Construction and production of Foxp3- Fc [IgG] DNA vaccine/fusion protein

Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell [DC] based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc[IgG] can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I [cross priming]

Methods: DNA construct containing fragment C [Fc] portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli [E. coli] strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining

Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification

Conclusion: Due to successful expression of Foxp3-Fc [IgG], it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means

Venom components of Iranian scorpion hemiscorpius lepturus inhibit the growth and replication of human immunodeficiency virus 1 [HIV-1]

Background: During the recent years, significant progress has been achieved on development of novel anti-viral drugs. Natural products are assumed as the potential sources of novel anti-viral drugs; therefore, there are some previous studies reporting the anti-viral compounds from venomous animals. Based on the significant value for tracing of non-toxic anti-viral agents from natural resources, this study was aimed to investigate the anti-viral activity of some HPLC purified fractions derived from the venom of Iranian scorpion, Hemiscorpius lepturus, against human immunodeficiency virus 1 [HIV-1] and herpes simplex virus 1 [HSV-1]

Methods: H. Lepturus crude venom was subjected to reverse phase HPLC analysis to determine its active components precisely where four dominant fractions obtained at retention time of 156-160 minutes. The phospholipase A2 and hemolytic activities of the purified fractions were first evaluated. Then the anti-viral activity was measured using single cycle HIV [NL4-3] replication and HSV [KOS] plaque reduction assays

Results: The H. lepturus crude venom inhibited HIV replication by 73% at the concentration of 200 micro g/ml, while it did not show significant anti-HSV activity. It also inhibited the cell-free viral particles in a virucidal assay, while it showed no toxicity for the target cells in a proliferation assay. The four HPLC fractions purified from H. lepturus inhibited HIV with IC50 of 20 micro g/ml

Conclusion: H. lepturus venom contains components with considerable anti-HIV activity insofar as it has virucidal activity that offers a novel therapeutic approach against HIV infection. Our results suggest a promising pilot for anti-HIV drug discovery with H. lepturus scorpion venom

[One-step synthesis of anionic PEG-citrate G2 dendrimer and evaluation of its cytotoxicity in two cell lines]

Objective: Dendrimers are three-dimensional nanostructures that have numerous applications in medicine, including drug delivery and imaging. Although anionic dendrimer polyethylene glycol-citrate has a high potential to increase solubility of waterinsoluble drugs and drug delivery, its multi-step synthesis procedure is time consuming. In addition, toxic substances such as dichloromethane are used in its synthesis procedure. In this study, we have developed a simple one-step synthesis method using green chemistry

Methods: We examined four different methods to improve the synthesis method of this dendrimer. Products were characterized by FTIR, LC-MS and DLS. Cytotoxicity was assessed by the XTT method

Results: We synthesized a G2 polyethylene glycol-citrate dendrimer in one-step without purifying G1. This process was chosen as a beneficial method for synthesis of the G2 dendrimer. When compared with previous methods, this procedure had higher efficiency and greatly reduced response. This procedure used nontoxic materials. XTT assay results showed that this dendrimer created by green chemistry had no cytotoxicity in Hela and Vero cells up to a concentration of 800 microM

Conclusion: One-step synthesis of anionic polyethylene glycol-citrate G2 dendrimer is a simple, beneficial production method. The dendrimer is biocompatible and can be used as a suitable carrier for drug delivery purposes

Induction of IFN-gamma cytokine response against hepatitis B surface antigen using melittin

In this study we co-administered melittin along with HBsAg/alum vaccine to investigate if it helps elicitation of Th1/Th2 response. Hepatitis B virus [HBV] infection is a life-threatening liver infection, which can lead to chronic liver disease. Vigorous T cell responses are stimulated at acute, self-limiting HBV infection, while chronic HBV infection elicits very weak T cell responses. The prevalence of HBV infection has been decreased by the approved vaccination approach using recombinant HBs antigen [HBsAg] and alum i.e. HBV vaccine. Alum, a strong Th2 stimulator, is usually used as adjuvant to increase HBsAg immunogenicity. The present vaccine does not induce protective and/or prophylactic immune response in some groups. Melittin, major active component in the venom of honeybee, induces Th1 development. Experimental mice were immunized with melittin plus hepatitis B vaccine on day 0 following by two booster doses with the same injections. Lymphocyte proliferation, IFN-gamma, and IL-4 level, total antibody and isotyping of IgG1, IgG2a IgG2b, and IgM were measured using ELISA. Administration of melittin and HBV vaccine had no effect on lymphoproliferation and total antibody responses, but increased IFN-gamma response and induced Th1 response. The present study proposed that administration of melittin along with conventional vaccine shifts T cell responses towards Th1/Th2 dominated with Th1 response. The resultant immune response leads to activation of both cell-mediated and humoral immune responses, both of which required for clearance of HBV infection.

