Iranian Journal of Basic Medical Sciences
Volume:17 Issue: 11, Nov 2014

Macrophages accumulate in poorly vascularised and hypoxic sites including solid tumours, wounds and sites of infection and inflammation where they can be exposed to low levels of oxygen for long periods. Up to date, different studies have shown that a number of transcription factors are activated by hypoxia which in turn activate a broad array of mitogenic, pro-invasive, pro-angiogenic, and pro-metastatic genes. On the other hand, macrophages respond to hypoxia by up-regulating several genes which are chief factors in angiogenesis and tumorigenesis. Therefore, in this review article we focus mainly on the role of macrophages during inflammation and discuss their response to hypoxia by regulating a diverse array of transcription factors. We also review the existing literatures on hypoxia and its cellular and molecular mechanism which mediates macrophages activation.

Candidiasis infection caused by Candida albicans has been known as a major problem in patients with immune disorders. The objective of this study was to genotype the C. albicans isolates obtained from oral cavity of patients with positive human immunodeficiency virus (HIV+) with or/and without oropharyngeal candidiasis (OPC). A total of 100 C. albicans isolates from Iranian HIV+patients were genotyped using specific PCR primers of the 25S rDNA and RPS genes. The frequencies of genotypes A, B and C which were achieved using 25S rDNA, were 66, 24 and 10 percent, respectively. In addition, genotypes D and E were not found in this study. Each C. albicans genotype was further classified into four subtypes (types 2, 3, 2/3 and 3/4) by PCR amplification targeting RPS sequence. In general, genotype A3 constituted the majority of understudy clinical isolates obtained from oral cavity of Iranian HIV+ patients.

Production of a recombinant and immunogenic antigen using dengue virus type-3 envelope protein is a key point in dengue vaccine development and diagnostic researches. The goals of this study were providing a recombinant protein from dengue virus type-3 envelope protein and evaluation of its immunogenicity in mice. Multiple amino acid sequences of different isolates of dengue virus type-3, corresponding to the envelope protein domain III, were achieved from GenBank. Clustal V alignment tool was used to provide a consensus amino acid sequence. Nucleotide sequence of the coding gene was optimized using “Optimizer”. The origami (DE3) strain of Escherichia coli was used as the host in order to express the protein. A commercial affinity chromatography method was used to purify the recombinant protein. Immunogenicity of the recombinant protein was evaluated in mice using ELISA, MTT and cytokine assays. A consensus amino acid sequence corresponding to the most important region of dengue virus type-3 envelope protein (domain III) was provided. A high concentration (≥ 20 mg/L culture medium) of soluble recombinant antigen (EDIII3) was achieved. Immunized mice developed specific antibody responses against EDIII3 protein. The splenocytes from EDIII3-immunized mice showed a high proliferation rate in comparison with the negative control. In addition, the concentrations of two measured cytokines (IFN-γ and IL-4) were increased markedly in immunized mice. The results showed that the expressed recombinant EDIII3 protein is an immunogenic antigen and can be applied to induce specific immune responses against dengue virus type-3.

Adipose derived stem cells (ADSCs) can be engineered to express bone specific markers. The aim of this study is to evaluate repairing tibia in animal model with differentiated osteoblasts from autologous ADSCs in alginate scaffold. In this study, 6 canine’s ADSCs were encapsulated in alginate and differentiated into osteoblasts. Alkaline phosphatase assay (ALP) and RT-PCR method were applied to confirm the osteogenic induction. Then, encapsulated differentiated cells (group 1) and cell-free alginate (group 2) implanted in defected part of dog's tibia for 4 and 8 weeks. Regenerated tissues and compressive strength of samples were evaluated by histological and Immunohistochemical (IHC) methods and Tensometer Universal Machine. Our results showed that ADSCs were differentiated into osteoblasts in vitro, and type I collagen and osteocalcin genes expression in differentiated osteoblasts was proved by RT-PCR. In group 2, ossification and thickness of trabecula were low compared to group 1, and in both groups woven bone was observed instead of control group's compact bone. Considering time, we found bone trabeculae regression and ossification reduction after 8 weeks compared with 4 weeks in group 2, but in group 1 bone formation was increased in 8 weeks. Presence of differentiated cells caused significantly more compressive strength in comparison with group 2 (P-value ≤0.05). This research showed that engineering bone from differentiated adipose-derived stem cells, encapsulated in alginate can repair tibia defects.

