Advanced Pharmaceutical Bulletin
Volume:5 Issue: 5, Dec 2015

Type 1 diabetes (T1D) is a pancreatic beta cell specific autoimmune disease. One of the most significant current discussions in T1D studies is therapy. Since the conventional therapy, islet transplantation and external insulin, e.g., cannot prevent the destructive autoimmune process against original beta cells and persistent hyperglycemia remains, so recent developments in the field of T1D therapy paved the way to a renewed interest in immunotherapy based on the disease process, especially monoclonal antibody therapy. Due to encouraging laboratory results, cytokine antibody-based drugs could be effective in the clinical direction of the T1D disease process. Hence, implementation of this approach can be useful to improve clinical and laboratory manifestations of T1D.

Human Toll-like receptors (TLRs) are a family of transmembrane receptors, which play a key role in both innate and adaptive immune responses. Beside of recognizing specific molecular patterns that associated with different types of pathogens, TLRs may also detect a number of self-proteins and endogenous nucleic acids. Activating TLRs lead to the heightened expression of various inflammatory genes, which have a protective role against infection. Data rising predominantly from human patients and animal models of autoimmune disease indicate that, inappropriate triggering of TLR pathways by exogenous or endogenous ligands may cause the initiation and/or perpetuation of autoimmune reactions and tissue damage. Given their important role in infectious and non-infectious disease process, TLRs and its signaling pathways emerge as appealing targets for therapeutics. In this review, we demonstrate how TLRs pathways could be involved in autoimmune disorders and their therapeutic application.

Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cell (HSC) transplantation for the treatment of patients with leukemia if matched donor is not available. CD34 is a pan marker for human hematopoietic stem cells, including umbilical cord blood stem cell. In comparison to other sources, cord blood CD34 cells proliferate more rapidly and produce large number of progeny cells. For ex vivo expansion of Umbilical Cord Blood- HSCs/HPCs, different combinations of cytokines have been used in many laboratories. IL2rg cytokines, including IL2, IL7 and IL15, are key cytokines in the regulation of differentiation, proliferation and survival of immune cells. IL2 is important cytokine for T cell survival and proliferation, IL7 involve in B cell development and IL15 is a key cytokine for NK cell development. In this study we evaluated the generation of T cells derived from CD34 and CD34- cord blood mononuclear cells by using combination of cytokines including IL2, IL7 and IL15.
Cultured cord blood mononuclear cells were evaluated at distinct time points during 21 days by using flow cytometry.
Present study showed that differentiation of T cells derived from CD34 cord blood mononuclear cells increased by using IL2 and IL7 at different time points. In the other hand IL15 did not show any significant role in generation of T cells from CD34 cord blood mononuclear cells.
Taken together, our data illustrated that either IL2 or IL7 versus other cytokine combinations, generate more T cell from cord blood CD34 cells, probably this cytokines can be the best condition for ex vivo expansion of UCB HSCs.

Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody.
To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively.
SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively.
The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.

The direct transmission of avian influenza viruses to human and increasing drug resisted strains posing new threats for public health. Therefore, development of efficient vaccines is needed to generate protective and persistent immunity to the viruses.
Three motifs of Mx protein sequence in human, mouse and poultry located in interferon induced (GTP ase) domain were candidate as biologic adjuvant for enhancing the immune responses against influenza virus. Chimera proteins composed with the conserved HA2 subunit of influenza virus and the Mx motifs named HA2/Mx were modeled and evaluated by in silico analysis includes bioinformatics algorithms in order to explore biological characteristics of these peptides.
Amongst the predicted models, HA2/Mx1 peptide showed the better results following protein structures prediction, antigenic epitopes determination and model quality evaluation. Comparative homology modeling was performed with Swiss Model and the model was validated using ProSA. Epitope predictions revealed the construct could induce both B and T cell epitopes that expect a high immune response.
Taken together, these data indicate that the HA2/Mx1 chimera peptide can be potentiated for developing an adjuvant-fused influenza vaccine capable of stimulating effective immune response.

