[Cytotoxicity evaluation of taverniera spartea on human cancer cell lines]

For centuries, plants have been a major source for drug discovery. Some examples of anticancer agents developed from plants are the vinblastine, vincristine, taxol and camptothecin. Breast cancer is one of the most commonly diagnosed cancers among women and prostate cancer remains a considerable health problem for men around the world. The purpose of this study was cytotoxicity evaluation of Taverniera spartea on human cancer cell lines. Methods: In the present study, we determined the cytotoxic effects of total methanol extracts and their fractions of Taverniera spartea on MCF-7 and BT-474 human breast cancer cells and also PC-3 and Du-145 prostate cancer cells. Cytotoxicity was evaluated by MTT assay and flow cytometry analysis. The chloroform fraction of Taverniera spartea showed the highest toxicity MTT assay. The IC50 value of this fraction was 70.69 mg/ml for MCF-7 breast cancer cell line after 48 h of exposure. Chloroform fraction showed necrotic effects on MCF-7, BT-474 and PC-3 in contrast apopthotic induction on Du-145 in flow cytometry analysis Taverniera spartea has cytotoxic effects. Further investigation is needed to determine chemical characterization of the active principles and the molecular mechanisms mediated anticancer activities of Taverniera spartea

Production and purification of anti-rhombomys opimus immunoglobulins

Zoonotic cutaneous leishmaniasis [ZCL] is an increasing public health problem in some endemic regions. Horseradish peroxidase [HRP] conjugated rabbit anti-Rhombomys opimus [R. opimus] Ig is needed for immunoblotting and ELISA tests used to explore the immune response of the rodents against the sand fly saliva. In this study, the production of HRP conjugated rabbit anti-R. opimus Ig was conducted for the first time. Rhombomys opimus Ig was purified from serum by protein G affinity chromatography column and injected into rabbit to produce anti-R. opimus Ig antibody. The titration of antibody against R. opimus Ig in rabbit serum was checked using indirect ELISA. Rabbit anti-R. opimus Ig was purified by Sepharose-4B-R. opimus Ig column. Reactivity of this antibody was assessed by indirect ELISA and was conjugated to HRP by periodate method. Approximately 3.5 mg Ig was purified from 1 ml R. opimus serum using protein G affinity chromatography column. The molecular weight of purified R. opimus Ig was estimated about 150 kDa by SDS-PAGE. Nearly 2.3 mg rabbit anti-R. opimus Ig was purified from 1 ml immunized rabbit serum. The purified antibody was conjugated to HRP and the optimum titer of HRP conjugated rabbit anti-R. opimus Ig was determined as 1:8000 using direct ELISA. HRP conjugated rabbit anti-Gerbil IgG has been produced by a few companies, but to our knowledge HRP conjugated rabbit anti-R. opimus Ig is not commercially available. Production of HRP conjugated rabbit anti-R. opimus Ig is considerably helpful for immunological studies of R. opimus, the main reservoir host of ZCL in Iran as well as some other countries

Short term reactogenicity of a triple diphtheria-tetanus-whole cell pertussis vaccine in Iranian infants

Immunization against diphtheria, tetanus and pertussis [DTP] has long been applied in Iran using whole cell vaccine. Despite the role of whole cell DTP [DTwP] vaccine in reduction of mortality as a result of disastrous diseases such as diphtheria, tetanus, and pertussis, serious local and systemic complications have been attributed to these vaccines. This study was performed to determine the complications of DTwP vaccine in infants attending some of the health centers of Tehran in 2006-2007. In this prospective study, 330 infants were injected with DTwP vaccine manufactured by Razi Institute of Iran. All subjects received DTwP vaccine at 2, 4, and 6 months of age following the national vaccination schedule of Iran. Reactogenicity was assessed by the parents for 7 days post-vaccination using diary cards. Of the 279 infants who completed the vaccination study, pain was the most frequent local reaction after the primary vaccination [68.1-75.3%]. The mean diameters of the redness and swelling at first day post-vaccination were 2.81 +/- 6.91 and 2.60 +/- 7.93 mm in the first dose, 2.40 +/- 6.25 and 1.94 +/- 5.74 mm in the second dose and 2.24 +/- 5.66 and 2.16 +/- 6.03 in the third dose, respectively. Fever [axillary temperature > 37.5°C] was the most frequently reported systemic reaction during the primary vaccination [53.8-58.8%]. All systemic reactions observed after each dose were either reduced or completely disappeared during a week. The high incident of complications observed following vaccination with this cellular triple vaccine may be related to the formulation or the bacterial cell fragments used in vaccine production

