on AMPA receptor subunits expression in rats’ hippocampus.Methods: This experimental study was done in Physiology Research Center, Kashan University of Medical Sciences at winter 2014 on male Wistar rats. Animals were divided to 2 groups (n=36 for each) were kept in standard 12 hours light/12 hours dark condition (light reared, LR) or in complete darkness (dark reared, DR) from birth to the end of the experiments. Using reverse transcription polymerase chain reaction (RTPCR) and Western blotting techniques respectively, expression of mRNA and protein of GluR1 and GluR2 subunits was evaluated in rats’ hippocampus at ages 2, 4 and 6 weeks in both groups. After quantification of the expressions, the data were compared by two way analysis of variance.Results: The relative expression of GluR1 subunit decreased about 24% (P=0.004) in the hippocampus of 6 WLR rats in comparison to 2 WLR ones. The relative expression of the other AMPA receptor subunit, GluR2, also increased about 190% in the hippocampus of the 6WLR animals when compared to the 2 WLR rats (P<0.0001). Dark rearing increased the relative expression of both subunits of AMPA receptors, GluR1 and GluR2, about 20 percent (P=0.01) in the hippocampus of 6 WDR rats in comparison to 2 WLR animals.Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

Background: Sciatic pain in association with lumbar disc herniation may require surgical intervention in the form of lumbar discectomy. Yet, the optimal time for this operation has not been specified in medical literature.Methods: In a Cross-sectional study, 147 patients (100 men and 47 women) with radiological and clinical signs of L4-L5 or L5-S1 disc herniation were entered to our registry prior to March 2009. They were all examined, diagnosed and operated on (lumbar discectomy) in Ghaem General Hospital, Mashhad, Iran. Patients were all subsequently followed for one year. The follow-up continued in a number of 126 cases, whose satisfaction was rated via phone interview for an extra year. The patients’ assessments were implemented employing three scaling systems, both before and following lumbar discectomy, to name the Modified Oswestry Disability Index (MODI), Visual Analogue Scale (VAS) and Prolo Functional Economic Outcome Rating Scale (PORS). The former two were used for pre-operative and post-operative assessments whereas the latter was applied during the first year of follow-up. The focus of the investigation during second year was on patients’ satisfaction.Results: Mean age of our cases were 34±7.4 years. According to the duration of the sciatica, patients were divided into 4 groups. <3 months, 3 to <6 month, 6 to <12 months and >12 months. Statistical analysis revealed a significant difference between patients having undergone lumber discectomy with a history of sciatica for less and more than three month before the operation in terms of pre-and post-operative visual Analogue Scale (P=0.022). However, there could be found no such disparity in other clinical scores (P=0.63 for MODI, P=0.85 for Prolo scale and P=0.73 for satisfaction).Conclusion: Patients with less than three months of sciatica may seem to enjoy a better clinical outcome after one year, there could be found no correlation between the duration of sciatica and the satisfaction after two years.

Background: Hyperemesis gravidarum (HG) starts between four and seven weeks after the first day of the last period, and ends at twenty weeks of pregnancy. The etiology of HG, is unknown. Recent studies worldwide show the Helicobacter pylori (H. pylori) infection as a possible cause of the severe nausea and vomiting in pregnancy, recent studies showed H. pylori to have a role in occurrence of it during pregnancy. The current study assessed the immunoglobulin G (IgG) and immunoglobulin M (IgM) titer to H. pylori in pregnant women with HG.Methods: This is a case-control study of the pregnant patients of a gynecologist's office in the Marvdasht city from April to September 2013. One hundred and twenty three patients were randomly chosen based on their conditions and were divided into two groups (case n1= sixty three and control n0= sixty). The IgG and IgM titers against H. pylori were measured by ELISA method. Data analysis was performed using Chisquare test, Mann-Whitney U-test, and T-test in SPSS software, version 20 (SPSS, Inc., Chicago, IL, USA). The significant level of the test was considered (P=0.05).Results: Totally, 123 pregnant women were evaluated; 63 women with hypermesis gravidarum and 60 without HG. Forty nine women out of 63 in HG group and 48 women in the control group were IgG positive for H. pylori. Also, mean serum level of IgG was 51.6 in the HG group (P=0.685). Twelve women out of 63 in HG group and 20 women in the control group were IgM positive for HP. Also, mean serum level of IgM was 27.7 in the HG group (P=0.675) Except for the mother's blood type, there were no other statistically significant differences found between the two types of antibodies against H. pylori.Conclusion: There is no relationship between helicobacter pylori and hyperemesis gravidarum in pregnant woman. Considering the high prevalence of H. pylori infection in our country, there is a need for studies with more samples and more diagnostic methods.

