Design and production of a novel chimeric human growth hormone superagonist fused to human Fc domain.

Background and purpose: Growth hormone (GH) has been known as a crucial metabolic hormone expressed at the pituitary and the other number of cells and tissues and responsible for body growth. Because of the short half-life of GH, daily subcutaneous injections were shown to be more effective for GH therapy. This represents a burden for patients. So, there is a strong effort from the industry to create a long-acting form of GH and lots of technologies like GH fusion proteins are used to increase GH half-life. Experimental approach: In this study, the Fc domain of human IgG1 with serine-glycine linkers was attached to the C-terminal of a GH superagonist via molecular cloning. The presence of recombinant vector in E. coli host was confirmed by PCR. SDS-PAGE and western blot analysis showed the expression of recombinant proteins in the bacterial lysate. The binding ability to growth hormone receptors is determined by ELISA. Findings / Results: Our results showed that the novel SupGH-Fc has a good binding affinity to its receptor in ELISA in comparison to standard GH, although it has a big size. Conclusion and implications: Our data in this study clearly demonstrated the expression of the SupGH-Fc in a recombinant protein expression system. It is an introduction to the production of the new recombinant GH, which can bind to its receptor more effectively than commercial growth hormones and also might have a longer half-life.

Lactococcus-based vaccine against brucellosis: IgG immune response in mice with rOmp16-IL2 fusion protein.

This study was designed to introduce the recombinant Lactococcus lactis MG1363 as a cell factory candidate for production of recombinant Brucella melitensis Omp16-Human IL2 (r-Omp16-IL2) and to suggest it as a promising safe, non-pathogenic mucosal live vaccine against brucellosis. Three groups of BALB/c mice (10 mice per group) were intragastrically administrated with phosphate-buffered saline (PBS), L. lactis harboring the empty pAMJ2008 plasmid and with L. lactis expressing rOmp-IL2. The first two groups were classified as control groups and the third one is indicated as treatment group. Another group was injected by the intraperitoneal (i.p.) route with purified rOmp16-IL2 protein. The total serum IgG of each group was assessed with indirect ELISAs at two days before immunization and also two weeks after the last immunization. Results showed that BALB/c mice intragastrically administrated with L. lactis expressing rOmp-IL2 had dominant IgG response compared to the control (PBS administrated) group (P < 0.05). The level of IgG was significantly increased by intraperitoneally injection of recombinant Omp-IL2 in adjuvant compared to the intragastrically administration of PBS and L. lactis/pAMJ2008 as control groups, and also compared to L. lactis/pAMJ2008-rOmp-IL2 (P < 0.05). Our findings provide the use of L. lactis rOmp16-IL2 as a new promising alternative safe strategy than presently live attenuated vaccines toward developing an oral vaccine or subunit-based vaccine against brucellosis.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis.

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Expression of Helicobacter pylori CagL gene in Lactococcus lactis MG1363 and evaluation of its immunogenicity as an oral vaccine in mice.

Helicobacter pylori is a gram negative pathogen which commonly colonizes in the human gastric mucosa from early childhood and persists throughout life. CagL is a 27-kDa protein that is located at the tip of T4SS pili and highly conserved among pathogenic H. pylori strains. Lactic acid bacteria especially Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. In this study H. pylori CagL gene was cloned in pAMJ2008 vector and transferred to Lactococcus lactis MG1363 as the host for CagL antigen production. This recombinant bacterium was orally subjected to mice, and the immune response to CagL was evaluated by ELISA. Intracellular expression of CagL protein was confirmed by Western blot analysis. Mucosal immunization of mice with the recombinant L. lactis significantly stimulated CagL-Specific antibodies: IgA, IgG, cytokine IL-17 and IFN-γ. Moreover, the specific anti-CagL IgA response was detected in the feces of immunized mice. These results indicate that CagL of H. pylori was successfully expressed in L. lactis and the recombinant bacteria can be potentially used as an edible vaccine against H. pylori infection.

