Iranian

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The characteristics of cellular and molecular mechanisms associated with cell proliferation and differentiation is important to understand malignancy. In this report we characterise a leukemic model, D5A1, to study the action of differentiation agent, cellular events and gene expression of the selected transcription factors. Cells induced with 4 mM hexamethylene bisacetamide (HMBA) caused signs of erythroid differentiation (changes in morphology and size, haemoglobinisation) and cessation of proliferation including accumulation of cells in G0/G1. Treatment with HMBA caused a time-related decrease of tumorigenicity detectable by 48 hour. Northern-blotting showed induction of -amino levulinic acid synthase-erythroid (ALAS-E) mRNA at 48 hours and appeared in a strong level subsequently. C-myc (myelocytomatosis) and c-myb (myeloblastoma) mRNA levels decreased transiently in early hours returning to control values by 24 hour and decreased again. Stem cell leukaemia (SCL) and GATA-1 mRNA were markedly down regulated in early hours and then returned back. A later time point, upregulation of GATA-1 and SCL was relevant to maturation phenotype. These data provide a useful model to study the cellular and molecular events in leukomogenesis and action of differentiation therapy in leukaemia

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The hypotensive effect of garlic has been well-documented in human subjects and animals. Since endothelial activity regulates vascular reactivity in physiological and pathophysiological conditions, the aim of the present study was to investigate the effect of garlic on endothelium-dependent and independent relaxation of rat aorta for elucidation of mechanism of the garlic anti-hypertensive effect. Four and eight weeks after treatment with garlic extract, aortic rings were studied for relaxation response to acetylcholine and isosorbide dinitrate. The obtained results showed that endothelium-dependent relaxation response of aortic rings to acetylcholine, from rats treated with garlic for 4 and 8 weeks, increases about 5-24% and 3-27%, respectively compared to controls. But, endothelium-independent relaxation response to isosorbide showed no difference in all groups. Moreover, the relaxant effect of garlic extract was time-dependent. The obtained findings strongly suggest that garlic exerts its relaxant effect through endothelium by production and/or releasing of endothelium-derived relaxing factor

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Decreased cardiac responsiveness to adrenergic stimulation has been observed in cholestatic liver disease, but the cause remains unclear. Previous reports have suggested that nitric oxide overproduction might have a role in cholestasis-induced bradycardia via inhibition of L-type calcium channels. In the present study, the digoxin has been used to increase cardiac Ca2+ transient in male Sprague-Dawley rats with obstructive cholestasis and the chronotropic responsiveness to adrenergic stimulation was evaluated. Cholestasis was induced by surgical ligation of the bile duct under general anesthesia and sham-operated animals were considered as control. The animals were divided into two groups, which received either digoxin (10, 20 mg/kg/day) or saline. One week after the operation, spontaneously beating atria were isolated and chronotropic responses to epinephrine were evaluated in a standard oxygenated organ bath. The basal spontaneous beating rate of the atria in the cholestatic animals was not significantly different from that of sham-operated rats in vitro. Meanwhile, cholestasis induced a significant decrease in chronotropic effect of epinephrine. This effect was corrected by daily administration of digoxin (20 mg/kg/day). The results also showed that plasma alkaline phosphatase activity was increased by bile-duct ligation, and digoxin treatment had no effect in the elevation of this marker of liver damage. The protective effect of digoxin on impaired chronotropic responsiveness to adrenergic stimulation in cholestatic rats might be related to increase of Ca2+ transient. However, further studies are necessary to confirm the molecular basis of this effect