[p1] This study highlights xylanase overproduction from Bacillus mojavensis via UV mutagenesis and optimization of the production process. Bacillus mojavenis PTCC 1723 underwent UV radiation. Mutants’ primary screening was based on the enhanced Hollow Zone Diameter/ Colony Diameter Ration (H/C ratios) of the colonies in comparison with the wild strain on Xylan agar medium. Secondly, enzyme production of mutants was compared with parental strain. Optimization process using lignocellulolytic [AGA2] wastes was designed with Minitab software for the best overproducer mutant. H/C ratio of 3.1 was measured in mutant number 17 in comparison with the H/C ratio of the parental strain equal to 1.6. Selected mutant produced 330.56 IU/ml xylanase. It was 3.45 times more enzyme than the wild strain with 95.73 IU/ml xylanase. Optimization resulted 575 IU/ml xylanase, with wheat bran as the best carbon source, corn steep liquor as the best nitrogen source accompanied with natural bakery yeast powder, in a medium with pH 7, after 48 hr incubation at 37°C, and the shaking rate of 230 rpm. Optimum xylanase activity was assayed at pH 7 and 40°C. Enzyme stability pattern shows it retains 62% of its initial activity at pH 9 after 3 hr. It also maintains up to 66% and 59% of its initial activity after 1 hr of pre-incubation at 70°C and 80°C. Mutation and optimization caused 5.9 times more enzyme yield by mutant strain. Also this enzyme can be categorized as an alkali-tolerant and thermo-stable xylanase.

Protective effects of diuretics, particularly of hydrochlorothiazide (HCT), for the development of seizure attacksepilepsy have been described in vivo. However, itsthe mechanism of action of HCT is unknownneeds to be elucidated. Extracellular field potentials were recorded from the CA1- and CA3-subfields of the hippocampus of rats. Epileptiform discharges were induced by omission of Mg2+ from the artificial cerebrospinal fluid (ACSF). HCT was added to the ACSF at a concentration of 2 mmol/l (n=5), 0.2 mmol/l (n=5) or 0.02 mmol/l (n=5). Frequency, amplitude and duration of the epileptiform discharges were evaluated. Long-term potentiation (LTP) was induced with and without the presence of HCT (n=6; 2 mmol/l). In addition, rats were injected with HCT (n=4) or saline (n=2), and the brain tissue was analyzed using HPLC. Application of 0.02, 0.2, and 2 mmol/l HCT accelerated the frequency of discharges by 50%, 91%, and 100%, respectively. The amplitude of burst discharges also increased by 9%, 54%, and 300%, and the duration of epileptiform discharges increased by 10%, 30% and 120%. All parameters returned close to the basal levels after 60min washout of the substance. HCT increased the electrical evoked potentials but did not affect the LTP in hippocampal tissues. There was no evidence of HCT in the rat brain after intraperitoneal injection. Exposure of hippocampal slices to HCT enhanced epileptiform activity in a dose-dependent manner. In addition, HCT does not seem to cross the blood brain barrier in rats. Thus, the anticonvulsive effect of HCT most likely is not through direct neuronal effect.

