Cytotoxic and apoptotic properties of a novel nano-toxin formulation based on biologically synthesized silver nanoparticle loaded with recombinant truncated pseudomonas exotoxin A.

Bacterial toxins have received a great deal of attention in the development of antitumor agents. Currently, these protein toxins were used in the immunotoxins as a cancer therapy strategy. Despite the successful use of immunotoxins, immunotherapy strategies are still expensive and limited to hematologic malignancies. In the current study, for the first time, a nano-toxin comprised of truncated pseudomonas exotoxin (PE38) loaded silver nanoparticles (AgNPs) were prepared and their cytotoxicity effect was investigated on human breast cancer cells. The PE38 protein was cloned into pET28a and expressed in Escherichia coli, BL21 (DE3), and purified using metal affinity chromatography and was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AgNPs were biologically prepared using cell-free supernatant of E. Coli K12 strain. Nanoparticle formation was characterized by energy dispersive spectroscopy, transmission electron microscopy, and dynamic light scattering. The PE38 protein was loaded on AgNPs and prepared the PE38-AgNPs nano-toxin. Additionally, in vitro release indicated a partial slow release of toxin in about 100 hr. The nano-toxin exhibited dose-dependent cytotoxicity on MCF-7 cells. Also, real-time polymerase chain reaction results demonstrated the ability of nano-toxin to upregulate Bax/Bcl-2 ratio and caspase-3, -8, -9, and P53 apoptotic genes in the MCF-7 tumor cells. Apoptosis induction was determined by Annexin-V/propidium flow cytometry and caspases activity assay after treatment of cancer cells with the nano-toxin. In general, in the current study, the nano-toxin exhibit an inhibitory effect on the viability of breast cancer cells through apoptosis, which suggests that AgNPs could be used as a delivery system for targeting of toxins to cancer cells.

Alloimmunization against platelets, granulocytes and erythrocytes in multi-transfused patients in Iranian population.

BACKGROUND: This study aims at alloantibody screening, determination of the types of these antibodies in multiple-transfused patients with chronic hematologic diseases. PATIENTS AND METHODS: This descriptive study was performed on 240 patients with chronic hematological diseases referred to public hospitals in Iran. Single blood sample was taken and tested for the presence of antibodies. In case of a positive antibody screening, antibody identification was performed using granulocyte agglutination test (GAT), granulocyte indirect immunofluorescence test (GIIFT), platelet indirect immunofluorescence test (PIIFT), monoclonal antibody-specific immobilization of platelet antigen (MAIPA) and panel cells. RESULTS: Out of 240 patients, 105 patients (43.75 %) had been alloimmunized. The incidence of alloantibodies against red blood cells (RBCs) in positive alloantibodies patients were 84.76% (89/105). The most common alloantibody was against antigens of the kell (anti-K) and Rh (anti-E) and (anti D) systems (46.66%, 18.09% and 11.43% respectively). The overall incidence of anti- human leukocyte antigen (HLA) antibodies were 65.7% (69/105). Polymorphonuclear (PMN)-specific antibodies were found in 6.66% (7/105). Also from 105 patients, 14 patients had alloantibodies against platelet. DISCUSSION: In general, it is recommend that to decrease the rate of alloantibody synthesis, the packed cells should be cross matched for minor blood groups especially for Rh (E) and kell. In addition, the use of leukodepleted blood products can decrease the frequency of alloimmunization against platelet (PLT), PMN and HLA antigens.