The aim of this study was to verify the density of mast cells (MCs) and microvessels in odontogenic cysts. Furthermore, the correlation between MCs and microvessels was evaluated to assess the contribution of MCs to angiogenesis and growth of odontogenic cysts. This approach may be a basis for the development of future pharmaceuticals addressed to MCs performance to manage odontogenic cysts. To our knowledge, no study investigating the correlation between MCs and microvessels has been performed to date.
60 cases of odontogenic cysts consisting of 20 radicular cysts (RCs), 20 odontogenic keratocysts (OKCs) and 20 dentigerous cysts (DCs) were included in this study. Five high power fields in superficial connective tissue and five high power fields in deep connective tissue were counted for each sample. Moreover, a total mean of ten fields was calculated.
RC showed the highest mean numbers of MCs and microvessels (p Although the number of MCs was not significantly associated with microvessels, these cells may be related to the growth of odontogenic lesions, particularly RCs. Further studies on the in vivo functions of MCs will make the concept more clear.

Toll-like receptors (TLR) are well known components of the innate immune system. Among them, TLR4 is related to the inflammatory processes involved in atherosclerotic plaque formation. Our purpose was to compare the monocytic expression of TLR4 following implantation of drug-eluting (DES) and bare stents (BMS).
In this study, patients with chronic stable angina undergoing elective percutaneous coronary intervention (PCI) in ShahidMadani Heart Hospital, Tabriz, Iran were included. Ninety-five patients receiving DES and 95 patients receiving BMS were selected between 2012 and 2014.Everolimus eluting stents were implanted for DES group. Both groups received similar medications and procedure. Blood samples were taken before PCI, 2 hours and 4 hours after termination of PCI. Expression of TLR4 on monocytes was measured using flowcytometry. Patients were matched for age, sex and coronary artery disease risk factors, but not for TLR4 expression rate before PCI.
A significant difference was seen between DES and BMS in TLR4 expression before (21.3±2.8% vs. 15.5±2.7%; P Our findings suggest thateverolimuseluted from the stents can decrease PCI induced increase in the TLR4 expression on the surface of monocytes.

Target therapy against molecular markers of EGFR family has been recently used in the treatment protocol of several diseases. However, the role of Her-2/neu in OSCC is a matter of controversy and more studies are required to clarify the role of Her-2/neu in OSCC. We aimed to investigate the role of Her-2/neu in the process of carcinogenesis in oral epithelium as ELP is a premalignant and OSCC is a malignant lesion, but RLP shows no evidence of premalignancy and malignancy.To our Knowledge, this is the first study comparing Her-2/neu expression in erosive lichen planus (ELP), reticular lichen planus (RLP), and oral squamous cell carcinoma (OSCC).
60 samples, including 20 cases of RLP, 20 cases of ELP, and 20 cases of OSCC were evaluated immunohistochemically in this study.
Our findings showed that there was not a significant increase in Her-2/neu expression from RLP to ELP and from ELP to OSCC. Therefore, Her-2/neu had no role in differentiating between RLP, ELP and OSCC and this protein does not contribute to the process of carcinogenesis in the oral epithelium.
The lack of Her-2/neu overexpression indicates that molecular targeting of Her-2/neu protein is not recommended as an adjuvant therapy in OSCC and OLP.

