Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein).

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP's influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands. KEY POINTS: • YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity. • YAP5SA regulates genes involved in proliferation, survival, and mTOR activation. • YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

Evaluation of the safety and efficacy of biosimilar recombinant growth hormone in children with growth hormone deficiency: non-inferiority, randomized, parallel, multicentric and Phase III trial.

OBJECTIVES: This study is designed in order to compare the efficacy and safety of recombinant human growth hormone (rhGH) with the reference brand. METHODS: According to the inclusion criteria, 85 people in 13 Iranian centers were randomly selected to receive biosimilar Somatropin (Somatin®) (44 people) and reference Somatropin (Norditropin®) (41 people) at a dose of 35 µg/kg/d, seven days/week for 12 months. The primary outcomes included height velocity (HV) was measured during 12 months of treatment. RESULTS: The two intervention groups' Height changes were similar. The mean HV was 10.96 cm/year in the biosimilar group and 10.05 cm/year in the reference groups after 12 months. Estimates of the lower bounds of 95% CI for mean height differences in the biosimilar intervention group compared to the reference intervention group did not exceed the 2 cm margin. Therefore, the non-inferiority of biosimilar intervention compared to the brand product is verified. Common ADRs in both groups were nausea in two patients (2.4%), diarrhea in two patients (2.4%), increased body temperature in one patient (1.2%), and headache in one patient (1.2%). CONCLUSIONS: The finding of this study indicated that Somatin® and Norditropin® have comparable efficacy and safety profiles. CLINICAL TRIAL REGISTRATION: www.IRCT.irIRCT20171122037571N1.

Association of rs5051 and rs699 polymorphisms in angiotensinogen with coronary artery disease in Iranian population: A case-control study.

Coronary artery disease (CAD) is the third most common cause of mortality globally (with 17.8 million deaths annually). Angiotensinogen (AGT) and polymorphisms in this gene can be considered as susceptibility factors for CAD. We performed a retrospective case-control study to determine the correlation of AGT rs5051 and rs699 polymorphisms with CAD in an Iranian population. We genotyped 310 CAD patients and 310 healthy subjects using polymerase chain reaction-based methods. To confirm the accuracy of the screening approach, 10% of genotyped subjects were validated using gold-standard Sanger Sequencing. To evaluate the effect of the candidate polymorphisms, white blood cells were randomly purified from the subjects and AGT expression was measured by quantitative reverse transcriptase-polymerase chain reaction. Sex stratification indicated a significant correlation between CAD and male sex (P = .0101). We found a significant association between the rs5051 A allele (P = .002) and the rs699 C allele, and CAD (P = .0122) in recessive and dominant models. Moreover, our findings showed a significant association of the haplotype, including the rs5051 A/A and rs699 T/C genotypes, with CAD (P = .0405). Finally, AGT mRNA levels were significantly decreased in patients harboring the candidate polymorphisms (P = .03). According to our findings The AGT rs5051 A and AGT rs699 C alleles are predisposing variants of CAD risk and severity in the Iranian population.

Enhancing protein production and growth in chinese hamster ovary cells through miR-107 overexpression.

Chinese Hamster Ovary (CHO) cells are widely employed as host cells for biopharmaceutical production. The manufacturing of biopharmaceuticals poses several challenges, including restricted growth potential and inadequate productivity of the host cells. MicroRNAs play a crucial role in regulating gene expression and are considered highly promising tools for cell engineering to enhance protein production. Our study aimed to evaluate the effects of miR-107, which is recognized as an onco-miR, on erythropoietin-producing CHO cells (CHO-hEPO). To assess the impact of miR-107 on CHO cells, a DNA plasmid containing miR-107 was introduced to CHO-hEPO cells through transfection. Cell proliferation and viability were assessed using the trypan blue dye exclusion method. Cell cycle analysis was conducted by utilizing propidium iodide (PI) staining. The quantification of EPO was determined using an immunoassay test. Moreover, the impact of miR-107 on the expression of downstream target genes was evaluated using qRT-PCR. Our findings highlight and underscore the substantial impact of transient miR-107 overexpression, which led to a remarkable 2.7-fold increase in EPO titers and a significant 1.6-fold increase in the specific productivity of CHO cells (p < 0.01). Furthermore, this intervention resulted in significant enhancements in cell viability and growth rate (p < 0.05). Intriguingly, the overexpression of miR­107 was linked to the downregulation of LATS2, PTEN, and TSC1 genes while concurrently driving upregulation in transcript levels of MYC, YAP, mTOR, and S6K genes within transgenic CHO cells. In conclusion, this study collectively underscores the feasibility of utilizing cancer-associated miRNAs as a powerful tool for CHO cell engineering. However, more in-depth exploration is warranted to unravel the precise molecular intricacies of miR-107's effects in the context of CHO cells.