Mutant forms FMS-like tyrosine kinase-3 (FLT3), are reported in 25% of childhood acute lymphoid leukemia (ALL) and 30% of acute myeloid leukemia (AML) patients. In this study, drug response, growth promoting, and protein trafficking of FLT3 wild-type was compared with two active mutants (Internal Tandem Duplication (ITD)) and D835Y. FLT3 was expressed on factor-dependent cells (FDC-P1) using retroviral transduction. The inhibitory effects of CEP701, imatinib, dasatinib, PKC412 and sunitinib were studied on cell proliferation and FLT3 tyrosine phosphorylation. Total expression and proportion of intracellular and surface FLT3 was also determined. FDC-P1 cells became factor-independent after expression of human FLT3 mutants (ITD and D835Y). FDC-P1 cells expressing FLT3-ITD grow 3 to 4 times faster than those expressing FLT3-D835Y. FD-FLT3-ITD cells were three times more resistant to sunitinib than the FD-FLT3-WT cells. The Geo means for surface FLT3 expression in FD-FLT3-ITD and –D835Y were 65 and 70% less than the FD-FLT3-WT cells. About 40% of expressed FLT3 was detected as intracellular in FD-FLT3-D835Y cell compared to 4 and 4.5% in FD-FLT3-WT and –ITD cells. Retention of D835Y FLT3 mutant protein may cause altered signaling, endoplasmic reticulum stress and activation of apoptotic signaling pathways leading to lower proliferation rate in FD-FLT3-D835Y than the FLT3-WT and ITD mutant., these may also also contribute, along with the preferential affinity, to the increased sensitivity of D835Y of CEP701 and PKC412. Studying these genetic variations can help determining the prognosis and designing a therapeutic plan for the patients with FLT3 mutations.

To examine the expressions of CD11a, CD11b, and CD11c integrins in the myocardial tissues of rats with isoproterenol-induced myocardial hypertrophy. This study also provided morphological data to investigate the signal transduction mechanisms of myocardial hypertrophy and reverse it. A myocardial hypertrophy model was established by subcutaneously injecting isoprenaline in healthy adult Sprague-Dawley rats. Myocardial tissues were obtained, embedded in conventional paraffin, sectioned, and stained with hematoxylin. Pathological changes in myocardial tissues were then observed. The expressions and distributions of CD11a, CD11b, and CD11c integrins were detected by immunohistochemistry. Changes in the mRNA expressions of CD11a, CD11b, and CD11c in the myocardial tissues of rats were detected by RT-PCR. Image analysis software was used to determine the expressions of CD11a, CD11b, and CD11c integrins quantitatively. Immunohistochemical results showed that the positive expressions of CD11a, CD11b, and CD11c integrins increased significantly in the experimental group compared with those in the control group. The mRNA expressions of CD11a, CD11b, and CD11c in the myocardial tissues of rats were consistent with the immunohistochemical results. The increase in the protein expressions of CD11a, CD11b, and CD11c integrins may have an important role in the occurrence and development of myocardial hypertrophy.

To clarify the protective effects of Cichorium glandulosum (CG) extracts on thioacetamide (TAA)-induced rat hepatic fibrosis. The dry roots of CG were smashed and percolated with 95% ethanol, and the residual was prepared into petroleum ether extract (CG-V), ethyl acetate extract (CG-VI) and n-butyl alcohol extract (CG-VII). Thirty-six Wistar rats were randomly divided into a normal group, a model group, a CG-V group (15 mg/kg), a CG-VI group (3 mg/kg), a CG-VII group (6 mg/kg) and a positive drug group (silibinin capsule, 8 mg/kg). Organ indices and serum levels of glutamic-oxaloacetic and glutamic-pyruvic transaminases of intragastrically administered rats were obtained. Expressions of FN, Smad3, IGFBPrP1 and TGF-β1 genes were detected by Western Blot and immunohistochemical assays. Apoptosis was examined by TUNEL assay. Hepatic fibrosis of treatment groups was evidently mitigated. Expressions of FN, Smad3 and TGF-β1 in administration groups were higher than those in normal group, and moreover were significantly higher in CG-V and CG-VII groups than those of model group. Apoptotic index of model group was significantly higher than that of normal group, but indices of CG-V and CG-VII groups were significantly lower than that of model group. Significantly more FN, Smad3 and IGFBPrP1 were expressed in treatment groups than those in normal group. CG extracts may function by altering TGF-β/Smads signal transduction pathway.