Thymol and carvacrol, two main components of thyme, have shown anti-inflammatory effects. The aim of this study was to assess the effects of these components on Jurkat leukemia cells as an in vitro T cell model and their molecular mechanisms of activity.
Cells were cultured in the presence of components and subsequently stimulated with phorbol-12-myristate-13-acetate (PMA)/calcium ionophore for evaluating interleukin (IL)-2 and interferon (IFN)-γ production. The activation of T cell transcription factors that included nuclear factors of activated T cells (NFATs), activator protein-1 (AP-1; c-Jun/c-Fos), and nuclear factor (NF)-KB were examined by Western blot analysis.
Thymol and carvacrol at 25 µg/ml significantly reduced IL-2 levels from 119.4 ± 8pg/ml in control cells treated only with PMA/Calcium ionophore and the solvent to 66.9 ± 6.4pg/ml (thymol) and 32.3 ± 3.6pg/ml (carvacrol) and IFN-γ from 423.7 ± 19.7pg/ml in control cells to 311.9 ± 11.6pg/ml (thymol) and 293.5 ± 16.7pg/ml (carvacrol). Western blot analyses of nuclear extracts showed that the same concentrations of components significantly reduced NFAT-2 to 44.2 ± 2.7% (thymol) and 91.4 ± 2.3% (carvacrol) of the control (p Thymol and carvacrol could contribute to modulation of T cell activity by reducing IL-2 and IFN-γ production possibly through down regulation of AP-1 and NFAT-2 transcription factors suggesting their potential usefulness for reduction of T cell overactivity in immune-mediated diseases.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology.
Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment.
Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody.
The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α.

TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology.
The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column.
The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein.
In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality.

The purpose was to design a new construction containing influenza virus (H1N1) M2e gene and HA2 gene by bioinformatics approach, cloning the construct in to Escherichia coli and produce M2e-HA2 peptide.
The procedure was done by virus cultivation in SPF eggs, hemagglutination assay (HA), RNA isolation, RT-PCR, primers designed (DNAMAN 4 and Oligo7), virtual fusion construction translation (ExPASy), N-Glycosylated sites prediction (Ensemblegly-Iowa), complete open reading frame (ORF), stop codon studied (NCBI ORF Finder), rare codon determination (GenScript), Solvent accessibility of epitopes (Swiss-PdbViewer), antigenic sites prediction (Protean), fusion PCR of M2e-HA2 gene, sequence analysis, nested PCR, gel electrophoresis, double digestion of pET22b() plasmid and the fusion construct, ligation of them, transformation of the ligated vector (pET22b-M2e-HA2) to E.coli (BL21), mass culture the cloned bacterium ,induction the expression by isopropyl-beta-D-thiogalactopyranoside (IPTG), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), purification the fusion peptide by Ni-NTA column, western blot to verify the purification.
In this study we developed a new approach for fusion of Influenza virus M2e (96 nucleotides) and HA2 (663 nucleotides) genes based on fusion PCR strategy and produced a fused fragment with 793 nucleotides. The construct was successfully cloned and expressed.
This construct is a 261 amino acid chimeric fusion peptide with about 30 KD molecular weight. According on the latest information; this is the first case of expression and purification M2e-HA2 fusion chimeric peptide, which could be used for development of a recombinant M2e-HA2 fusion protein vaccine.

To assess the immunohistochemical profile of the atypical nuclei in leiomyoma with bizarre nuclei and compare with benign and malignant counterparts.
26 cases of uterine smooth muscle tumors including 12 leiomyosarcoma(LMS), 10 leiomyoma with bizarre nuclei (LBN) and 4 smooth muscle tumor with uncertain malignant potential (STUMP) were selected using whole tissue sections for this study and analysis. Six cases of ordinary leiomyoma were included as benign control group. All representative section were stained for P53, Ki67 , estrogen receptor and progestrone receptor. Analysis was carried out using SPSS 16.0 for windows software.
Six out of 12 cases of LMS showed strong and diffuse nuclear staining with p53 antibody (50%). In contrast none of STUMPs and only one case of LBN cases showed focal positive reaction with P53. Percentage of positive cells for ki67 in LMS was 14.92 while only 0.85% of cells in LBNs was labeled with Ki67 proliferative marker. (P Loss of inhibitory function of wild type P53 gene in leiomyosarcoma is an essential event that discriminate frankly malignant tumors from STUMP and atypical leiomyoma.