Biochemical and Biological Evaluation of an L-Asparaginase from Isolated Escherichia coli MF-107 as an Anti-Tumor Enzyme on MCF7 Cell Line

Background: One of the most widely used anticancer agents is microbial L-ASNase. Herein, we assessed the biochemical and biological properties of an isolated L-ASNase from a Gram-negative bacteria strain, Escherichia coli MF-107. Methods: Using garden asparagus, we obtained several bacterial isolates. These strains were further screened for L-ASNase activity. A promising bacterial isolate was selected for L-ASNase production and subsequent purification. The molecular weight of purified L-ASNase was determined. The MTT assay was applied to assess the cytotoxic effect of the purified enzyme. Also, for caspase activity determination and the apoptotic effect of purified enzyme on in cells, we conducted a real-time PCR method. Results: The molecular weight of the enzyme was approximately 37 kDa. In the pH range of 7.5 to 8, the enzyme had considerable stability. At 35 °C, the purified L-ASNase optimum activity was recorded. The cytotoxic effect of the enzyme on treated cells was dose-dependent with an IC50 value of 5.7 IU/ml. The Bax gene expression considerably raised by 5.75-fold (p < 0.001) upon L-ASNase treatment. On the other hand, the anti-apoptotic Bcl-2 gene expression showed a 2.63-fold increase compared to the control (p < 0.05). It was detected that the mRNA levels of caspase-3 and p53 were considerably upregulated (5.93 and 1.85-fold, respectively). We did not find any alternation in the caspase-8 activity of the treated cells compared to untreated cells. Conclusion: In this research, the proliferation of the breast cancer cells remarkably inhibited via the cytotoxic effect of isolated L-ASNase from microbial sources.

Evaluation of Inflammasome Activation in Peripheral Blood Mononuclear Cells of Hemodialysis Treated Patients with Glomerulonephritis.

Recently, it has been found that abnormal activation of inflammasomes, the intracellular multiprotein complexes, plays an important role in the pathogenesis and the development of inflammatory diseases. To determine whether the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is involved in chronic inflammatory condition reported in glomerulonephritic- hemodialysis (HD) patients, we investigated the mRNA levels of NLRP3, CASP-1, ASC, IL-1ß, IL-18, NLRC4, and P2X7 in human peripheral blood mononuclear cells (PBMCs) collected from 28 glomerulonephritic-HD patients. To confirm the mRNA quantification results, we investigated the IL-1ß content and Caspase 1 activity in serum and PBMC lysates, respectively. Compared with PBMCs derived from healthy subjects, genes encoding proinflammatory cytokines such as IL-1ß and IL-18 as well as NLRP3, ASC, CASP-1 were markedly overexpressed in those derived from patients. Moreover, there was no significant difference between the expression level of P2X 7 mRNA in PBMCs isolated from glomerulonephritis-HD patients and controls. The serum level of active IL1-ß and cell lysate CASP-1 activity were up-regulated in patients compared to controls. We also revealed that PBMCs isolated from glomerulonephritis-HD patients had elevated mRNA levels of NLRC4 compared to controls, suggesting the priming of NLRC4 inflammasome. These results revealed that the NLRP3-ASC-caspase-1 axis might have a role in increased inflammation severity reported in glomerulonephritic patients undergoing hemodialysis. These findings provide new insights into molecular mechanisms underlying chronic inflammation in HD- glomerulonephritic patients. Additionally, the NLRP3 inflammasome pathway can be attractive as a potential therapeutic target for complication avoidance in HD- glomerulonephritic patients.

Cytotoxic and apoptotic properties of silver chloride nanoparticles synthesized using Escherichia coli cell-free supernatant on human breast cancer MCF 7 cell line.

Synthesis of silver and silver based nanoparticles using microorganisms has received profound interest because of obtaining nanoparticles with unique physicochemical and biological properties. In the current study, for the first time, synthesis of silver chloride nanoparticles (AgClNPs) using cell-free supernatant of Escherichia coli culture is reported. Prepared AgClNPs were characterized by EDS, XRD and FESE. Data revealed the synthesized nanoparticles, mostly, have a spherical shape with an average size of 13 nm. Additionally, MTT assay elucidated a dose-dependent cytotoxicity of AgClNPs against MCF-7 cells (IC50 = 44 µg/mL). Quantitative real-time reverse transcription-PCR and colourimetric assays were employed to investigate the mechanism of cell toxicity in several cell death pathways. The results revealed the ability of AgClNPs to upregulate Bax/Bcl-2 ratio and p53 at mRNA level. Moreover, other apoptotic factors such as caspase-3, 8 and 9 were also upregulated at both mRNA and proteome levels. Finally, apoptosis induction was confirmed by Annexin-V/PI detection assay. Based on the obtained data, biosynthesized AgClNPs using E. coli cell-free supernatant exhibit a cytotoxic effect on human breast cancer cells through up-regulation of apoptotic factors, which suggest them as anti-tumour agents for further investigations.