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Angiotensin II is a major endocrine hormone that affects directly both vascular smooth muscle and endothelial cells. Since vascular reactivity to angiotensin II changes in more physiological and pathophysiological conditions, the present study was performed to investigate the effect of intraperitoneal administration of angiotensin-converting enzyme inhibitor and captopril (30 and 50 mg kg-1, once daily for 8 weeks) on contractile response of rat aorta. After 8 weeks, the treated rats were anesthetized, their thoracic aortas were excised and placed in a Petri dish filled with Krebs solution for recording of contraction and relaxation response. The obtained results showed that captopril did not modify body weight gain and food or water intake but contractile response of aortic rings to phenylephrine in treated rats with 30 and 50 mg kg-1 captopril, in the presence of endothelium, decreases about 11-22% and 29-32% (P<0.05-P<0.01), respectively, when compared to the controls. Denuded aortic rings from 30 and 50 mg kg-1 captopril-treated rats showed 11-21% and 7-11% decrease in contractile response, respectively. There was a marked endothelium-dependent relaxation response to acetylcholine in 50 mg kg-1 captopril-treated rats compared to the controls (P<0.05). Endothelium-independent relaxation response to isosorbide dinitrate showed no significant difference in all groups. According to these results, it is suggested that captopril exerts its relaxant effect directly and/or indirectly through endothelium by production and releasing of endothelium-derived relaxing factors

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Background: When fetal red cells enter the maternal circulation from placenta, an event would be happened that is described as feto-maternal hemorrhage (FMH). This life-threatening condition could be detected by using RBC antigens (surface antigens and intracellular antigens). Therefore, the measurement of fetal RBC in an artificial model would be useful to calculate FMH and consequently the dosage of Rhogam for prophylaxis. The aim of the present study was to evaluate FMH in an artificial mixture model. Methods: A series of 40 artificial specimens were prepared consisting of Rh(D) negative adult blood (non-immunized) spiked with varying amounts of Rh(D) positive cord blood from mothers between 20-30 years old in Shahid Beheshti Hospital, Tehran, Iran. Monoclonal anti-D and anti-HbF (fetal hemoglobin) were used for detection of fetal RBC in artificial mixture sample modeling. Results: This study showed that the percentage of fetal cells in artificial sample for anti-D antigen is in ranges of 0.28%-0.32% for a 0.25% dilution mixture, and 1.3%-2.05% for the mixture with dilution 2%. In addition, the ranges of data for anti-HbF staining was obtained 0.2%-0.34% for the 0.25% dilution sample, and the ranges of 1.04-1.8% for the 2% dilution. The regression analysis indicated that the correlation of anti-D assessment with expected standard method was r2 = 0.9672 and anti-HbF assessment was r2 = 0.8842. Conclusion: Although both molecule targets could be used for detection of fetal RBC, in this model, anti-D staining was more accurate than anti-HbF staining. However, since anti-D can not be utilized for low-density or weak phenotype and other incompatibility, the anti-HbF labeling could be used for all FMH.

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Background: Although opioids suppressive effects on immune system function have been reported, this study demonstrates inflammatory reactions, such as production of pro-inflammatory cytokines and suppression of anti-inflammatory cytokines, are the main causes at organ’s allotransplantation rejection in chronic morphine-treated recipients. Methods: 28 rats were categorized in 4groups through intra-peritoneal administrations: control, sham, morphine treated animals (20 mg/kg injected of morphine daily until biopsy day), morphine and naloxane treated animals (20 mg/kg morphine and 2 mg/kg naloxane daily injected until biopsy day), which their donors were normal rats. The grafts were done at the 14th day of the experiment. Plasma interleukins levels (IL-6 and IL-10) in three sampling times were measured by ELISA. With almost 80% of macroscopic rejection signs in rats of one group, full thickness skin biopsy has been taken and histological parameters like perivascular infiltrates, epidermal changes, and stromal changes were detected. The statistical significance differences between the control and experimental groups were analyzed using the Kruskal-Wallis, followed by ANOVA post hoc test. Results: Accelerated skin allograft rejection by chronic morphine consumption can be resulted of increased IL-6 concentration and decreased IL-10. The enhancing effects of morphine on the graft inflammation were partially antagonized by Naloxane. It can illustrate the complexity of opiates and immune system connections and should be considered during organ transplantation of opiate addicts. Conclusion: Expansion of skin cells in recipient with chronic morphine administration history may be resulted in failure.

