Development of Nano-ELISA Method for Serological Diagnosis of Toxoplasmosis in Mice.

Toxoplasmosis is a widespread parasitic disease caused by a protozoan parasite Toxoplasma gondii. Currently, nanotechnology has been used for the diagnosis of many infectious diseases. It could be due to the fact that nanoparticles play an important role in accurate and fast diagnosis. The purpose of this study was to design a Nano-enzyme linked immunosorbent assay (Nano-ELISA) kit using excreted/secreted (E/S) antigens to have higher sensitivity and specificity than those reported for the designed enzyme-linked immunosorbent assay (ELISA) kit for the diagnosis of Toxoplasmosis in mice. Firstly, the serum samples were collected from 15 infected mice with T. gondii and 15 healthy ones. Then, E/S antigens were separated from parasite tachyzoites and used for designing an ELISA kit. In addition, the mice sera were evaluated using the designed ELISA kit. Finally, the serum samples were assessed by Nano-ELISA kits designed with E/S antigen and conjugate of gold nanoparticles. The obtained results of the present study showed that the sensitivity and specificity of the designed ELISA kit were reported as 80% and 86.66%, respectively, that both improved to 93.33% in these sera with the designed Nano-ELISA kit. This finding revealed the significant improvement of sensitivity and specificity using gold nanoparticles in designing the ELISA kit. Furthermore, according to the literature, the use of E/S antigens in designing recognizable ELISA kits has been always highlighted considering the presence of numerous antigens in T. gondii. The results of this study revealed that the use of E/S antigens in the preparation of an ELISA kit was very effective. This is very important, especially in the lower titers of antibody requiring a more accurate diagnosis. On the other hand, the Nano-ELISA method designed with E/S antigens can be more sensitive and specific than ELISA for the diagnosis of Toxoplasmosis and can be the basis for further studies in this regard.

Seroprevalence and molecular detection of Toxoplasma gondii in healthy blood donors in southwest Iran.

BACKGROUND: Toxoplasmosis is a cosmopolitan parasitic disease caused by Toxoplasma gondii (T. gondii). Blood transfusion is a probable route of T. gondii transmission. Due to lack of information about seroprevalence of T. gondii in healthy blood donors, this study was aimed to determine the chronic and acute infection using serological and molecular methods. MATERIAL AND METHODS: In this cross-sectional investigation, 380 samples were collected from donated bloods. Anti-Toxoplasma IgG and IgM antibodies were examined using enzyme-linked immunosorbent assay (ELISA). Also, all IgG positive samples were tested by IgG avidity test. Eventually, to detection of active infection, DNA was extracted from IgM positive and low IgG avidity samples and then tested using nested-polymerase chain reaction (PCR). RESULTS: Among 380 blood donors, 131 (34.47%) were positive for only anti-T. gondii IgG, 2 (0.5%) were positive for only anti-T. gondii IgM, and 11 (2.9%) were positive for both IgG and IgM antibodies. Then, 142 samples (131 IgG + and 11 IgG +IgM +) were evaluated using IgG avidity test. Of these, 115 (81%) had high avidity IgG indicates past infection; 16 (11.26%) had low avidity IgG representing recent infection, and 11 (7.74%) were equivocal. With nested PCR, 20 samples of 50 seropositive samples were diagnosed positive. CONCLUSION: Detected active infection using nested-PCR draws attention to the possibility of T. gondii infection via blood transfusion which emphasizes the importance of parasite DNA screening before donation of blood in high risk groups such as: multi-transfused persons, immunosuppressed patient, and pregnant women.

Quercetin induces apoptosis and necroptosis in MCF-7 breast cancer cells.