Background: In the last decade due to emerge and remerge of influenza viruses, quality improvement of vaccines to increase immune responses in target populations have been more necessary. The potential of biologic adjuvant to stimulate and induce immune system is the basis of modern researches in prevention and controlling program of infectious diseases. In this study, the effect of the coding sequence of cellular Myxovirus resistance (Mx) protein as a biological adjuvant for inducing humoral immune response against influenza virus was investigated.Methods: The experimental study was performed on Balb/c mice in Razi Vaccine and Serum Research Institute from June to November 2014. Three conserved motifs of Mx were selected following sequence alignment between human, mouse and bird species. Potential of the motifs for stimulation immune responses against influenza virus were evaluated using in silico analysis. Based on the immune informatics data Mx1 sequence was the best immune inducer and cloned into pcDNA3.1 vector. Then formulated with inactivated H9N2 influenza antigen as adjuvant and injected to mice groups. The sera of vaccinated mice were collected prior to priming and boosting injections and also at defined weeks and analyzed with serological assays. Histopathological examination was done for evaluation of the vaccine and adjuvant safety.Results: The mean weight of the Balb/c mice in all control and treatment groups was similar and ranged from 21 to 37 gr (P=0.05). The difference in increasing antibody titers against influenza virus in immunized mice who received Mx1-adjuvanted vaccine especially in second boosting was significant (P=0.01) compared to the vaccine alone group. More than 78% of the immunized mice receiving two-time boosting have the mean antibody titer of >6 (Log2) which was higher (P=0.001) comparing to the mice with one booster injection.Conclusion: These data revealed that Mx1 as biological adjuvant was able to increase antibody titer and induction memory immune responses against influenza immunization without causing any side effects.

Background: Diabetic foot infections a common complication of diabetes. Staphylococcus aureus is most common pathogen associated with diabetic foot infection. Frequency of methicillin-resistant Staphylococcus aureus (MRSA) associated with diabetic foot infection at other country is 15-30% and important cause at hospital acquired infection. The aim of this study was to evaluate the prevalence of pathogenic organisms and the prevalence and contributing factors of MRSA in patients with diabetic foot infection.Methods: A cross-sectional study from 70 diabetic foot infection at Imam Khomeini Hospital, Tehran, Iran from March 2013 to September 2014. A retrospective analysis of wound swabs taken from infected foot ulcers in diabetic patient, selected from an outpatient diabetic foot. Swabs were used to obtain tissue fluid from the base after debriding the ulcer with a sterile scalpel. Sixty patients with positive wound swabs were included. Size of ulcer and ulcer grade were measured in all patients.Results: A total of 61 microorganisms were isolated. The mean number of isolate was 0.87. Corynebacterium spp were the sole pathogen in three cases than taked into normal flora. There wasn’t significant P-value between demographic subjects and MRSA frequency. In this study, P=0.05, CI: 95% were considered statistically significant. Gram positive aerobic bacteria were the most common micro-organism isolated (52.54%) followed by gram-negative aerobic bacteria (44.26%). among the gram-positive aerobic organisms, Staphylococcus aureus was found most frequently and 10% were MRSA that confirmed by E-test. There wasn’t significantly different in measurement of the MRSA positive patients compared to MRSA negative patients (P>0.05).Conclusion: In this study, MRSA prevalence was similar to other Asian studies and different from west countries study. There wasn't significant difference between gram positive and gram-negative microorganisms that look out to change of microbiological profile in diabetic foot infections and creation multi-drug resistant bacteria. MRSA infections and other multi drug resistant organisms is a serious problem and increasing problem in diabetic foot infections. Further studies are required to assess the need for antibiotics in treating foot ulcers in diabetes and to assess the optimal therapeutic management.