Study of non-coding RNAs is considerable to elucidate principal biological questions or design new therapeutic strategies. miRNAs are a group of non-coding RNAs that their functions in PI3K/AKT signaling and apoptosis pathways after T cell activation is not entirely clear. Herein, miRNAs expression and their putative targets in the mentioned pathways were studied in the activated CD4+T cells. Herein, proliferation rate and IL-2 secretion were measured in treated and untreated cells by IL-2. Putative targets of up-regulated miRNAs were predicted by bioinformatics approaches in the apoptotic and PI3K/AKT signaling pathways. Then the expression of two putative targets was evaluated by quantitative RT-PCR. Proliferation rate of treated cells by IL-2 increased in a dose- and time- dependent manner. Naive and activated CD4+T cells induced by different dose of IL-2 secreted abundant amounts of IL-2. Also, in IL-2 un-induced cells (IL-2 depleted cells) after 3 days, decrease of proliferation has been shown. In silico analysis predicted putative targets of up-regulated miRNAs such as AKT1, AKT3 and apoptotic genes in the activated cells induced or un-induced by IL-2. Decrease of AKT3 was shown by Q-RT-PCR as a potential target of miRNAs overexpressed in IL-2 depleted cells. But there was no significant difference in AKT1 expression in two cell groups. Our analysis suggests that decrease of AKT3 was likely controlled via up-regulation of specific miRNAs in IL-2 depleted cells. Also it seems that miRNAs play role in induction of different apoptosis pathways in IL-2 induced and un-induced cells.

Some histopathological alterations take place in the ischemic regions following brain ischemia. Recent studies have demonstrated some neuroprotective roles of crocin in different models of experimental cerebral ischemia. Here, we investigated the probable neuroprotective effects of crocin on the brain infarction and histopathological changes after transient model of focal cerebral ischemia. Experiment was performed in four groups of rats (each group; n=8), sham, control ischemia and ischemia treated rats. Transient focal cerebral ischemia was induced by 80 min middle cerebral artery occlusion (MCAO) followed by 24 hr reperfusion. Crocin, at doses 50 and 80 mg/kg, was injected at the beginning of ischemia (IP injection). Neurologic outcome (Neurological Deficit Score, NDS scale), infarct volume (TTC staining) and histological studies were assessed 24 hr after termination of MCAO. Treatment with crocin, at doses 50 and 80 mg/kg, significantly reduced the cortical infarct volume by 48% and 60%, and also decreased striatal infarct volume by 45% and75%, respectively. Crocin at two different doses significantly improved the NDS of ischemic rats. At histological evaluation, crocin, at dose 80 mg/kg more than 50 mg/kg, decreased the number of eosinophilic (prenecrotic) neurons and reduced the fiber demyelination and axonal damage in ischemic regions. Our findings indicated that crocin effectively reduces brain ischemia-induced injury and improves neurological outcomes. Crocin also is a potent neuroprotective factor that can be able to prevent histopathological alterations following brain ischemia.

Cardiomyocytes have small potentials for renovation and proliferation in adult life. The most challenging goal in the field of cardiovascular tissue engineering is the creation of an engineered heart muscle. Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to Cardiomyocyte creation applications. Human adipose-derived stem cells (ASCs) are good candidate for use in stem cell-based clinical therapies. They could be cultured and differentiated into several lineages such as cartilage, bone, muscle, neuronal cells, etc. In the present study, human ASCs were cultured on random and aligned polycaprolactone (PCL) nanofibers. The capacity of random and aligned PCL nanofibrous scaffolds to support stem cells for the proliferation was studied by MTT assay. The cardiomyocyte phenotype was first identified by morphological studies and Immunocytochemistry (ICC) staining, and then confirmed with evaluation of specific cardiac related gene markers expression by real-time RT-PCR. The proliferation rate of ASCs on aligned nanofibrous PCL was significantly higher than random nanofibrous PCL. ICC and morphological studies results confirmed cardiomyocyte differentiation of ASCs on the nanofibrous scaffolds. In addition, the expression rate of cardiovascular related gene markers such as GATA-4, α-MHC and Myo-D was significantly increased in aligned nanofibrous PCL compared with random nanofibrous PCL. Our results show that the aligned PCL nanofibers are suitable physical properties as polymeric artificial scaffold in cardiovascular tissue engineering application.