OBJECTIVE: This study investigated the quercetin (Que) effects on growth of MCF-7 human cancer breast cell line and its cellular death mechanism. BACKGROUND: Quercetin has been found to be very efficacious against many different types of cancer cells. However, the study is not sufficiently powered to demonastrate anticancer mechanisms. METHODS: MCF-7cells were treated by 50 µM/ ml of Que for 48 hours. MCF-7 cells were also pretreated with 10 Μm ZVAD (apoptosis inhibitor) or 3 mM Nec-1 (necroptosis inhibitor) for evaluation of cell death induced by apoptosis or necroptosis. RESULTS: MTT and clonogenicity assays revealed that the Que induced a significant increase in cell viability and proliferation in presence of Nec-1 in comparison to the presence of ZVAD (p < 0.05). Que also increased apoptosis as revealed by DAPI staining and morphology evaluations. Following Que treatment Bcl-2 expression was significantly decreased while Bax expression was significantly increased. Que in presence of Nec-1 decreased expression of Bax gene, reduced apoptotic index, increased cell viability and proliferation of MCF-7 cells in comparison to absence of Nec-1. MCF-7 cells showed a significantly increased expression of RIPK1 and RIPK3 in response to Que plus ZVAD in comparison to absence of ZVAD. CONCLUSION: Our results revealed that the high Que toxicity for breast cancer cells depends on multiple cell death pathways, which involve mainly necroptosis (Fig. 6, Ref. 21).

Very sensitive electrochemical determination of diuron on glassy carbon electrode modified with reduced graphene oxide-gold nanoparticle-Nafion composite film.

In this work, a very sensitive electrochemical sensor based on glassy carbon electrode (GCE) modified with reduced graphene oxide-gold nanoparticles/Nafion (rGO-AuNPs/Nafion) composite film was applied to determine diuron. Synthesized GO was characterized using X-ray diffraction (XRD) and UV-visible spectroscopy. The surface morphology of the rGO-AuNPs/Nafion film was also characterized using scanning electron microscopy and electrochemical impedance spectroscopy. Cyclic voltammetry (CV) and adsorptive differential pulse voltammetry (AdDPV) were applied to investigate the electrochemical response of the diuron on the modified electrode. The electrode showed a linear response at 1.0×10-9-1.0×10-7 M and a detection limit of 0.3nM under the optimized conditions. The effect of some other species on the determination of diuron was investigated and the sensor showed good selectivity for determination of diuron. The constructed sensor was applied to determine diuron in enriched samples of orange juice, mineral and tap water which statistical t-test showed accuracy of method. Also the sensor was applied to obtain diuron content in the tea sample. The reliability of the proposed sensor was confirmed after comparing the results with those obtained using high performance liquid chromatography (HPLC) as a comparative method.

Human Split-Thickness Skin Allograft from Brain-Dead Donors.

BACKGROUND: Looking for an appropriate skin substitute for temporary and permanent coverage of wounds remains one of the main obstacles of medical researchers. OBJECTIVE: To investigate the rate of inflammation, symbiosis, and survival of grafted allograft skin from brain-dead donors (BDDs) in rabbits. METHODS: After receiving negative serologic tests of BDDs, we prepared partial thickness skin grafts. They were then used in treating wounds of 5 rabbits in comparison with split-thickness skins taken from cardiac dead donors. RESULTS: On histopathological examinations, we found no difference between the skins. All samples were separated from the baseline in 15-20 days. CONCLUSION: Gamma-irradiated freeze-dried human split-thickness skin taken from BDDs is safe and can be used for the treatment of deep skin burns.

Preventive effects of procyanidin A2 on glucose homeostasis, pancreatic and duodenal homebox 1, and glucose transporter 2 gene expression disturbance induced by bisphenol A in male mice.

Procyanidins (PCs) as oligomeric compounds with antidiabetic properties formed from catechin and epicatechin molecules. Bisphenol A(BPA) is a common chemical material use in food and beverage packaging. The aim of this study was to explore the protective effects of procyanidin A2 (PCA2) against glucose homeostasis disturbance and gene expression of pancreatic and duodenal homebox 1 (Pdx1) as well as glucose transporter 2 (Glut2) induced by BPA in male mice. First tested these five concentrations of PCA2 (3 - 300 µM) alone and in combination with BPA(100 µg/L), on insulin secretion from isolated islets at in vitro condition. Next, examined the influence of BPA and PCA2 on islet apoptosis using flowcytometry. At in vivo condition, the BPA (100 µg/kg) and PCA2 (10 µmol/kg) administered for 20 days then, blood glucose and insulin, Pdx1 and, Glut2 genes expression, and oxidative stress markers examined. The results indicated that PCA2 strongly prevents islet cells apoptosis induced by BPA and, co-administration of PCA2 and BPA modified hyperglycemia. BPA reduced Pdx1 and Glut2 mRNA expression and antioxidant level in pancreas tissue, whereas PCA2 prevented from these effects. The findings from these studies suggest that use of PCA2 rich plants have preventive effects on hyperglycemia, and type 2 diabetes.

