Avicenna Journal of Medical Biotechnology
Volume:10 Issue: 3, Jul-Sep 2018

Schizophrenia is a chronic debilitating psychiatric illness that accounts for a significant portion of the burden caused by mental illnesses worldwide. Primary negative symptoms of schizophrenia are not secondary to extrapyramidal, depressive or positive symptoms 1,2. Negative symptoms are the core features of the illness which are associated with long-term functional disability and poor outcome 1,3. These symptoms include deficits in social and emotional functioning, blunted affect and lack of spontaneity. There is a growing body of evidence for the role of inflammation and immune system dysregulation in psychiatric disorders 4. Although the precise pathophysiology of schizophrenia is not completely known, a number of recent studies support the probable pathologic role of immunologic dysfunction in this disorder. Assessing serum cytokine levels such as interleukin 1 (IL-1), IL-2, IL-6, and chemokine CCL11 in schizophrenic patients demonstrates profound alterations compared to healthy matched controls 4. Furthermore, increased cyclooxygenase-2 (COX-2) expression as well as prostaglandin E2 production in schizophrenia, are among other postulated etiologies supported by recent studies 4. On the other hand, it has been shown that immune response imbalance is associated with decreased activity of indoleamine 2, 3-dioxygenase enzyme which subsequently leads to accumulation of kynurenic acid, an endogenous antagonist of glutamate N-methyl-D-aspartate (NMDA) receptor. Compared with anit-inflammatory agents like celecoxib and NAC, monoclonal antibodies also have more potent anti-inflammatory properties. Indeed, COX-2 inhibitors and N-acetylcysteine have moderate efficacy in treatment of schizophrenia and autism 1,2,5. British scientists have begun testing a radically new approach to treating schizophrenia based on emerging evidence that it could be a disease of the immune system. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted 6,7. Then key evidence for immune dysfunction in patients with schizophrenia is considered. A collaboration between the Medical Research Council (MRC) and King’s College London, is based on emerging evidence that schizophrenia may be an immune disease. The drug, natalizumab, works by targeting microglia, a type of immune cell residing in the brain which are thought to be overactive in people at risk of developing schizophrenia 6,7.

Recently, Phosphatidylcholine (PC) has been used as an off-label treatment for lipolysis injection, which is associated with inflammatory reaction due to sodium deoxycholate, an emulsifier, so that inflammation as side effect occurs in those patients. Liposome formulation from soybean lipid was thought to be a better and safer alternative. This study aimed to analyze the mechanism of Liposomal Soybean Phosphatidylcholine (LSPC) extract from Indonesian soybeans (containing 26% PC) to induce Adipose-derived Stem Cells (ASCs) death in vitro.
Liposomes were prepared using thin film hydration method followed by a stepwise extrusion process to produce a small amount of 41.0-71.3 nm. Liposomal soybean phosphatidylcholine extract (LSPCE), liposomal purified PC (LPCC), and solution of PC were used for comparison. Annexin V fluorescein Isothiocyanate/ Propidium Iodide (FITC/PI) double staining by flow cytometry and also measurement of caspase-3 activity using ELISA were used to quantify the rate of apoptosis. ASCs viability was measured using MTT assay after induction with liposomes. Morphological changes were shown using a phase-contrast, inverted microscope and Transmission-Electron Microscope (TEM).
The flow cytometry results showed that cells treated with both LSPCE and LPCC showed increase in early apoptosis beginning at 6 hr after incubation, which was confirmed by caspase 3 measurement. MTT assay showed that both LSPCE and LPCC could decrease viability of cells. Cells treated with LSPCE and LPCC showed some rounded cells, which was an early sign of cell death. Cells treated with SD showed extensive membrane damage with necrosis features using TEM.
The results above demonstrated that LSPCE induced apoptosis of ASCs.

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which is an obligate intracellular parasite in the infected host. Individuals who have been recovered from clinical leishmaniasis develop strong immunity against reinfection. DNA vaccines are the new type of vaccines that induce expression of protein eukaryotic cells. DNA vaccines can be stimulated by the cellular and humoral immune responses using one or several genes.
A DNA vaccine containing plasmids encoding the pcLACKꗄ genes of Leishmania major (L. major) (MHRO/IR/75/ER) in the vicinity of IL-12 gene expression was made and then its protective efficacy in comparison with single-gene of LACK was evaluated. Also, BALB/c mice were immunized intramuscularly three times. The humoral and cellular immune responses were evaluated after immunization with pcLACK, pcLACKꗄ툃aIL12, and then challenged with L. major.
Humoral response and IFN-γ values were significantly higher than control groups after immunization with pcLACK, pcLACKꗄ툃aIL12 and challenge with L. major (p≤0.05). IL-4 values were increased in the control groups in such a way that they were remarkably higher than the pcLACK, pcLACKꗄ툃 pCAGGS-IL12 groups (p≤0.05) after immunization and challenge with L. major.
The survival time of the immunized mice with pcLACK, pcLACKꗄ툃 pCAGGS-IL12 groups was higher than the control groups. Then, DNA vaccine of pcLACK appeared to be likely able to induce more protection against infection with L. major in mice. Therefore, cocktail DNA is effective to enhance specific immunity.

CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20. In previous studies, a Rituximab based humanized single chain variable fragment (scFv) antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies (IBs) in Escherichia coli (E. coli) cytoplasm. The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv in E. coli.
For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b () and transformed into the E. coli BL21 (DE3) was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated.
Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325 µg/ml soluble anti-CD20 scFv.
The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody in E. coli.

Biosynthetic teriparatide (1-34) (TPD) is a N-terminally truncated version of human parathyroid hormone (hPTH). The recombinant form of this polypeptide has been expressed in Escherichia coli (E. coli) and approved as the first anabolic treatment of osteoporosis in the EU and the USA. Feasibility of expression and secretion of a tag- fused form of TPD into Bacillus subtilis (B. subtilis) was examined due to several advantages of B. subtilis over E. coli in production of recombinant proteins with pharmacological activities.
A codon optimized gene containing TPD open reading frame carrying enterokinase site in its upstream was fully synthesized. According to our cloning scheme, this synthetic polynucleotide was used as a template for PCR amplification using engineered primers in such a way that a polyhistidin tag was added in frame to the upstream of the amplicon as well as two restriction sites at its ends. The resulted amplicon, a cassette containing His-tag, enterokinase site and TPD, from 5’ to 3’, was cloned into pTZ57R/T vector and subjected to sequencing.The cassette was then subcloned into pHT43 shuttle vector and transformed into B. subtilis. Expression of target protein was analyzed by SDS-PAGE and western blotting upon induction by IPTG.
The accuracy of construction of pHT43-TPD was confirmed by sequencing and restriction map analyses. SDS-PAGE and western blotting results showed that the recombinant fusion form of hPTH was successfully expressed and secreted into cytoplasm and extracellular medium.
TPD may be successfully expressed and secreted in B. subtilis; however, optimization of expression conditions is required for enhancing target protein yield.

Hepatitis C (HCV) is known as a serious blood-borne disease that infects millions of people globally. NS3 is a conserved non-structural sequence of hepatitis C virus which has a major role in activating specific CTL responses. As known, there is no effective vaccine against HCV infection, thus it is required to design a specific regimen of vaccination. Recently, the strong immunological properties of Heat shock proteins (Hsps) led to their use as immunomodulators and an antigen carrier for subunit vaccine candidates. In the current study, the role of Hsp20 was evaluated as a HCV NS3 gene carrier in mammalian cell line.
At first, the recombinant plasmids of pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3 were constructed and their accuracy was confirmed by digestion and sequencing. Then, all recombinant plasmids were transfected into HEK293T cells by Lipofectamine and TurboFect gene delivery systems. Finally, the expression of proteins was assessed by fluorescent microscopy, western blotting, and flow cytometry.
In western blotting, the 47, 59, and 79 kDa bands were detected for pEGFP-Hsp20, pEGFP-NS3, and pEGFP-Hsp20-NS3, respectively. The percentage of NS3-Hsp20-GFP protein expression was ~67% by TurboFect and ~50% by Lipofectamine indicating high potency of TurboFect delivery system. Furthermore, the expression of Hsp20 (~83%) was higher than NS3 (~58%) in the cells transfected by TurboFect using flow cytometry analysis. This result was confirmed in the expression of Hsp20-NS3 fusion (~67%) in which Hsp20 increased the delivery of HCV NS3 in vitro. The same data were obtained by Lipofectamine transfection reagent.
Briefly, our data confirmed the role of Hsp20 as a suitable antigen carrier for DNA vaccine design.

The purpose of this study was to investigate the sterol profiling and predict the pharmacological potential of marine gastropod Telescopium telescopium (T. telescopium), collected from mangrove ecosystem in the South west coast of India.
Sterol fractions were separated from the crude lipids using 15% ethyl acetate. Ethyl acetate fractions were dried under ultrahigh purity N2 and analyzed using GC-MS. The biological activity was predicted using the software CLC-Pred; In silico predictions of cytotoxicity for tumor and non-tumor cell lines and PASS.
This study proved the existence of four sterols, of which cholesterol was abundant. It was found that most of the steroids profiled from T. telescopium displayed activity against reproductive system as well as skin related diseases.
The predicted anti infertility and skin related activity of the steroids identified from the marine gastropod T. telescopium is useful to attract industrial interest towards this species which will be helpful in rising new combinations with added therapeutic and nutritional worth.