[Determination of antigenic determinants in the C-terminal 311 amino acids of haps adhesion from nontypeable haemophilus influenza]

Haps protein plays central role in initial interaction of nontypeable Haemophilus influenzae [NTHi] with human respiratory epithelial cells. While other surface-exposed proteins of NTHi are highly variable, The HapS domain is highly conserved among H. influenzae strains. Recent studies demonstrated that HapS adhesive activity resides within the C-terminal 311 amino acids of the protein and also they showed that the C-terminal 311 amino acids of HapS [C-Haps] are capable of eliciting a protective immune response against NTHi colonization. The pET24a-chaps plasmid harboring c-haps sequence from NTHi PTCC1766 was constructed. The amino acid sequences of C-Haps of this study was aligned with C-Haps of three NTHi strains [N187, TN106, P860295] and antigenicity plot of studied rC-Haps was done bioinformatic software. The pET24a-chaps expression was conducted in E.coli BL21 [D3E] and its expression was confirmed by SDS-PAGE and Western blotting methods. The rC-Haps was purified via immobilized metal affinity chromatography. Amino acid sequence alignment of rCHaps sequence of current study and rC-Haps from the NTHi strains N187, TN106, P860295 showed more than%97 identity. Antigenicity plot identified 9 common highly antigenic domains that were located exactly in conserved regions among 4 different NTHi strains. Due to presence of highly conserved antigenic epitopes among C-Haps of NTHi PTCC1766 and other NTHi strains, rC-Haps of current study could be theoretically a vaccine candidate against NTHi strains of different geographical areas

Induction of strong and specific humoral and T-helper 1 cellular responses by HBsAg entrapped in the Methanobrevibacter smithii archaeosomes

Application of adjuvants with microbial origins is a recently highlighted approach in the vaccinology trials. Archaeosomes are among these microbial compounds with both adjuvant and liposomal activities and features. In the present study, recombinant HBsAg encapsulated into Methanobrevibacter smithii [M. smithii] archaeosomes. Balb/c mice immunized with this compound and humoral and cytokine secretion pattern of immunized models analyzed. Frequency of IFN-gamma secreting cells in the HBsAg-containing archaeosomes group was significantly higher than HBsAg and HBsAg+C/IFA groups [p

Overexpression and purification of c-terminal fragment of the passenger domain of hap protein from nontypeable haemophilus influenzae in a highly optimized escherichia coli expression system

Nontypeable Haemophilus influenzae [NTHi] is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae [H. influenzae] Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called "HapS" and a 45 kDa Cterminal translocator domain called "Hapbeta". Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain [CBD] which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24alpha-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography [IMAC] using Ni-NTA columns. The highest expression level of target protein was achieved when CBD expressing E. coli BL21 [DE3] cells were grown at 37°C in 2xTY medium with 1.0 mM IPTG at mid-log phase [OD[600 nm] equal to 0.6] for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than%97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies

Selective cyclooxygenase-2 inhibitor compound 11B improves haloperidol-induced catatonia by enhancing the striatum dopaminergic neurotransmission

The aim of this research was to investigate the Cyclooxygenase-2 [COX-2] selective inhibition effect on haloperidol-induced catatonia. In this study, the effect of orally, acutely and Sub-chronically administrations of compound 11b [1-[phenyl]-5-[4-methylsulfonylphenyl]-2-ethylthioimidazole] [2, 4 and 8 mg/kg], a newly selective COX-2 inhibitor, was investigated against the haloperidol-induced catatonia phenomenon comparing to the standard drug scopolamine [1 mg/Kg] followed by microdialysis analysis of Striatum dopaminergic neurotransmission. The results showed a great potency for compound 11b in improvement of catalepsy followed by enhancing the dopaminergic neurotransmission p < 0.05. In addition, our statistical analysis showed that the protective effect of compound 11b against haloperidol-induced catatonia was both dose- and time-dependent. These findings are additional pharmacological data that suggest the effectiveness of compound 11b in treatment of schizophrenic drug overdoses and also Parkinson's disease [PD] affiliated rigidity