Combination effect of melatonin and dexamethasone on liver ischemia/reperfusion injury.

OBJECTIVE: Liver failure following ischemia-reperfusion (I/R) injury is a major concern in liver surgery. The purpose of this study was to evaluate combination pretreatment with melatonin (MEL) and dexamethasone (DEX) on liver I/R model. Male Wistar rats (n = 60) were assigned to 5 groups of 12 animals each: (1) Sham: laparotomy without I/R; (2) I/R: hepatic I/R; (3) I/R+MEL: hepatic I/R+melatonin injected intraperitoneally (20 mg/kg); (4) I/R+DEX: hepatic I/R+ dexamethasone injected intravenously (10 mg/kg); (5) I/R+MEL+DEX: hepatic I/R+ melatonin injected intraperitoneally+dexamethasone injected intravenously. The liver was subjected to ischemia by clamping the portal triad for 30 minutes and then reperfused for 6 hours after ischemia by removing the clamps. RESULTS: The levels of glutathione peroxidase (GPx) and superoxide dismutase (SOD) decreased after hepatic I/R in all groups. Levels of GPx and SOD were higher in I/R+MEL+DEX group compared to I/R, I/R+MEL and I/R+DEX groups and they were significantly higher in I/R+MEL group compared to I/R and I/R+DEX groups (p < 0.05). Levels of ALT, AST, TNF-α, hepatic tissue malondialdehyde (MDA), liver injury index, and apoptotic index increased after hepatic I/R. Levels of ALT, AST, tissue MDA, tissue injury index and apoptotic index were lower in I/R+MEL+DEX group compared to those in I/R, I/R+MEL and I/R+DEX groups, and in I/R+MEL they were significantly lower than in I/R+DEX group (p < 0.05). TNF-α level was lower in I/R+MEL+DEX group compared to other groups and it was significantly lower in I/R+DEX group than in I/R+MEL and I/R groups (p < 0.05). CONCLUSION: Combination therapy with melatonin and dexamethasone had better results in decreasing the liver injury compared to when each of them was administered alone (Tab. 3, Ref. 58).

Assessment of changes in expression and presentation of NKG2D under influence of MICA serum factor in different stages of breast cancer.

Breast cancer is the most common cancer in women worldwide. In this study, we correlated the serum level of major histocompatibility complex class I-related chain A (sMICA) with expression and presentation of NKG2D receptors on NK cells among patients with breast cancer. Peripheral blood (PB) samples were collected from 49 healthy and 49 breast cancer patients before surgery and chemotherapy. The expression and presentation of NKG2D were assessed using qRT-PCR and flow cytometry, respectively. Furthermore, sMICA levels were determined using ELISA. In flow cytometry, whole blood samples were stained with anti-CD56/NKG2D/CD3 and the obtained results were analyzed using WinMDI software. In addition, SPSS software was used for statistical analysis of data. Significantly higher levels sMICA were detected in the sera of the majority of cancer patients in contrast to healthy volunteers (P < 0.001). The expression and presentation of NKG2D receptor were significantly lower than those in healthy persons, and with an inverse correlation to sMICA and positively correlated with tumor stage. Our study showed that sMICA may have an important role in diminishing the expression and presentation of NKG2D receptor in breast cancer patients and proposes the notion that sMICA can be a target candidate for treatment of breast cancer.

An in vitro study on suppressive effects of Leishmania major on IL-2Rα expression on peripheral human T lymphocyte.

Leishmania sp. is an intracellular protozoan parasite that causes significant morbidity and mortality in many parts of the world. The parasite can escape from host immune system by several mechanisms. Understanding biological behavior of the parasite can help us to control and treatment leishmaniasis. Therefore current study was conducted to determine suppresive effect of Leishmania major on IL-2Rα expression in the human peripheral T Lymphocytes. Human peripheral T Lymphocyte were co-cultured with standard strain of Leishmania major (MRHO/IR/75/EK) in RPMI1640 medium. Infected cells were stained with FITC-labelled anti-CD25 (IL-2Rα chain MAb) and Picoerithrin-labelled anti-CD4 (CD4 MAb) and analyzed by flow cytometry. The results showed that L. major suppressed IL- 2Rα expression in activated T cells as well as inhibited lymphocyte proliferation 6h after infection and was increased up to 36 hour later. This finding also indicated that suppressed IL- 2R expression was increased when the number of promastigote was added up to 7.5×10(6) cells/ml. Inhibition of IL-2R expression by the parasite might play a critical role for escaping from host immune system. Understanding biological characterization of the Leishmania can be useful for vaccine development and also cytokine therapy.