Production and characterization of anti-Her 2 monoclonal antibodies

Breast cancer is the most common cancer among women in the world. Early diagnosis of this cancer is a key element for its treatment. One of the approaches for diagnosis of breast cancer is detection of its tumour-associated markers. Hence, Her2 has been the main focus of the researches in the field. For diagnosis of Her2 overexpression, monoclonal antibodies [mAb] reacting against Her2 were produced in this study. For this purpose, two peptides from extracellular domain of Her2 were selected and the mAbs reacting against them were produced by hybrodoma technology. Reactivity of these antibodies were then evaluated in different immunological assays including ELISA, Immunoflurescence [IF], western blot [WB] and immunoprecipitation [IP]. Total of 5 clones were produced from two separate fusions, and antibody isotyping revealed that all clones were IgM. These mAbs showed appropriate reactivities in the following assays: ELISA, immunofluresence by staining of breast cancer cell line [SKBR3], WB and IP by detecting the 185 KD band of Her2. In conclusion, it seems that the mAbs are useful diagnostic tools for detection of Her2 expression in patients with breast cancer

[Detection of Legionella pneumophila by nested PCR-RFLP and ELISA on urine specimens of pregnant women with respiratory infections]

Pneumonia during pregnancy can induce serious consequences to the mother and the fetus, therefore its diagnosis and therapy is very important. There are few published articles on Legionella infection prevalence during pregnancy. In patients with Legionellosis, bacterial LPS and DNA are excreted into urine for extended periods, so combination of PCR and ELISA methods would be a good diagnostic tool. This research was done to determine the prevalence of L. pneumophila in pregnant women with respiratory infections. This is a cross-sectional study on 95 pregnant women with respiratory infection carried out during winter to summer 2006. Presence of Legionella infection was con-firmed by nested PCR-RFLP and antigen detection in urine specimens by ELISA method. The data were analyzed by SPSS, version 13, by using independent t tests, Fisher's exact test, ?2, a logistic model and McNemar's test, while considering p<0.05 as significant. The prevalence of infection using PCR was 22.1% [CI=14.1%-30.1%] and by ELISA it was 4.2% [CI=2%-8.2%]; this difference was statistically significant [p<0.005]. The most pre-valent clinical features were Cough [56.8%], headache [54.7%], abdominal pain [38.9%], chills [35.8%], fever [22.1%] and diarrhea [8.4%]. There were significant statistical relationships bet-ween cases with a positive CRP and fever, chills and abdominal pain and previous liver or renal problems [p<0.05, p<0.001]. There were significant relationships between fever and chills with ELISA results [p<0.05] but no relationships with other variables. There was a considerable prevalence of this infection in the studied population [22.1%]. It seems that performing PCR and ELISA tests on urine sample is suitable in detecting Legionella species and it can provide results in a less than a day_ a great help in the diagnosis and treatment of pneumonia especially during pregnancy

expression profile of indolamine 2,3-dioxygenase in murine endometrium during estrous cycle

Activation of Indolamine 2,3-dioxygenase [IDO], an enzyme responsible for tryptophan catabolism, has been reported to be a necessary requirement to achieve immunological tolerance against the fetus and protection against intracellular and extracellular pathogens. The objective of this study was to evaluate the expression of IDO gene in murine endometrium and its expression rate in different phases of estrous cycle. Noticing the role of this enzyme especially in the survival of a semi-antigenic embryo, the results of this study may be used as a basis for practical studies on the immunologic bases of recurrent abortions. In this experimental study, we studied the expression of IDO in the female BALB/c mice endometrium during four stages of estrous cycle. The phases of estrous cycle were determined by examining vaginal cytology .At each phase, endometrium was pealed away and the relative expression of IDO mRNA was detected by semi-quantitative RT-PCR using specific primers to IDO and mGAPDH as a housekeeping gene. The specificity of reaction was confirmed by enzymatic digestion of amplicon which yielded to 138bp and 259bp fragments. Our results showed, for the first time, that IDO is expressed in the endometrium of cycling mice during all stages of estrous cycle. The expression of IDO was highest at estrus and lowest at diestrus [p<.001]. Expression of IDO in endometrium during all phases of estrous cycle reveals that this enzyme as an effective arm of innate immune system may serve a role in protecting the female reproductive tract against ascending infections. Also regarding the fact that, mating only occurs at estrus phase, the high expression of IDO in this phase, may act as the main mechanism in inducing immunological tolerance to the fetus

Intrafollicular fluid antigamete antibodies in infertile patient candidates for ICSI