Prediction of the Omp16 Epitopes for the Development of an Epitope-based Vaccine Against Brucellosis.

BACKGROUND: Brucellosis is an infectious disease caused by Brucella bacteria that cause disease in animals and humans. Brucellosis is one of the most common zoonotic diseases transmitted from animals-to-human through direct contact with infected animals and also consumption of unpasteurized dairy products. Due to the wide incidence of brucellosis in Iran and economical costs in industrial animal husbandry, Vaccination is the best way to prevent this disease. All of the available commercial vaccines against brucellosis are derived from live attenuated strains of Brucella but because of the disadvantage of live attenuated vaccines, protective subunit vaccine against Brucella may be a good candidate for the production of new recombinant vaccines based on Brucella Outer Membrane Protein (OMP) antigens. In the present study, comprehensive bioinformatics analysis has been conducted on prediction software to predict T and B cell epitopes, the secondary and tertiary structures and antigenicity of Omp16 antigen and the validation of used software confirmed by experimental results. CONCLUSION: The final epitope prediction results have proposed that the three epitopes were predicted for the Omp16 protein with antigenicity ability. We hypothesized that these epitopes likely have the protective capacity to stimulate both the B-cell and T-cell mediated immune responses and so may be effective as an immunogenic candidate for the development of an epitope-based vaccine against brucellosis.

Green and Facile Synthesis of Highly Photoluminescent Multicolor Carbon Nanocrystals for Cancer Therapy and Imaging.

Carbon dots (CDs), as a new generation of fluorescent nanoparticles, have been greatly considered for different biomedical applications. In the present study, a one-pot hydrothermal method was developed for the synthesis of a series of carbon dots (CDs) for cancer imaging and therapy. Taxane diterpenoids were utilized as the carbon source, different diamines were used as the nitrogen source, and folic acid was used as a targeting agent. High-quality photostable and multicolor (blue and green) carbon nanocrystals with a hexagonal shape, a narrow size distribution of less than 20 nm, and high fluorescence quantum yield of up to 50.4% were obtained from taxanes in combination with m-phenylenediamine and folic acid to give the best results. The nanoparticles displayed a potent anticancer activity with IC50 values of 31.3 ± 2.7 and 34.1 ± 1.1 µg mL-1 for the human MCF-7 and HeLa cancer cell lines, respectively, and IC50 value of 120.5 ± 3.8 µg mL-1 on the normal human fibroblast cells. The flow cytometry studies determined apoptosis-mediated cell death as the main anticancer mechanism of CDs, and the molecular studies revealed the induction of both extrinsic and intrinsic apoptosis pathways. The overall results indicated the great potential of synthesized CDs for the simultaneous cancer imaging and therapy.

Common microbial causes of significant bacteriuria and their antibiotic resistance pattern in the Isfahan Province of Iran.

Urinary tract infections (UTIs) are considered the most common community-acquired infections worldwide, which have possible complications along with significant economic impact on national healthcare systems. The aim of this study was to identify the most common causes of significant bacteriuria and to assess their antimicrobial resistance pattern in the Isfahan province of Iran. In this cross-sectional study, 11,678 urine samples of the patients referred to Mahdieh Medical Diagnostic Centre Charity were examined over a period of 10 months (from September 2015 to June 2016). Among the cases, 6.85% were positive for bacteriuria (F/M = 11.3). Escherichia coli (62%) was the most frequently isolated bacteria, followed by Staphylococcus epidermidis (13.9%) and Staphylococcus aureus (6.8%). E. coli was more prevalent among patients with diabetes mellitus. E. coli isolates showed the highest resistance to nalidixic acid, Trimethoprim/Sulfamethoxazole and Cefixime. Our results revealed that broad-spectrum antibiotic resistance is frequent among isolated uropathogens in Isfahan, Iran.

Taguchi Experimental Design for Optimization of Recombinant Human Growth Hormone Production in CHO Cell Lines and Comparing its Biological Activity with Prokaryotic Growth Hormone.