Antibiotic-coated pins for prevention of pin-tract infection: a rabbit study.

PURPOSE. To evaluate the efficacy of antibiotic-coated pins for prevention of pin tract infection in a rabbit model. METHODS. 10 rabbits were divided into 2 groups. A unilateral external fixator was applied to the tibia with 4 self-taping 1.8-mm pins. In the test group, pins were coated with hydroxyapatite and antibiotic. In the control group, pins were not coated. All pins were then placed in Staphylococcus aureus- containing media. At postoperative day 5, all 40 pin sites were subcutaneously inoculated with S aureus. The sites were clinically examined for signs of pin tract infection. Nine days later, a piece of soft tissue around the pin site was harvested for microbiologic examination. RESULTS. In the test group, all except one pin sites appeared clean and without clinical infection, and the culture media remained clear. In the control group, all pin sites showed evidence of clinical infection and yielded positive cultures, and the culture media became dark indicating growth of S aureus. CONCLUSION. Antibiotic-coated pins were effective in preventing pin tract infection.

Lipoic acid prevents hepatic and intestinal damage induced by obstruction of the common bile duct in rats.

INTRODUCTION: Cholestatic liver diseases are characterized by impaired hepatocellular secretion of bile, resulting in intracellular accumulation of bile acids which result in a shift in the oxidant/prooxidant balance in favor of increased free radical activity and injury of different tissues including liver and intestine. The aim of this research was to study protective effect of lipoic acid (LA) as a potent antioxidant in cholestsis induced hepatic and intestinal injury in rats. MATERIALS AND METHODS: Forty five adult male Wistar rats were randomly assigned to four groups each containing fifteen rats as follows: sham operation (SO) (control), bile duct ligating (BDL), and BDL+LA (25 mg/kg). After fourteen days hepatic and intestinal tissue sampled and blood serum sampled for pathologic and biochemical studies. RESULTS: Levels of SOD and GPx antioxidant enzymes were higher in BDL+LA group comparing to BDL group, levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyltranspeptidase (GGT), and pathologic scores in liver and intestine were lower in BDL+LA group comparing to BDL group significantly, but there is no significant difference in concentrations of total bilirubin between groups. CONCLUSIONS: Our results showed the protective potential of LA with liver and intestine damage. Despite improvements in operative technique and the development of potent, broad-spectrum antibiotics, biliary tract surgery in patients with obstructive jaundice is still associated with high morbidity and mortality rates In summary, our results show that BDL induced hepatic and intestinal injury were significantly attenuated by LA administration and the administration of LA could effectively diminish this damage.

Rate of family refusal of organ donation in dead-brain donors: the Iranian tissue bank experience.

BACKGROUND: The growing gap between organ supply and demand remains a worldwide serious problem. Losing dead-brain donor organs can be attributed to several reasons including un-recognition of potential donor by ICU staff, death before official declaration of brain death and high refusal rate of deceased donors' families. OBJECTIVE: To study the trend of dead-brain patients' relatives refusal of organ donation from 2007 to 2011. METHODS: This study was a retrospective review of all patients who had been introduced as brain death to the organ procurement unit (OPU) of Iranian Tissue Bank between April 2007 and April 2012 according to preliminary neurological exam performed in the hospital of origin. The refusal rate of dead-brain patients' families and its reasons was evaluated. RESULTS: A total of 874 ICU admitted patients with severe brain injury (Glasgow coma score <7) was introduced to our center and were visited by the coordinator team during April 2007 to April 2012. 412 (47%) patients were excluded from the study mainly due to unsuitability for donation according to the approved medical protocols (n=205) and not fulfilling the brain death criteria (n=66). The families of the remaining cases (n=462) had been interviewed 343 (74.2%) of whom permitted donation. The mean±SD age of donors was 29.8±13.2 years; the male/female ratio was almost 2. The most common reason of brain death was traffic collision (n=120; 56.3%) and cerebrovascular accidents (n=40; 18.8%). The refusal rate from 2007 to 2011 has decreased respectively, from 30.4% to 20% in Tehran, and from 57.1% to 51.6% in other cities. The overall refusal rate was 25.8%. CONCLUSION: Our study confirmed that more level of expertise of the coordinator team and continuous public education, would result in higher rate of consent to organ donation.