[Designing and construction of bicistronic plasmid pIRESIgk/ mIL18/Fc: potential implications for vaccine investigations]

IL18 is a cytokine that plays an important role in the T-cell-helper type 1 [Th1] response and hence, plasmid-encoded IL18 is considered as a potent genetic adjuvant for DNA vaccine studies. In this study, a bicistronic eukaryotic plasmid capable of secreting a more stable mouse IL18 [fused with Fc gamma 2a fragment] was constructed and expression of this chimer cytokine was also assessed. RNA purified from stimulated mouse spleenocytes and then cDNA corresponding to mouse IL18 [mIL18] and Fc gamma 2a fragments were constructed by RT-PCR. Sequential subcloning of mIL18 and IgG2aFc fragments first into pSL1180 and then pSecTag2 plasmids resulted in the fusion of mIL18/Fc and addition of immunoglobulin kappa signal sequence [Igk/mIL18/Fc], respectively. Final cloning of Igk/mIL18/Fc sequence downstream of CMV promoter into the NheI/XmaI sites of pIRES2-GFP plasmid and led to the construction of pIRES-Igk/mIL18/Fc plasmid, which was transfected to HEK293T cell line by Turbofec Transfection reagent and expression analysis, was evaluated by ELISA assay. Restriction enzyme analysis of pSL-mIL18 pSL-mIL18/Fc pSec-mIL18/Fc and pIRESIgk/ mIL18/Fc plasmids with the enzymes that were applied for clonings led to the isolation of fragments with expected size and then plasmid of pIRES-Igk/mIL18/Fc was also confirmed following sequencing reactions. Moreover, expression and secretion of mIL18 to the medium was evidenced in transfected 293T cells, compared to non-transfected controls. pIRES-Igk/mIL18/Fc plasmid possesses the capacity of the cloning and expression of putative antigen gene under the direction of IRES sequence, and also expression of mIL18 as a great secretive genetic adjuvant. This results can be useful to design an efficient DNA vaccine especially for inducing host cellular immune response, moreover, cab be considered a promising for accessing to new generation of DNA vaccine

Effect of dexamethasone on striatal neurotransmissions in the rats subjected to Parkinson's disease animal model

The aim of this study was to evaluate the effects of dexamethasone on striatal dopaminergic, glutamatergic and gamma amino butyric acid [GABA] ergic neurotransmission in normal and parkinsonian rats. Dexamethasone [0.15, 0.30, 0.60 and 0.8 mg/kg] was administered to normal or parkinsonian rats [i.p.] followed by the analysis of the striatal neurotransmitters concentrations. Additionally, the effect of dexamethasone on the damaged Substantian nigra pars compata [SNc] neurons has been investigated. Dexamethasone resulted in decreased level of striatum glutamatergic-GABAergic and enhanced dopaminergic neurotransmission in normal and parkinsonian rats. In addition, acute treatment with dexamethasone did not improve the lesion at all. These findings suggest the new therapeutic mechanism of action for dexamethasone in Parkinson's disease animal model

[Production and physicochemical evaluation of Neisseria meningitidis serogroup A outer vesicle as a vaccine candidate]

Neisseria meningitidis serogroup A Polysaccharides vaccines have been available for many years, but these vaccines have many disadvantages due to their induction of T-Cell independent responses. To overcome these problems, many researches have been focused on other parts of bacterial cell component such as OMV [Outer membrane vesicle]. In this study, OMV containing PorA were extracted and evaluated by biological and immunological methods. OMV were extracted by siadat, et al method. Physicochemical properties of extracted OMV were analyzed by electron microscopy and SDS-page. The toxicity of LPS content in OMV was assayed by LAL test. The Presence of PorA was confirmed by western blot. Antibodies synthesis after immunization by OMV was evaluated using ELISA method. The content of extracted protein was 0.1 mg/ml. Size of OMV was between 50 and 150 nanometer. SDS-PAGE showed that PorA was located in 35-40 kDa. LAL test showed that the endotoxin activity was ranged in 126EU/ml which is safe for using. The ELISA test showed that the total IgG titer was elevated after first injection. The results showed that the conformation of extracted OMV was stable, and there were no progeny determinants in OMV. Also, OMV elicited high level of specific antibodies against Neisseria meningitidis serogroup A. These results indicate that the OMV can be used as a meningococcal vaccine after further investigations