Epigenetic regulation of gene expression can be carried out through chromatin remodeling enzymes such as SWI/SNF. Brg1 also known as SMARCA4 is a catalytic subunit of SWI/SNF, which is necessary for MMPs expression. Matrix metalloproteinases (MMPs) are known as important player enzymes during tumor progression and metastasis. Aberrant epigenetic modification of chromatin should be precisely clarified to reveal probable unknown pathways in ESCC progression. Probable role of Brg1 in ESCC tumorigenesis and metastasis was studied through the assessment of Brg1 mRNA expression in KYSE30, and further evaluation about the biology of Brg1 was performed through the Brg1 silencing. Level of Brg1 mRNA expression in KYSE30 was compared to normal tissues using the real time polymerase chain reaction (PCR). Moreover, KYSE30 cells were transfected with Brg1-siRNA to silence the Brg1. Our results showed for the first time that Brg1 mRNA expression was increased in KYSE30 cell line (ESCC cell line) compared with normal esophageal tissue of ESCC patients. Rate of transfection in KYSE30 was also between 40 to 50%, using the pSilencer-Brg1shRNA (1:1 ratio). Our data indicated that chromatin remodeling machinery is a novel aspect in tumor biology of ESCC, and overexpression of Brg1 as an important member of SWI/SNF might be involved in the migration and invasion of ESCC tumoral cells.

Although recent investigations have shown chronic inflammation and inflammation-associated diseases might be ameliorated by exercise; little is known about the relation between exercise training with anti/pro-inflammatory cytokines. This cross sectional study was conducted to compare interleukin-4 (IL-4), IL-6, IL-10, IL-12, IL-13, interferon gamma (IFN-γ) levels in serum, and their in vitro production by whole blood (WB) cells and by peripheral blood mononuclear cells (PBMCs) in response to mitogens lipopolysaccharide and phytohemagglutinin. Twelve elite wrestlers with history of three times per week exercise training for about 9.5 years, and thirteen healthy silent controls were recruited. To analysis the cytokines by enzyme linked immunosorbent assay (ELISA), the blood samples were taken 24 hr after the last training session from the wrestlers. Serum analysis for IL-4, IL-6, IL-10, IL-12, IL-13 and IFN-γ indicated no statistical difference between the two groups. Meanwhile, 48 hr in vitro activation of WB and PBMCs by the mitogens revealed that IL-6 production was elevated in both WB and PBMCs. Whereas, IL-12 and IL-13 were decreased in supernatant of PBMCs and WB cells cultures, respectively. It seems that wrestling cause immune system cells to produce anti-inflammatory myokine IL-6 and decrease production of pro-inflammatory cytokine IL-12 and IL-13.

P In vitro chemosensitivity and resistance assays (CSRAs) are a promising tool for personalized treatment of glioblastoma multiform (GBM). These assays require a minimum of 1 to 2 g of tumor specimen for testing, but this amount is not always accessible. We aimed to assess the feasibility and validity of utilizing stereotactic biopsies of GBM in CSRAs. Single cell suspension was prepared from 1 g weight explants of the established xenograft tumor of GBM. Also, primary culture was carried out on 35 mg weight specimens, as a surrogate for stereotactic biopsies. Then, chemoresponse profile of cells obtained by direct cell disaggregation and primary culture was determined using temozolomide and carmustine by clonogenic assay[AGA1]. There was no statistically significant difference in the cytotoxicity of temozolomide and carmustine between cells obtained from both methods. This work supports the feasibility of using stereotactic biopsies of GBM in CSRAs.