Protective effect of pentoxyfilline in renal toxicity after methotrexate administration.

INTRODUCTION: Nephrotoxicity is an important side-effect of treatment with Methotrexate (MTX). Pentoxifylline (PTX) is an anti-inflmmatory and anti-oxidant agent. We hypothesized that pentoxifylline may affords renal protection by downregulating TNF-alpha as well as by improving cellular anti-oxidant activity. MATERIALS AND METHODS: Forty five male Wistar rats were assigned to 3 groups of 15 animals each: Group 1: control group (0.9% saline). Group 2: MTX; injected with 20 mg/kg MTX intraperitoneally (i.p.). Group 3: MTX + PTX injected i.p. MTX (20 mg/kg) + PTX (50 mg/kg) i.p. PTX was administered since 3 days before MTX administration and continued for 6 days. After 6 days rats were anesthetized and serum sampled and renal tissue removed for biochemical and histological evaluation. RESULTS: Data showed that glutathione peroxidase (GPx), superoxide dismutase (SOD) activities were lower in PTX + MTX group comparing to MTX group significantly (p < 0.05). Renal tissue injury index and percent of TUNEL positive cells, renal tissue malondialdehyde (MDA) levels, serum BUN (Blood Urea Nitrogen), creatinine (Cr) and TNF-alpha levels were higher in MTX group comparing to MTX+PTX group significantly (p < 0.05). CONCLUSIONS: In this study, the increased level of tissue MDA and serum TNF-alpha level together may be suggested that the underlying mechanism is related to direct toxicity of MTX rather than blockage in folate synthesis in kidneys. PTX administration also attenuated renal tissue injury and number of apoptic cells and suppressed the elevation of BUN and Cr levels. However, further studies are essential to elucidate the exact mechanisms of MTX-induced renal toxicity, and protection and the effect of PTX.

CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model.

Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.

Sodium alginate as a novel therapeutic option in experimental colitis.

The potential therapeutic effect of low-viscosity sodium alginate (LVA) was studied in a rat model of acute colitis induced by intracolonic administration of acetic acid. This experimental model produced a significant ulcerative colitis. Induction of colitis also significantly enhanced the serum and colonic mucosal cytokine (IL-6 and TNF-alpha) and eicosanoid (LTB4 and PGE2) levels, which paralleled with the severity of colitis. LVA solution was administered orally as drinking water at concentration of 0.5% (W/V) for 1 week. The tolerability and inhibitory effect of LVA on matrix metalloproteinase-2 (MMP-2) were tested using WEHI-164 cell line and zymography method. The results showed that LVA therapy is able to significantly reduce colonic damage score, histological lesion, serum and colonic mucosal IL-6, TNF-alpha, LTB4 and PGE2 levels in treated group compared with nontreated controls. Moreover, in vitro examinations revealed that treatment with LVA could diminish MMP-2 activity. It is concluded that LVA is able to suppress acetic acid-induced colitis in rats. Some of the action of LVA may be associated with its inhibitory effects on cytokine and eicosanoid production and MMP-2 activity. Our data suggest that LVA could potentially be a novel therapeutic option for inflammatory bowel disease.

Branch-site selection in a group II intron mediated by active recognition of the adenine amino group and steric exclusion of non-adenine functionalities.

The 2'-hydroxyl on a specific bulged adenosine is the nucleophile during the first step of splicing by group II introns. To understand the means by which the ribozyme core recognizes this adenosine, it was mutagenized and effects on catalytic activity were quantified. The results indicate that a low level of mutational variability is tolerated at the branch-site of group II introns, with no apparent loss of fidelity. Analyses of mutant and modified nucleotides at the branch-site reveal that adenine is recognized primarily through the N6 amino group and by steric exclusion of functionalities found on other bases. The mutational and single atom effects reported here contrast with those observed during spliceosomal processing, suggesting that there are important differences in adenosine recognition by the two systems.