Antagonistic Property of G2013 (α-L-Guluronic Acid) on Gene Expression of MyD88, Tollip, and NF-κB in HEK293 TLR2 and HEK293 TLR4.

INTRODUCTION: Inhibition of Toll-like receptors (TLRs) signaling plays a crucial role in suppressing the inflammation and available data presenting G2013 as an immunomodulatory agent, therefore, we designed this study to answer whether G2013 can affect the signaling pathway of TLR2 and TLR4. METHODS: Cytotoxicity study of G2013 was performed by MTT assay. HEK293 TLR2 and HEK293 TLR4 cell lines were cultured and treated with low dose (5µg/ml) and high dose (25µg/ml) of G2013 for 24 hours. Gene expressions of MyD88, Tollip, and NF-κB were defined by quantitative real-time PCR. RESULTS: The cytotoxicity assay showed that the concentrations lesser than 125µg/ml of G3012 had no apparent cytotoxicity, however, the concentrations of 5µg/ml and 25µg/ml could suppress the mRNA expression of MyD88, Tollip and NF-κB in HEK293 TLR2 and HEK293 TLR4 cell lines. CONCLUSION: in our study, we verified the linkage between the immunosuppressive property of G2013 and TLR2, TLR4 signaling cascade; but so far, the specific target of G2013 and its molecular mechanism has not been detected yet. We recommend further studies on other Patten Recognition Receptors (PRRs)and other mechanisms of inflammation like oxidative stress to be conducted in the future.

Association between thrombophilia gene polymorphisms and recurrent pregnancy loss risk in the Iranian population.

Miscarriage is the most common complication in pregnancy. Considering the importance of the problem thrombophilia in pregnant women and its association with recurrent pregnancy loss (RPL), analysis of polymorphisms of genes involved in thrombophilia can be useful. We investigated the frequency and association between ten polymorphisms of seven thrombophilia genes and RPL in an Iranian population. This case-control study was conducted on 200 women with recurrent pregnancy loss and also on 200 women with at least one successful pregnancy as the control group. Using PCR-RFLP, DNA from samples were analyzed for carrying A5279G, A4070G, and FV Leiden of factor V; FXIII (Val34Leu); FII (A20210G); BF (-455 G/A); ITGB3 (1565T/C); 677C/T and 1298A/C of MTHFR; and PAI-1 (-675 I/D, 5G/4G) polymorphisms. The BF(-455 G/A), MTHFR (677 C/T, 1298A/ C), PAI-1 (-675 I/D,4G/ 5G), FV Leiden, FV (A5279G), FXIII (Val34Leu) polymorphisms, which had shown positive relation, and ITGB3 1565T/C were the polymorphisms with negative relation to RPL. But in this study it is indicated that there is no significant association between FII (A20210G) and FV (A4070G) polymorphism and RPL. All the data acquired from the RPL patients in this experiment illustrate the importance of screening thrombophilia. Nevertheless, more studies on large-scale populations may be needed to identify novel genetic variants. ABBREVIATIONS: ASRM: American Society of Reproductive Medicine; HHCY: hyperhomocysteinemia; MTHFR: methylenetetrahydrofolate reductase; PCR: polymerase chain reaction; PAGE: poly-acrylamide gel electrophoresis; RPL: recurrent pregnancy loss.

Evaluation of G2 Citric Acid-Based Dendrimer as an Adjuvant in Veterinary Rabies Vaccine.