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Background: Endometriosis is a common chronic inflammation causing major problems including infertility. The role of omega-3 and omega-6 fatty acids as their potential anti-inflammatory effects in endometriosis needs to be further explored. The objective of this study was to compare serum phospholipid fatty acid profile in endometriosis patients with controls, and to explore the correlation of this profile with the severity of the disease. Methods: Sixty-four endometriosis patients and 74 control women, in reproductive age, participated in this study. Among the endometriosis patients, 19 cases were in stage I, 27 cases in stage II, 8 cases in stage III, and 10 cases in stage IV. Each patient underwent laparoscopy. Before surgery, 5 ml of blood was obtained. After extraction of the total lipids, serum total phospholipid fraction was isolated by thin layer chromatography. Fatty acid composition of the phospholipid fraction was determined by gas chromatography and the resulted profile was compared in endometriosis patients and controls. The profile was also compared in the endometriosis group based on the severity of disease. Results: Stearic acid was significantly lower in the endometriosis group as compared to controls (P= 0.030). No other fatty acid compositions were significantly different between patients and controls. Serum ratio of eicosapentaenoic acid (EPA) to arachidonic acid (AA) was in reasonable correlation with the severity of endometriosis (r = 0.34, P = 0.006). Conclusion: According to these findings, levels of fatty acids in serum total phospholipids seem not to be a marker for endometriosis, but the EPA to AA ratio was a relevant factor indicating severity of illness.

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Background: Testosterone and its metabolites have important roles in learning and memory. The current study has conducted to assess the effect of pre-training, post-training and pre-probe trial intrahippocampal CA1 administration of 3 alpha-anderostanediol (one of the metabolites of testosterone) and indomethacin (as 3 alpha-hydroxysteroid dehydrogenase enzyme blocker) on acquisition, consolidation and retrieval in Morris water maze (MWM) task. Methods: Adult male rats were bilaterally cannulated into CA1 region of hippocampus and then received 3 alpha-diol (0.2, 1, 3 and 6 μg/0.5 μl/side), indomethacin (1.5, 3 and 6 μg/0.5 μl/side), indomethacin (3 μg/0.5 μl/side) + 3 alpha-diol (1 μg/0.5 μl/side), 25-35 min before training, immediately after training and 25-35 min before probe trial in MWM task. Results: Our results showed that injection of 3 alpha-diol and indomethacin significantly increased the escape latency and traveled distance to find hidden platform in acquisition and consolidation stage, but did not have any effect on retrieval of spatial learning as compared with the control group. Conclusion: It is concluded that intra-CA1 administration of 3 alpha-diol and indomethacin could impair spatial learning and memory in acquisition and consolidation stage. Also, intrahippocampal injection of indomethacin + 3 alpha-diol could not change spatial learning and memory impairment effect of indomethacin or 3 alpha-diol in MWM task.

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Background: Ritalin has high tendency to be abused. It has been the main indication to control attention deficit hyperactivity disorder. The college students may seek for it to improve their memory, decrease the need for sleep (especially during exams), which at least partially, can be related to serotonergic system. Therefore, it seems worthy to evaluate the effect of Ritalin intake on mature brain. There are many studies on Ritalin effect on developing brain, but only few studies on adults are available. This study was undertaken to find Ritalin effect on serotonin transporter (SERT) density in medial frontal cortex (MFC) of mature rat. Methods: Thirty male Wistar rats were used in the study. Rats were assigned into five groups (n = 6 per group): one control, two Ritalin and two vehicle groups. Twelve rats received Ritalin (20 mg/kg/twice a day) orally for eleven continuous days. After one week of withdrawal and another two weeks of rest, in order to evaluate short-term effects of Ritalin, six rats were sacrificed. Another six rats were studied to detect the long-term effects of Ritalin therefore, they were sacrificed 12 weeks after the previous group. The immunohistochemistry was performed to evaluate the results. Results: Immunohistochemistry studies showed a higher density of SERT in both 2 and 12 weeks after withdrawal from Ritalin intake in MFC of rat and there was no significant difference between these two groups. Conclusions: Our findings demonstrated both short- and long-term effects of Ritalin on frontal serotonergic system after withdrawal period.

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Background: Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors. Methods: In this experimental study, endometrial biopsies (n = 7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48 or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student’s t-test, and P < 0.05 was considered statistically significant. Results: Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P = 0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P < 0.05). Conclusions: Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.