Background: Sexual health is one component of reproductive health that has great impact on the health of individual and society. Sexual health has expressed as a strategy to achieve the millennium development goals. To date, many Iranian studies have addressed the issue of reproductive and sexual health but no study has addressed the definition of sexual health and described its components. Therefore, this study aimed to explore the definition of sexual health and provide a description of its components.Methods: This qualitative study was conducted from June 2009 to November 2013 in Tehran, Iran. Participants were Iranian sexual health experts selected by targeted sampling. Data was collected through semi-structured interviews. All interviews were audio- taped and transcribed verbatim with participants’ permission and then coded by researcher. Development of codes and themes was inductive and arose from the interviews. Data collection was stopped when data saturation was reached. Data was analyzed through conventional content analysis.Results: The theme of individual and family health was extracted from data. From this theme and its categories, the definition of sexual health and its components was extracted including knowledge and information (training needs), physical and mental health, economic needs, social and cultural values and religious values. Based on the results, the sexual health of adults is satisfying from sexual needs by establishing a healthy and socially defined relationship with someone of the opposite sex.Conclusion: According to this study, sexual health has a special definition in each age group and also has different requirements. To start a healthy sexual relationship, at first a correct relationship between a woman and a man must shape. A relationship that is based on customary, religious and social values. Sexual health is a system that consists of abstract components and without proper formation of each of these components we will not be able to access sexual health completely. It seems one of the most important components of sexual health is the knowledge and information about sexual issues. However, further studies are needed to confirm this result.

Background: The relationship of the base of appendix to the cecum remains constant, whereas the tip can be found in a retrocecal, pelvic, subcecal, preileal, or right pericolic position. These anatomic considerations have significant clinical importance in the context of acute appendicitis. The knowledge about the correct anatomical position of appendix may facilitate in generating an accurate diagnosis of appendicitis as well as assist in achieving a better prognosis and early treatment. The present study aimed to determine the anatomical location of the appendix in Iranian cadavers.Methods: This descriptive cross-sectional study was conducted on 200 cadavers who were referred to the Forensic Center of Tehran from March to September 2013. The data including age, sex, weight, and appendix length and position were collected and analyzed using SPSS software, version 16 (SPSS, Inc., Chicago, IL, USA).Results: In the present study, 200 cadavers were evaluated accidentally, of which 173 (86.5%) were males and 26 (13%) were females, and the mean age was 39.96 years±16.31 (SD). The mean wall thickness of the appendix was 9.78 cm±16.31 (SD). The mean appendix length was 9.86 cm±1.79 (SD) in men and 9.30 cm±1.56 (SD) in women. The appendix height was long in 20 cadavers (10%), short in 3 cadavers (1.5%), and moderate in 177 cadavers (88.55%) cadavers. The appendix position was posterior in 120 (60%), ectopic in 32 (16%), and pelvic in 48 (24%) cadavers.Conclusion: Majority of appendices examined in the present study were positioned at the posterior (Retrocecal) of pelvis. According to different positions of appendices in different populations and different races, the knowledge of appendix position in various populations is necessary for early diagnosis and treatment and fewer complications for related disease.

Background: Spices (flavorings) are contaminated to microbial agents such as filamentous and yeast fungi during production processing. Almost these are grown and harvested in warm and humid areas of the world where the growth of a wide variety of organisms is readily supported. The aim of this study was to assess the fungal contaminations of three current spices including turmeric, black pepper and cinnamon from some supermarkets in Tehran and evaluating of their hygienic quality.Methods: In this cross- sectional study that was performed in laboratory of Medical Mycology, School of Public Health in Tehran University of Medical Sciences from December 2012 to September 2014, 165 packed spices including 55 samples from each 11 valid brands of cinnamon, turmeric and black pepper were selected from different regions of Tehran. Culture was performed on many different fungal media from 10-1 to 10-4 dilutions of their samples. The fungal colonies obtained from cultures were studied by traditional laboratory methods. On the other hand, the number of unknown possible colonies was identified by molecular methods and then all identified colonies were counted.Results: Totally, from 165 packed spices, 4317 colonies include 29 different fungal species were isolated and identified from cinnamon (1520), turmeric (1373) and black pepper (1424). The etiologic agents were mainly including Aspergillus niger (7.3%), Penicillium spp. (4.1%), Paecilomyces spp. (2.8%) and Aspergillus flavus (2.3%), respectively. Non-parametric Kruskal-wallis test indicated that there was no significant difference statistically among brands at each level. Also the present study showed P=0.0003 among under study spices. The most contaminated spices were cinnamon, while turmeric had the lowest contamination rate.Conclusion: The obtained results of this cross-sectional study and the available proofs in community indicate that, there are the high levels of fungal contaminations in current used spices. Therefore, it is necessary to control the production units.