For induction of an appropriate immune response, especially in the case of an inactivated vaccine, the use of an adjuvant is crucial. In this study, adjuvanticity effect of G2 dendrimer in veterinary rabies vaccine has been investigated. A nonlinear globular G2 dendrimer comprising citric acid and polyethylene glycol 600 (PEG-600) was synthesized and the toxicity was studied in vitro on the J774A.1 cell line. The adjuvanticity effect of the dendrimer was then investigated on rabies virus in NMRI mice as a model. Different concentrations of dendrimer were used to determine the best formulation for the survival of the mice after virus challenge. The rise of neutralizing antibody was also checked by rapid fluorescent focus inhibition test (RFFIT). The relative potency of the prepared formulation was finally calculated using standard NIH test and the results were compared (and discussed) with the commercially available rabies vaccine. The accuracy of dendrimer synthesis was confirmed using Fourier transform infrared (FT-IR), size, and zeta potential analysis. The in vitro toxicity assay revealed that no significant toxic effect is observed in cells when data are compared with the control group. The in vivo assay showed that a higher survival rate in the mice received a special formulation due to adjuvanticity effect of dendrimer, which is also confirmed by RFFIT. However, the relative potency of that formulation does not give expected results when compared with the alum-containing rabies vaccine. In the current investigation, the adjuvanticity effect of G2 dendrimer was demonstrated for the first time in rising of neutralizing antibodies against rabies virus. Our data confirm that nanoparticles can enhance immune responses in an appropriate manner. Moreover, engineered nanoparticles will enable us to develop novel potent multivalent adjuvants in vaccine technology.

Green synthesis and evaluation of silver nanoparticles as adjuvant in rabies veterinary vaccine.

BACKGROUND: Green synthesis of nanoparticles by plant extracts plays a significant role in different applications. Recently, several studies were conducted on the use of nanoparticles as adjuvant. The main aim of this study was to evaluate green synthesized silver nanoparticles (AgNPs) as adjuvant in rabies veterinary vaccine and compare the results with the existing commercially available alum adjuvant. MATERIALS AND METHODS: In the current study, AgNPs were prepared by the reduction of aqueous silver nitrate by leaf extract of Eucalyptus procera. The formation of AgNPs was confirmed by ultraviolet (UV)-visible spectrophotometer, scanning electron microscopy, dynamic light scattering, and X-ray diffraction analysis. Then, different amounts of AgNPs (200 µg, 400 µg, 600 µg, and 800 µg) were added to 1 mL of inactivated rabies virus. The loaded vaccines (0.5 mL) were injected intraperitoneally into six Naval Medical Research Institute mice in each group on days 1 and 7. On the 15th day, the mice were intracerebrally challenged with 0.03 mL of challenge rabies virus (challenge virus strain-11, 20 lethal dose [20 LD50]), and after the latency period of rabies disease in mice (5 days), the mice were monitored for 21 days. Neutralizing antibodies against rabies virus were also investigated using the rapid fluorescent focus inhibition test method. The National Institutes of Health test was performed to determine the potency of optimum concentration of AgNPs as adjuvant. In vitro toxicity of AgNPs was assessed in L929 cell line using MTT assay. In addition, in vivo toxicity of AgNPs and AgNPs-loaded vaccine was investigated according to the European Pharmacopeia 8.0. RESULTS: AgNPs were successfully synthesized, and the identity was confirmed by UV-visible spectrophotometry and X-ray diffraction analysis. The prepared AgNPs were spherical in shape, with an average size of 60 nm and a negative zeta potential of -14 mV as determined by dynamic light scattering technique. The highest percentage of viability was observed at 15 mg/kg and 20 mg/kg of AgNPs-loaded vaccine concentrations after injecting into the mice. The calculated potencies for alum-containing vaccine and AgNPs-loaded vaccine (dose 15 mg/kg) were 1.897 and 1.303, respectively. MTT assay demonstrated that alum at the concentration of 10 mg/mL was toxic, but AgNPs were not toxic. The in vivo toxicity also elucidated the safety of AgNPs and AgNPs-loaded vaccine in mice and dogs, respectively. CONCLUSION: In the current study, for the first time, the adjuvanticity effect of green synthesized AgNPs on veterinary rabies vaccine potency with no in vivo toxicity was elucidated according to the European Pharmacopeia 8.0.