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Backgrund: Imprinted genes are a unique subset of few genes, which have been differentially methylated region (DMR) in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies heve shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception microenvironments often occur by assisted reproduction techniques (ART). This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART. Method: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites (CpGs) located in this region. Result: Results showed that the overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85±4.87% and 43.75±5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined (49.52±1.86% and 50%, respectively). Conclusion: Considering the absence of in vivo produced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks.

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Background: There is a controversy about the relation between anti-hepatitis B (anti-HBs) antibody level and obesity. We designed this study to compare the vaccine efficacy in obese/overweight and non-obese cases. Methods: In this cross-sectional study, 242 obese/overweight and 85 non-obese individuals were participated. Cases were selected from a referral clinic for obesity and a referral hepatology clinic, both in Tehran, Iran.
Results: Obese cases had lower percentage of liver diseases (66.9% vs. 100%, P<0.001) but higher hepatitis B vaccination history (74.9% vs. 51.2%, P<0.001). Median±inter-quartile range of anti-HBs titer in obese cases was significantly lower than controls (48.5±194.5 vs. 100±557.6, P=0.012). Conclusion: The level of anti-HBs surface antigen antibody’s titer in obese cases without liver disease is lower than control group. Therefore, a suitable strategy is needed to overcome this problem, which can be the use of longer needles for vaccination.

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Background: Colon cancer-associated transcript 2 (CCAT2) is a newly recognized lncRNA transcribed from the 8q24 genomic region. It functions as an oncogene in various types of cancers including breast cancer, in which it affects Wnt/β-catenin pathway. Previous studies have shown a putative interaction between this lncRNA and MYC proto-oncogene. Methods: In the current study, we evaluated the expression of CCAT2 in breast cancer tissues with regards to the expression of its target MYC. In addition, we assessed the relationship between CCAT2 and MYC expression levels in tumor tissues and the clinical prognostic characteristics of breast cancer patients. Results: MYC expression levels were significantly up-regulated in tumor tissues compared with adjacent non-cancerous tissues (ANCTs), while such analysis showed no statistically significant difference between these two tissue types in CCAT2 expression. Starkly increased CCAT2 gene expression levels were found in 12/48 (25%) of cancer tissue samples compared with their corresponding ANCTs. Furthermore, significant inverse correlations were found between CCAT2 expression and stage, as well as lymph node involvement. Besides, a significant inverse correlation was found between the relative MYC expression in tumor tissues compared with their corresponding ANCTs and disease stage. Conclusions: These results highlight the significance of MYC and CCAT2 expressions in the early stages of breast cancer development and suggest a potentially significant role for CCAT2 in a subset of breast cancer patients, which could applied as a potential therapeutic target in these patients.

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BBackground: We have previously reported that immunization with GRA 2 antigen of Toxoplasma gondii induces protective immunity in CBA /J (H2k) and BALB/c mice (H2d). We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens (GRA2) combined with monophosphorryl lipid A (MPL) adjuvant elicits protective immune response against T. gondii. Methods: C57BL/6 (H2b haplotype) mice were immunized with GRA 2, formulated in MPL adjuvant. Results: Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-γ, was detected at three weeks post immunization. Mice immunized with GRA 2 had significantly (p < 0.01) fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-γ production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization. Conclusions: We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA 2 for incorporation into a multi-antigenic vaccine against T. gondii.

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Background: Design of experiments is a rapid and cost-effective approach for optimization of recombinant protein production process. In our previous study, we generated a potent dual-acting fusion protein, anti-CD22 scFv-apoptin, to target B-cell malignant cell lines. In the present investigation, we report the effect of different variables on the expression levels of this fusion protein. Methods: Four variables (cell optical density at induction, IPTG concentration, induction temperature, and induction time) were tested using experimental design. Results: Our findings demonstrated that among the examined variables, only the induction time had a significant positive effect on the protein expression yield. Conclusion: Experimental design was successfully applied in this study. The optimized condition obtained in the current study can be applied in future commercial production of this novel fusion protein.