Human arylamine N-acetyltransferase 2 (NAT2) gene has a key role in xenobiotic metabolism through the conjugation of acetyl group to xenobiotic substances. NAT2 has been suggested as a susceptibility factor in endometriosis; however, the results of studies have been controversial. In this study, the association of NAT2 polymorphisms with susceptibility to endometriosis was evaluated in an Iranian population.
This is an association study and totally 141 women with diagnosis of endometriosis and 158 healthy women as control group were analyzed for NAT2 gene polymorphisms (C481T, A803G, G857A and G590A) by PCR-RFLP methods.
The 590 GA genotype was significantly lower (p=0.001; OR=0.42, 95% CI: 0.25-0.71) in the patients (38.3%) than the control group (55.1%). The 590A allele was significantly lower (p=0.033; OR=0.69, 95% CI: 0.49-0.79) in the patients (31.2%) compared with the controls (39.6%). Analysis of haplotypes showed that NAT2 481C, 803A, 590A, 587A combination was significantly different between the case and control women (p= 0.029; OR=3.11, 95% CI: 1.13-8.52).
The NAT2 G590A SNP may be associated with susceptibility to endometriosis and the 590A allele may have a protective role in development of endometriosis. The NAT2 481C, 803A, 590A, 587A haplotype was associated with a higher risk of endometriosis in Iranian population.

Nonsyndromic cleft lip and/or palate (NSCL/P) is the most common orofacial birth defect, often attributed to ethnic and environmental differences. Up to now, linkage analyses and genome-wide association studies have identified several genomic susceptibility regions for NSCL/P. The WNT genes including WNT3 are strong candidates for NSCL/P, since they are involved in regulating mid-face development and upper lip fusion. This study tested association of the WNT3 polymorphisms, rs3809857 G/T and rs9890413 G/A, with the risk of NSCL/P in a population of Iranian infants.
The allelic and genotypic frequencies for each participant were determined in 113 unrelated Iranian subjects with NSCL/P and 220 control subjects using PCR and restriction fragment length polymorphism (RFLP) methods. A p-value of 0.05 was considered statistically significant.
The WNT3 rs3809857 GT genotype was significantly lower (p=0.039, OR=0.55, 95% CI=0.30-0.97) in the NSCL/P (21.2%) than the control group (30.42%). For the WNT3 rs9890413 G/A polymorphism, neither genotype nor allele frequencies were significantly different between the case and control groups.
Our results indicated that the WNT3 rs3809857 GT genotype may have a protective effect against NSCL/P in Iranian population.

Inflammatory cytokines have been known to be associated with Chronic Heart Failure (CHF). Given the importance of cytokines in the context of the failing heart, the prevalence of Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ) polymorphisms was studied in patients with CHF due to ischemic heart disease in a case-control study.
Fifty-six Iranian patients with CHF were enrolled in this study as the case group and compared with 139 healthy subjects, using polymerase chain reaction with sequence-specific primers method, so as to determine the frequency of alleles, genotypes and haplotypes of IFN-γ ( A/T) and IL-2 (-330 G/T, G/T) SNPs.
The GG genotype at IL-2 -330 in patients with CHF was significantly overrepresented in comparison with the control group (p=0.013). Such a positive genotypic association was also observed for IL-2 힮뽍 (p=0.022). Meanwhile, the GT genotype frequency at IL-2 -330/GT in the patient group was significantly lower than the one in healthy controls (p=0.049). No significant association was detected between the IFN-γ gene polymorphisms and individuals’ susceptibility to CHF.
Certain genotypes in IL-2 gene were overrepresented in patients with CHF, which could render individuals more vulnerable to this disease.

