Alpha alumina nanoparticle conjugation to cysteine peptidase A and B: an efficient method for autophagy induction

Background: autophagy as a cellular pathway facilitates several immune responses against infection. It also eliminates invading pathogens through transferring content between the cytosol and the lysosomal vesicles and contributes to the cross-presentation of exogenous antigens to T lymphocytes via MHC class I pathway. Autophagy induction is one of the main targets for new drugs and future vaccine formulations. Nanoparticles are one of the candidates for autophagy induction. Cysteine Peptidase A [CPA] and Cysteine Peptidase B [CPB] are two members of papain family [Clan CA, family C1] enzyme that have been considered as a virulence factor of Leishmania [L.] major, making them suitable vaccine candidates. In this research, Leishmania major cysteine peptidase A and B [CPA and CPB] conjugation to alpha alumina nanoparticle was the main focus and their entrance efficacy to macrophages was assessed

Methods: for this purpose, CPA and CPB genes were cloned in expression vectors. Related proteins were extracted from transformed Escherichia coli [E. coli] and purified using Ni affinity column. Alpha alumina nanoparticles were conjugated to CPA/CPB proteins using Aldehyde/Hydrazine Reaction. Autophagy induction in macrophages was assessed using acridine orange staining

Results: CPA/CPB protein loading to nanoparticles was confirmed by Fourier Transform Infrared Spectroscopy. alpha-alumina conjugated CPA/CPB antigen uptake by macrophages at different concentrations was confirmed using fluorescence microscope and flowcytometry. Highly efficient CPA/CPB protein loading to alpha-alumina nanoparticles and rapid internalization to macrophages introduced these nanocarriers as a delivery tool. Acridine orange staining demonstrated higher autophagy induction in CPA/CPB protein conjugated with alpha-alumina nanoparticles

Conclusion: alpha-alumina nanoparticles may be a promising adjuvant in the development of therapeutic leishmania vaccines through antigen delivery to intracellular compartments, induction of autophagy and cross presentation to CD[8] lymphocytes

Development of insulin resistance through induction of miRNA-135 in C2C12 cells

Objective: micro-RNAs [miRNAs] are a class of posttranscriptional regulators that play crucial roles in various biological processes. Emerging evidence suggests a direct link between miRNAs and development of several diseases including type 2 diabetes [T2D]. In this study, we aimed to investigate the effect of predicted miRNA and target genes on insulin resistance

Materials and Methods: this experimental study was conducted on the C2C12 cell line. Using bioinformatics tools miRNA-135 and two respective target genes-insulin receptor [Insr] and vesicle associated membrane protein 2 [Vamp2]- were selected as potential factors involved in insulin resistance process. Levels of glucose uptake miRNA expression and respective gene targets were determined after cell transfaction by miR-135

Results: it was determined that Insr gene expression was significantly down-regulated in miR-135 transfected C2C12 cell line [P

Conclusion: our data indicated no change on the Vamp2 expression level after miRNA transfection, while expression level of Insr was reduced and miR-135 expression was contrarily increased leading to poor stimulation of glucose uptake through insulin, and development of insulin resistance phenotype in C2C12 cell line

effects of plasma treated electrospun nanofibrous poly [epsilon-caprolactone] scaffolds with different orientations on mouse embryonic stem cell proliferation

Assessments of cell reactions such as motility, orientation and activation to the topography of the substratum will assist with the fabrication of a proper implantable scaffold for future tissue engineering applications. The current challenge is to analyze the orientation effect of elecrospun nanofibers of poly [epsilon-caprolactone] [PCL] on viability and proliferation of mouse embryonic stem cells [mESCs]. In this experimental study, we used the electrospinning method to fabricate nanofibrous PCL scaffolds. Chemical and mechanical characterizations were specified by the contact angle and tensile test. O2 plasma treatment was used to improve surface hydrophilicity. We used the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay to evaluate mESCs adhesion and proliferation before and after surface modification. The influence of the orientation of the nanofibers on mESCs growth was evaluated by scanning electron microscopy [SEM]. Statistical analysis was performed using one-way analysis of variance [ANOVA] With differences considered statistically significant at p

Comparative study of the effect of LPS on the function of BALB/c and C57BL/6 peritoneal macrophages

Macrophages influence their environment and surrounding immune cells as soon as stimulators affect them. Different sources of macrophages induce different reactions in their neighboring immune cells,which result in non-uniform immunologic outcomes. In this experimental research, we compare the behavior of peritoneal macrophages to lipopolysaccharide [LPS] stimulation from BALB/cmice as an indicator of a type 2 immune response and from C57BL/6 mice as an indicator of a type 1 immune response. In this experimental study, peritoneal macrophages prepared from thioglycolate stimulated BALB/c and C57BL/6 micewere treated with 1microg/ml LPS. At different time points after LPS treatment, nitric oxide [NO], interferon gamma [IFN-gamma]. interleukin 4 [IL-4],transforming growth factor beta[1] [TGF-beta[1]], interleukin 17 [IL-17], and interleukin 10[IL-10] production were measured in the supernatants of all macrophage cultures.Indoleamine 2, 3 dioxygenase [IDO] and phagocytic activitywere analyzed in the different experimental groups. The supernatant effects of LPS-treated macrophages on splenocyte proliferation was assessed by the colorimetric method using a 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide [MTT] reagent. According to cytokine analysis, different mouse strains show different cytokine patterns in response to LPS. C57BL/6 macrophages produced more IL-17, IL-10, and IFN-gamma while BALB/c macrophages produced more TGF-beta[1]., and IL-4. There was no significant difference in IDO activity between strains [p

Cloning and expression of leishmania infantum LPG3 gene by the lizard leishmania expression system

Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum [L.infantum]. The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 [rLPG3] to further research in vaccine designing against leishmaniasis