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Background: Cancer stem cells (CSCs) are a group of tumor cells with self-renewal property and differentiation potential. CSCs play a crucial role in malignant progression of several types of tumors. However, what is still controversial is the clinicopathological relationship between Nanog marker and its prognostic value in the patients with breast cancer. The expression of Nanog in the patients with breast cancer and its correlation with clinicopathological prognostic factors was explored in the present study. Methods: A sample of 120 breast cancer tissues was obtained from the patients who referred to Imam Khomeini Hospital in Sari city, Iran during January 2012 and December 2016. The associations between Nanog expression and clinicopathological factors were analyzed based on immunohistochemical analysis. Results: It was found that 67 (55.8%) patients had Nanog expression, and high expression rate was observed in 24 (36%) cases (staining index ≥3). Moreover, there was a statistically significant relationship between Nanog expression and clinicopathological factors, including tumor grade (p = 0.001), lymph node metastasis (p = 0.01), and the stage of the disease (p = 0.003). Conclusion: Findings of the study indicate that Nanog is a biomarker for prognostic prediction in patients with breast cancer. However, further studies of Nanog are suggested to provide novel therapeutic targets for curing breast cancer.

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Background: The innate immune system against malignancies is mainly orchestrated by natural killer cells, which carry out killing mechanisms by using their receptors, such as killer immunoglobulin-like receptors (KIRs). This study was designed to determine the diversity of KIR genes in non-melanoma skin cancers. Methods: A total of 160 subjects with skin cancer, including 60 cases of squamous cell carcinoma and 100 cases of basal cell carcinoma (BCC), and 270 healthy subjects formed the study groups. The sequence-specific polymerase chain reaction was carried out to detect the presence or absence of 16 KIR genes. Results: KIR3DL1 (p = 0.0381, OR = 4.78, 95% CI = 1.108 to 20.62) increased in BCC patients compared to healthy controls. Conclusion: We concluded that the higher frequency of KIR3DL1 in BCC patients compared with healthy controls may increase the probability of developing BCC in Iranians.

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Background: Hypercoagulability and hypofibrinolysis are among the symptoms exhibited by diabetic patients. Our study aimed to address the polymorphic nature of Alu DNA fragment in the human tissue plasminogen activator gene within diabetes mellitus (DM) Jordanian patients. Methods: Genomic DNA was isolated from 76 DM patients and 60 non-diabetic Jordanian individuals, and the Alu fragment was amplified using PCR. Results: The results showed that 80% of the non-diabetic Jordanian subjects were homozygotes for the deletion of the Alu fragment (Alu^-/-), 16.7% were homozygotes for its insertion (Alu^+/+), and 3.3% were heterozygotes (Alu^+/-). Besides, 36.8% of the diabetic patients exhibited the Alu^-/- or Alu^+/- genotype, and 26.3% were Alu^+/+. The Alu^-/- genotype occurred less frequently in the diabetic individuals. Conclusion: The high frequency of the Alu^-/- genotype constitutes a protective deletion with respect to DM within the normal subjects. 

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Aging, as a major risk factor of memory deficiency, affects neural signaling pathways in hippocampus. In particular, age-dependent androgens deficiency causes cognitive impairments. Several enzymes like protein kinase C (PKC) are involved in memory deficiency. Indeed, PKC regulatory process mediates α-secretase activation to cleave APP in β-amyloid cascade and tau proteins phosphorylation mechanism.  Androgens and cortisol regulate PKC signaling pathways, affecting the modulation of receptor for activated C kinase 1. Mitogen-activated protein kinase/ERK signaling pathway depends on CREB activity in hippocampal neurons and is involved in regulatory processes via PKC and androgens. Therefore, testosterone and PKC contribute in the neuronal apoptosis. The present review summarizes the current status of androgens, PKC, and their influence on cognitive learning. Inconsistencies in experimental investigations related to this fundamental correlation are also discussed, with emphasis on the mentioned contributors as the probable potent candidates for learning and memory improvement. 

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Background: The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Loss of supression (Los1) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends replicative lifespan. Herein, a los1-deficient strain of P. pastoris was generated and characterized. Methods: A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 single-chain variable fragment (scFv) expression were compared between the parent and mutant strains. Results: The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively. Conclusion: The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.