Immunologic disturbances must be considered as a major cause of infertility. Antigamete antibodies like antisperm antibodies [ASA] and to anti-zona antibodies [AZA] seem to be implicated in the etiology of infertility. These antibodies affect fertilization and embryo development. It is important to screen these antibodies in infertile women who are candidates for in-vitro fertilization [IVF], because the presence of these antibodies may switch the treatment from IVF to intra-cytoplasmic microinjection [ICSI]. The objective of this study was to determine the presence of ASA and AZA in the follicular fluids [FF] of women who sought candidacy for ICSI. In this prospective study, the follicular fluids of 96 infertile women [20 to 39 years old, mean 31.5 +/- 5.1], who were candidates for ICSI, were evaluated. According to the etiologies, 80 women had explained whereas 16 had unexplained infertility. All the follicular fluids were evaluated for the presence of ASA by ELISA and Sperm MAR test and also for the presence of AZA by ELISA. The data were analyzed by Chi-square test using SPSS soft-ware and the significance level was considered p<0.05. According to the results of ELISA and Sperm MAR test, none of the patients had ASA in their follicular fluids. However, twenty samples [20.8%] were positive for AZA. In patients with unexplained infertility, autoantibodies to zona pellucida were significantly higher in the follicular fluid than the group with proven etiologies for infertility [p=0.001]. The low incidence of ASA and the high incidence of AZA in the infertile women in this study, especially in women with unexplained infertility in Iran have to be considered seriously. Determination of AZA is highly recommended in the evaluation of infertile couples, especially those with unexplained infertility

effects of human chorionic gonadotropin on germ cell maturation and testosterone secretion in neonatal mouse testis

Human chorionic gonadotropin [hCG] as an LH agonist affects spermatogenesis and germinal cell numbers, and has extensive usages in infertility treatments. The aim of this study was to determine the effects of varied doses of hCG on germinal cell proliferation and androgenic status in mouse model. In this study, hCG dosages of 5 to 50 IU were injected into 18 mice in three experimental groups and 6 mice served as the control group [Group 1]. The mice in groups 2, 3 and 4 received subcutaneous injections of 5, 10 and 50 IU doses of hCG respectively, on days 15 and 25 of their lives. Blood samples were obtained from each mouse on days 28 and 65 for serum measurements of testosterone. One testis of each mouse was harvested for flow cytometric DNA analysis on day 65. Serum testosterone levels on day 28 were greater in groups 2, 3 and 4 compared to that of the control group. With increasing doses of hCG, the mean testosterone levels increased too and the highest values were observed in group 4. However, serum testosterone levels on day 65 were greatest in group 1 but progressively decreased in groups 2, 3 and 4, lowest in group 4, but there were no significant statistical differences among the groups. Groups 3 and 4 had a significantly reduced mean haploid cell numbers on day 65. The results of this study showed that testosterone production in neonatal mouse testis increases after hCG injection and there is a linear relationship between serum testosterone and hCG injections. With the passage of time and clearance of hCG, Leydig cell stimulation decreases and subsequently testosterone levels diminish too, especially in mice with highest doses of hCG injections. Therefore, for testosterone production in neonatal mouse testis, continuous stimulation of Leydig cells is essential

prevalence of chlamydia trachomatis infection by molecular analysis of urine samples in women attending OB and GYN clinics in Tehran

Chlamydia trachomatis is a common and curable STI which can be symptomatic or asymptomatic. Nowadays, PCR is a very sensitive diagnostic tool for detecting Chlamydia in urine and can be used in routine screening procedures as a noninvasive test. There are few studies on the prevalence of C. trachomatis in Iranian women and most of them have small sample sizes which are not suitable for epidemiological deductions. The aim of this study was to estimate the prevalence of urogenital C. trachomatis infections by PCR on urine samples of women in their fertility years and to evaluate the necessity of screening for asymptomatic infections in Iranian women. This WHO supported descriptive-analytical and cross-sectional study was performed on 1052, 15-49 year-old women. Participants were selected randomly from attendees of 5 Obstetric-Gynecologic clinics in Tehran during summer and fall of 2003. The research material consisted of a questionnaire and urine samples which were transported to Avesina Research Institute daily to extract their DNA and prepare them for PCR tests. The gathered data were analyzed by SPSS, version 11, and evaluated statistically by t-test, Chi-square, variance analysis and logistic regression, while considering p<0.05 as significant. The mean age of participants was 28.52 +/- 6.36 years. 56.2% of them had high school education, 94.2% were married, 91.8% were housewives, 32.5% were pregnant, 93.8% were sexually active, 99% of them were monogamous and 48.1% were on contraceptive methods. Among sexually active and non-pregnant participants, 10.4% were taking OCPs, 8.7% were using condoms, 16.3% had IUDs and the rest were on other contraceptive methods. In their reproductive history, 39% had vaginal discharges, 12.9% pelvic pains, 1% ectopic pregnancies, 21.2% abortions, 6.5% premature deliveries, 2.7% low birth weight infants and 7.2% were infertile. 129 subjects, [12.3%], had positive PCR tests. Statistically, there was no significant relationship between subjects, reproductive and personal histories of the subjects with the test results. Based on the estimated prevalence, it seems that chlamydial infection is prevalent in the studied population. In populations with prevalences higher than 4%, screening programs are recommended, so that Chlamydia screening can be considered as a part of health care programs in Iran to reduce the burden of the disease

nested-PCR assay for detection of cryptosporidium parvum oocysts in water samples