Ankylosing Spondylitis (AS) is a chronic autoinflammatory Spondyloar-thropathy (SpA) which is characterized by sacroiliitis, which progresses to the axial skeleton. It seems that non-Human Leukocyte Antigen (HLA) and also HLA-B27 are associated with the susceptibility and pathogenesis of the disease. The recent Genome-Wide Association Studies (GWASs) have reported intergenic rs6759298 to be associated with AS etiology. The aim of this study was investigation of the rs6759298 polymorphism in Iranian AS patients. In addition, probable correlations with clinical indices and manifestations were considered.
This study included 403 patients with AS. The control group consisted of 506 healthy individuals who were matched for sex, age, and ethnicity with AS group. Genotyping of rs6759298 was determined using the Amplification-Refractory Muta-tion System-Polymerase Chain Reaction (ARMS-PCR).
The GG genotype and G allele were found to be significantly more prevalent in the patient group in comparison to the control group [(p=2×10-6 and 7.44×10-9; OR (95% CI) =2.16 (1.56-2.98) and 1.73 (1.43-2.08)], respectively.
No associations were found between patients with three genotypes and any disease manifestations or clinical indices. This investigation confirmed a highly significant association of rs6759298 with disease susceptibility, with no effect on disease progress or clinical presentations. Since rs6759298 belongs to the 2p15 gene desert, further studies would elucidate the exact role of this polymorphism in the pathogenesis of AS.

It is more than sixty years that the concept of the fetal allograft and immunological paradox of pregnancy was proposed and in this context, several regulatory networks and mechanisms have been introduced so far. It is now generally recognized that mesenchymal stem cells exert potent immunoregulatory activity. In this study, for the first time, the potential impact of Menstrual blood Stem Cells (MenSCs), as surrogate for endometrial stem cells, on proliferative capacity of CD4 T cells was tested.
MenSCs and Bone marrow Mesenchymal Stem Cells (BMSCs) were isolated and assessed for their immunophenotypic features and multi-lineage differentiation capability. BMSCs and MenSCs with or without IFNγ pre-stimulation were co-cultured with purified anti-CD3/CD28-activated CD4 T cells and the extent of T cell proliferation at different MenSCs: T cell ratios were investigated by CSFE flow cytometry. IDO activity of both cell types was measured after stimulation with IFNγ by a colorimetric assay.
MenSCs exhibited dual mesenchymal and embryonic markers and multi-lineage differentiation capacity. MenSCs significantly increased proliferation of CD4 cells at ratios 1:2, 1:4 and 1:8. IFNγ pre-treated BMSCs but not MenSCs significantly suppressed CD4 T cells proliferation. Such proliferation promoting capacity of MenSCs was not correlated with IDO activity as these cells showed the high IDO activity following IFNγ treatment.
Although augmentation of T cell proliferation by MenSCs can be a basis for maintenance of endometrial homeostasis to cope with ascending infections, this may not fulfill the requirement for immunological tolerance to a semi-allogeneic fetus. However, more investigation is needed to examine whether or not the immunomodulatory properties of these cells are affected by endometrial microenvironment during pregnancy.

The Transmembrane Activator and Calcium modulator ligand Interactor (TACI), encoded by TNFRSF13B/TACI gene, is mutated in some patients with Common Variable Immunodeficiency (CVID) and IgA Deficiency (IgAD). The purpose of the study was to investigate for the first time in Turkish patients the prevalence of TNFRSF13B alterations in CVID, selective and partial IgAD patients.
Forty two CVID, 36 selective IgAD, 34 partial IgAD and 25 healthy controls were included. All patients were examined for TNFRSF13B gene mutations by PCR.
The percentages of TNFRSF13B mutations in CVID, selective and partial IgAD patients were 7.1, 2.7 and 2.9%, respectively. No disease causing TNFRSF13B mutation in healthy controls was found. Patients with TACI mutations had recurrent respiratory tract infections. None of them experienced autoimmunity, bronchiectasis or granulomatous disease. In conclusion, TNFRSF13B mutations were present not only in CVID patients, but also in IgAD cases.
Modifier genes as well as their combination with other genetic or environmental factors may play an important role in the development of the immunodeficiency phenotype.

Disruption of the cholinergic neurotransmitter pathway which is important for cognition, memory and learning abilities has been reported in Alzheimer’s Disease (AD) patients. The receptors involved include the Cholinergic Receptors Muscarinic (CHRM). CHRM2 gene has been associated with intelligence, personality traits, substance dependence and depression. CHRM3 has been found to heterodimerize with CHRM2.
DNA samples from 240 AD patients with SNPs rs6962027 of CHRM2 gene and rs7511970 of CHRM3 gene were amplified using PCR and genotyped using Restriction Fragment Length Polymorphism (RFLP). Chi-squared test was done to check if the genes are in Hardy-Weinberg equilibrium.
Results and Although the results did not show significant associations, these data denote plausible interaction between TT in SNP rs6962027 in CHRM2 gene and TT in SNP rs7511970 in CHRM3 gene affecting AD risk. SNP rs7511970 of CHRM3 gene may also exert an influence on late-onset AD.