Growth hormone deficiency results in growth retardation in children and the GH deficiency syndrome in adults and they need to receive recombinant-GH in order to rectify the GH deficiency symptoms. Mammalian cells have become the favorite system for production of recombinant proteins for clinical application compared to prokaryotic systems because of their capability for appropriate protein folding, assembly, post-translational modification and proper signal. However, production level in mammalian cells is generally low compared to prokaryotic hosts. Taguchi has established orthogonal arrays to describe a large number of experimental situations mainly to reduce experimental errors and to enhance the efficiency and reproducibility of laboratory experiments.In the present study, rhGH was produced in CHO cells and production of rhGH was assessed using Dot blotting, western blotting and Elisa assay. For optimization of rhGH production in CHO cells using Taguchi method An M16 orthogonal experimental design was used to investigate four different culture components. The biological activity of rhGH was assessed using LHRE-TK-Luciferase reporter gene system in HEK-293 and compared to the biological activity of prokaryotic rhGH.A maximal productivity of rhGH was reached in the conditions of 1%DMSO, 1%glycerol, 25 µM ZnSO4 and 0 mM NaBu. Our findings indicate that control of culture conditions such as the addition of chemical components helps to develop an efficient large-scale and industrial process for the production of rhGH in CHO cells. Results of bioassay indicated that rhGH produced by CHO cells is able to induce GH-mediated intracellular cell signaling and showed higher bioactivity when compared to prokaryotic GH at the same concentrations.

High dose Vitamin D intake and quality of life in relapsing-remitting multiple sclerosis: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Low level of vitamin D is associated with a more severe course and low quality of life in relapsing-remitting multiple sclerosis (RRMS). Low dose vitamin D intake has improved quality of life in RRMS patients. OBJECTIVE: This study explored the effect of high dose vitamin D intake on quality of life in RRMS patients in a double blind randomized clinical trial. METHODS: 94 RRMS patients were randomized to two groups. One group received 50,000 IU vitamin D3 every five days for 3 months. The other group received placebo. Interferon-ß (IFN-ß) continued as the main treatment in both groups. Quality of life was assessed using MSQOL-54 Persian version at the beginning and at the end of the study. RESULTS: After 3 months, the vitamin D group had a significant difference in mental health composite with placebo group, 62.41 ± 13.99 vs. 60.99 ± 17.99 (p-value = 0.041). Change in health was 75.74 ± 25.73 and 70.59 ± 26.45 in vitamin D and placebo group, respectively (p-value = 0.036). CONCLUSIONS: Mental QOL improved significantly after taking high dose vitamin D for 3 months in vitamin D group relative to placebo.

Short-term effect of high-dose vitamin D on the level of interleukin 10 in patients with multiple sclerosis: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Vitamin D has been related to the prevention of MS and to modulating its course. Recent studies have shown the safety of high-dose vitamin D in MS. OBJECTIVE: This study compared the effects of high-dose vitamin D on interleukin 10 (IL-10) levels in MS patients in a double-blind, randomized clinical trial. METHODS: Ninety-four patients with relapsing remitting MS (RRMS) were randomized into a treatment and a placebo group. Both groups received conventional MS treatment. The intervention group received 50,000 IU of vitamin D every 5 days for 3 months. IL-10 was measured at baseline and after 3 months. RESULTS: Serum levels of IL-10 were (median ± IQR): 12.58 ± 11.97 and 10.97 ± 9.97 pg/ml in the intervention and placebo groups, respectively, at baseline (p = 0.161); after 3 months, these levels were 13.76 ± 18.95 and 11.31 ± 19.63 pg/ml, respectively (p = 0.158). The IL-10 level increased significantly after receiving high-dose vitamin D for 3 months (ß = 0.737, p = 0.015 and R2 = 0.91). CONCLUSION: IL-10 levels increased significantly in RRMS patients after taking high-dose vitamin D3 for 3 months. High-dose vitamin D might be useful in promoting an anti-inflammatory state in RRMS patients.