Cryptosporidiosis is a gastroenteric disease caused by the protozoan parasite Cryptosporidium parvum. Water-borne transmission of this organism has become more prevalent in recent years. Current method for detection of C. parvum oocysts in water is immunofluoresence assay [IFA]. The method is time consuming, laborious and particularly not-specific. It cannot determine the infectivity of detected oocysts. We have evaluated a nested- PCR assay for sensitive detection of C. parvum oocysts in water samples. Water sample concentrates were spiked with Cryptosporidium oocysts and after DNA extraction and purification by QIAamp DNA mini kit, detection was achieved by nested PCR amplification of a 200 bp region of hsp70 gene specific for C. Parvum. The method could detect as few as one oocyst in seeded tap water samples. On the basis of these results, PCR could be a useful tool in the monitoring of water samples for the detection of Cryptosporidium oocysts

[Extraction and purification of ferritin from liver tissue]

Ferritin with molecular weight of 450 kDa is the most important iron storage protein and is made of 24 subunits consisting of light and heavy chains. Each ferritin molecule is able to store 4500 Fe[3+] molecules. The aim of this study was to determine the preparation of highly pure ferritin for usage in diagnostic and research systems. In this study, ferritin was extracted and purified by homogenizing liver tissue, heating at 75 degrees centigrade, ammonium sulfate fractionation and gel filtration chromatography on sephadex G-200 column. The purify of ferritin was improved by using recycling chromatography. Resulted protein was electrophoresed on polyacrylamide gel in the presence of sodium dodecylsulfate [SDS-PAGE]. Existence of ferritin was confirmed by ELISA test and potassium ferricyanide staining of gel. Silver nitrate staining of gel was used to confirm the purity of ferritin. Electrophoresis of ferritin under reducing conditions in presence of 2- mercapto ethanol was done to show the subunits [19 and 21 kDa] of ferritin. This purification method resulted in very pure ferritin and the yield was 100 microg/gr of wet liver tissue. Electrophoresis of ferritin under reducing conditions in presence of 2- mercapto ethanol showed the both subunits [19 and 21 kDa] of ferritin. Highly pure ferritin resulted by this method is appropriate for diagnostic and research purpose and the yield is reasonable comparing other studies

[Designing an ELISA method using sperm antigens for detection of antisperm antibody in comparison with SpermMar test

The role of antisperm antibodies with a prevalence of 6-26% is well known in immunological infertility. Thus, there is clinical importance to determine ASA levels in both male and female. Nowadays, one of the most important discussed controversies in the field of immunological infertility is establishing an standard method to determine ASA. It seems that ELISA method will be more sensitive, specific and more diagnostic in determination of ASA if sperm surface antigens could be used as coated antigens, with least contamination to sperm intracellular antigens and nonspermic antigens. So, the aim of this study is designing an ELISA method by using the best method of sperm antigens extraction with at least contamination. In this study we designed an ELISA method with three different extraction methods of sperm antigens including sonication method, using SDS detergent, and application of LIS detergent, then we compared ELISA method based on the three extraction methods as well as two similar commercial ELISA kit [IBL Co, and Bioserv Co] with SpermMar test. Comparing designed method with commercial kit indicated that among 28 sera which had 16 positive sera and 12 negative sera by SpermMar, 14 sera were true positive by LIS method and only 2 cases were false negative without any false positive results, whereas there were 5 true positive results and 11 cases false negative by the sonication method. The SDS method also had 13 true positive results with 3 false negative and 4 false positive results. In addition, two commercial kit had in turn 7 and 4 cases true positive and both of them had 1 case false positive and in turn 9 and 12 cases with false negative result. ELISA method designed by LIS detergent has adequate sensitivity [87.5%] with higher specificity [100%] and efficacy [92.8%] than other extraction methods. There is a significant correlation between this designed method and SpermMar test [r=0.572]. The results of this study indicated that ELISA method by LIS antigens has at least contamination with nonspermic antigens and it is better than other extraction methods and commercial ELISA kits for detection of antisperm antibody