In vitro antitumor activity of free and nano-encapsulated Na5[PMo10V2O40]·nH2O and its binding properties with ctDNA by using combined spectroscopic methods.

Free and nanosized starch and lipid encapsulated Na5[PMo10V2O40]·nH2O complexes (abbreviated as PMoV, SEP and LEP, respectively) have been prepared and structurally characterized by Fourier transform infrared (FT-IR) spectroscopy, inductively coupled plasma (ICP) analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. The results show that the PMoV retains its parent structure after encapsulation by starch and lipid nanoparticles. The in vitro antitumor activity of PMoV in its free and nano-encapsulated forms was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay that was carried out on two types of human cancer cells, MCF-7 (breast cancer cells) and HEK-293 (Human Embryonic Kidney). The results represent the enhancement of cell penetration and antitumor activity of PMoV due to its encapsulation in starch or lipid nanoparticles. However, this observed enhancement for the lipid relative to the starch nanocapsule can be attributed to its smaller size. In order to investigate the molecular nature of antitumor activity, the binding properties of PMoV with calf thymus DNA (ctDNA) were also comprehensively evaluated using UV-vis absorption spectroscopy, fluorescence quenching and fluorescence Scatchard plots. The results rule out the intercalating binding mode and propose the groove or outside stacking binding for PMoV. However, a biphasic binding behavior that is due to the change in the binding mode was observed by varying of [PMoV]/[ctDNA] mole ratio. The results of cell culture assay and DNA binding experiments represent that the rate of cell penetration is more important than DNA binding affinity in the antitumor activity for POM.

Optimizing conditions for production of high levels of soluble recombinant human growth hormone using Taguchi method.

Human growth hormone (hGH) is synthesized and stored by somatotroph cells of the anterior pituitary gland and can effect on body metabolism. This protein can be used to treat hGH deficiency, Prader-Willi syndrome and Turner syndrome. The limitations in current technology for soluble recombinant protein production, such as inclusion body formation, decrease its usage for therapeutic purposes. To achieve high levels of soluble form of recombinant human growth hormone (rhGH) we used suitable host strain, appropriate induction temperature, induction time and culture media composition. For this purpose, 32 experiments were designed using Taguchi method and the levels of produced proteins in all 32 experiments were evaluated primarily by ELISA and dot blotting and finally the purified rhGH protein products assessed by SDS-PAGE and Western blotting techniques. Our results indicate that media, bacterial strains, temperature and induction time have significant effects on the production of rhGH. The low cultivation temperature of 25°C, TB media (with 3% ethanol and 0.6M glycerol), Origami strain and a 10-h induction time increased the solubility of human growth hormone.

TNF-alpha Production by Peripheral Blood Monocytes in Multiple Sclerosis Patients and Healthy Controls.

BACKGROUND: Aberrant immune responses are evident in the pathogenesis of multiple sclerosis (MS) and it has been proposed that the spectrum of cytokines influence disease outcomes. Leptin and lipopolysaccharide (LPS) of Gram-negative bacteria are both potent cellular stimulators for production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α). The aim of this study was to compare the TNF-α production by peripheral blood monocytes from MS patients with healthy controls. METHODS: Peripheral blood samples were stimulated with LPS or leptin. After blocking the Golgi apparatus, intracellular cytokine production was assessed using a monoclonal antibody against human TNF-α by the flow cytometry technique. Moreover, plasma level measurement of cytokines was performed using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracellular levels of TNF-α were 16.80 ± 8.21 and 16.52 ± 8.23in MS patients and healthy controls which showed no statistically significant difference between them (p = 0.850). Leptin-stimulated and LPS-stimulated TNF-α production showed no significant difference between MS patients and the control group (p = 0.263 and p = 0.191, respectively). However, after treatment with leptin, a weak significant difference was shown between cases and control group (p = 0.049). There were significant differences between cases and controls regarding serum levels of IL-6 and Toll-like receptor-4 (TLR-4) before and after stimulation with leptin and LPS, separately (p < 0.05). CONCLUSION: Taken together, we cannot definitely conclude that TNF-α does not play an important role in pathogenesis of MS. However, other characteristics of monocyte activation such as IL-6 or TLRs can elucidate implication of peripheral blood monocytes in MS pathogenesis.

Effect of high dose vitamin D intake on interleukin-17 levels in multiple sclerosis: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Vitamin D has immunomodulatory effects in multiple sclerosis (MS). Vitamin D acts through various mechanisms such as secretion of cytokines. Interleukin-17 (IL-17) is a critical interleukin in inflammatory response in MS. OBJECTIVE: This study assessed the effect of oral high dose vitamin D intake on IL-17 levels in MS patients in a double blind randomized clinical trial. METHODS: 94 patients with a diagnosis of relapsing remitting multiple sclerosis (RRMS) were randomized to two groups. One group received 50,000 IU vitamin D3 every five days for 12weeks. The other group was given placebo. Both groups received interferon-ß (IFN-ß) treatment. Serum levels of IL-17 were measured at the beginning of the study and after 12weeks. RESULTS: IL-17 serum levels were 56.75±28.72pg/ml and 30.31±75.85pg/ml in the intervention and placebo group at the beginning of the study, respectively (Median±IQR, p=0.338). After 12weeks, IL-17 levels were 58.93±67.93pg/ml and 46.13±94.70pg/ml in the intervention and placebo group, respectively (Median±IQR, p=0.960). The multiple linear regression analysis indicated that the consumption of vitamin D3 was positively and significantly associated with the logarithm of IL-17 measures (ß=1.719; p=0.002 and R2=0.91), adjusted by EDSS scores. CONCLUSION: IL-17 levels showed significant change in RRMS patients after receiving high dose vitamin D3 for 12weeks.

Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-γ and TNF-α, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL.

Cytokines gene expression in newly diagnosed multiple sclerosis patients.

Multiple Sclerosis (MS) is characterized by multiple areas of inflammation, demyelination and neurodegeneration. Infiltrating Th1 CD4+ T cells secrete proinflammatory cytokines. They stimulate the release of some cytokines, expression of adhesion molecules and these cytokines may cause damage to the myelin sheath and axons. In this study, we analyzed plasma levels and gene expressions of five important cytokines in the new diagnosed MS Patients by ELISA and Real time PCR. PCR amplifications were performed to determine the IL-17, IL-23, IL-10, IL-27 and TGF-ß mRNA expression levels using the SYBR Green PCR Kit. Our results showed significant decrease in IL-10, IL-27 and TGF-ß but there was no significant difference in the IL-17 and IL-23 between patients and healthy controls. Altogether, our results indicated that dysregulation of cytokines, mainly increased expression of pro-inflammatory cytokines and decreased expression of inhibitory cytokines occurred in MS patients. This study may shed light to the probable role of these cytokines in neurodegeneration mechanism and current or future use of cytokines in managing and treatment of multiple sclerosis.

The beneficial effects of vitamin D3 on reducing antibody titers against Epstein-Barr virus in multiple sclerosis patients.

Recently, the relationship between immunoreactivity to Epstein-Barr virus (EBV) and hypo-vitamin D in multiple sclerosis (MS) patients has been described. The aim of this study was to investigate whether vitamin D3 supplementation in MS patients could influence the immune response against latent EBV infection. Forty MS patients were recruited in this study. Twenty-seven patients were supplemented with 50,000 IU/week of vitamin D3 for 6 months and thirteen enrolled as controls. 25-Hydroxyvitamin D (25OHD) levels and IgG titers against EBNA1 and VCA were determined pre- and post-supplementation. All the patients were seropositive for EBV prior to vitamin D supplementation. In this cohort, 22.5% and 47.5% of the MS patients had deficient and insufficient levels of 25OHD, respectively. Our findings confirm that antibody titers against EBV in MS patients rise after the onset of the disease and indicate that vitamin D3 supplementation could limit augmentation of these titers in MS patients.

Validation of serum IGF-I as a biomarker to monitor the bioactivity of exogenous growth hormone agonists and antagonists in rabbits.

The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, the most sensitive pharmacodynamic marker for the action of GH in humans, shows no response to treatment with recombinant human GH and there is little evidence for the effects of GHAs, except when administered at very high doses or when overexpressed. As an alternative, more suitable model, we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for the analysis of rabbit serum and tested precision, sensitivity, linearity and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with recombinant human GH or the GHA Pegvisomant. For a subset of samples, we used LC-MS/MS to measure IGF-I, and quantitative western ligand blot to analyze IGF-binding proteins (IGFBPs). Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, our results show that the sensitivity, precision (1.7-3.3% coefficient of variation) and linearity (90.4-105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I concentration in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple injections of recombinant human GH (IGF-I: 286±22 versus 434±26 ng/ml; P<0.01) and were highly correlated (P<0.0001). Treatment with the GHA lowered IGF-I levels from the fourth injection onwards (P<0.01). In summary, we demonstrated that the IDS-iSYS IGF-I immunoassay can be used in rabbits. Similar to rodents, rabbits display variations in IGF-I depending on sex, age and genetic background. Unlike in rodents, the IGF-I response to treatment with recombinant human GH or a GHA closely mimics the pharmacodynamics seen in humans, suggesting that rabbits are a suitable new model to test human GH agonists and antagonists.

Toll like receptor 2 and 4 expression in peripheral blood mononuclear cells of multiple sclerosis patients.

BACKGROUND: Multiple sclerosis (MS) is a T cell mediated autoimmune disease with unknown etiology. Appropriate MS therapeutic strategies need thorough understanding of both disease etiology and pathogenesis mechanisms. Ligation of TLR-2 and TLR-4 stimulates the production of several cytokines leading to CNS autoimmunity and neurodegenerative diseases. OBJECTIVE: To find a relationship between MS disability and TLR-2 and TLR-4 expression on mononuclear cells in the blood of MS patients. METHODS: Forty-five new case (NC) MS patients (33 females and 12 males) and 45 age and gender-matched healthy controls (HC) were recruited to the study. PBMCs were prepared and the expressions of TLR-2 and TLR-4 were assessed by flowcytometry technique using appropriate monoclonal antibodies. RESULTS: Our results showed that the expression of TLR-2 and TLR-4 proteins in the patients group was significantly higher than that of healthy controls. TLR-2 but not TLR-4 was correlated with expanded disability status scale (EDSS) scores. CONCLUSION: High expressions of TLR-2 and TLR-4 may represent a state of innate immune activation in patients with MS.

Transplantation of human adipose-derived stem cells enhances remyelination in lysolecithin-induced focal demyelination of rat spinal cord.

Adipose-derived stem cells (ADSCs) are a desirable stem cell source in neurodegenerative diseases treatment due to their ability to differentiate into different cell lineages. In this study, we transplanted human ADSCs (hADSCs) into a lysophosphatidylcholine (lysolecithin) model of multiple sclerosis (MS) and determined the efficiency of these cells in remyelination process. Forty adult rats were randomly divided into control, lysolecithin, vehicle, and transplantation groups, and focal demyelination was induced by lysolecithin injection into spinal cord. To assess motor performance, all rats were examined weekly with a standard EAE scoring scale. Four weeks after cell transplantation, to assess the extent of demyelination and remyelination, Luxol Fast Blue staining was used. In addition, immunohistochemistry technique was used for assessment of the presence of oligodendrocyte phenotype cells in damaged spinal cord. Our results indicated that hADSCs had ability to differentiate into oligodendrocyte phenotype cells and improved remyelination process. Moreover, the evaluation of rat motor functions showed that animals which were treated with hADSC compared to other groups had significant improvement (P < 0.001). Our finding showed that hADSCs transplantation for cell-based therapies may play a proper cell source in the treatment of neurodegenerative diseases such as MS.