The Effect of an Overhead External Load Lifting and Lowering on Dynamic Postural Control in Subgroups of Low Back Pain.

BACKGROUND: Understanding postural control in low back pain (LBP) subgroups can help develop targeted interventions to improve postural control. The studies on this topic are limited. Therefore, the primary purpose of this study was to compare the postural control of LBP subgroups with healthy individuals during overhead load lifting and lowering. METHODS: In this cross-sectional study, the participants were 52 with LBP and 20 healthy. The LBP patients were classified based on the O'Sullivan classification system into 21 flexion patterns and 31 active extension patterns. The participants lifted the box from their waists to their overheads and lowered it to their waists. Changes in postural control parameters were measured with a force plate system. RESULTS: The results of the analysis of variance showed that during load lifting, the mediolateral phase plane (p = .044) and the mean total velocity (p = .029) had significant differences between flexion patterns and healthy. Also, the load-lowering results showed that active extension patterns, compared with healthy, had significant differences in the anteroposterior-mediolateral phase plane (p = .042). The patients showed less postural sway than the healthy. CONCLUSIONS: The results in this work highlight the importance of identifying the homogenous subgroups in LBP and support the classification of heterogeneous LBP. Different subgroups exhibit different postural control behaviors. These behaviors can be due to the loading of various tissues during different tasks.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering.

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature. Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry. Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, "pB513-b CMV-P/4shRNAs/GFP" plasmid, "pB513-bHSP90-P/4sh-RNAs/GFP" and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days. Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Evaluation of the impact of polypeptide-p on diabetic rats upon its cloning, expression, and secretion in Saccharomyces boulardii.

This study was designed to evaluate the effectiveness of recombinant polypeptide-p derived from Momordica charantia on diabetic rats. In this research, the optimized sequence of polypeptide-p gene fused to a secretion signal tag was cloned into the expression vector and transformed into probiotic Saccharomyces boulardii. The production of recombinant secretion protein was verified by western blotting, HPLC, and mass spectrometry. To assay recombinant yeast bioactivity in the gut, diabetic rats were orally fed wild-type and recombinant S. boulardii, in short SB and rSB, respectively, at two low and high doses as well as glibenclamide as a reference drug. In untreated diabetic and treated diabetic + SB rats (low and high doses), the blood glucose increased from 461, 481, and 455 (mg/dl), respectively, to higher than 600 mg/dl on the 21st day. Whereas glibenclamide and rSB treatments showed a significant reduction in the blood glucose level. The result of this study promised a safe plant-source supplement for diabetes through probiotic orchestration.

Lenalidomide improves NKG2D-based CAR-T cell activity against colorectal cancer cells invitro.

Introduction: Although CAR-based immunotherapy is viewed as a promising treatment for tumors, particularly hematological malignancies, solid tumors can pose challenges. It has been suggested that the immunomodulatory medication Lenalidomide (LEN) may increase the effectiveness of CAR T cells in the treatment of solid tumors. The purpose of our study was to investigate the effect of NKG2D-based CAR T cell therapy on colorectal cancer cell lines, and then we assessed combinatorial therapy using NKG2D CAR T cells and lenalidomide in vitro. Methods and results: To prepare NKG2D CAR T cells, a second-generation NKG2D-CAR construct was designed and transfected into the T cells using a lentiviral system. The NKG2D CAR T cells showed significantly higher cytotoxic activity against colorectal cancer cell lines, HCT116 and SW480, compared to untransduced T cells. In addition, our data demonstrated that the cytotoxicity and cytokine secretion of NKG2D CAR T cells significantly increased in the presence of higher doses of lenalidomide. Conclusions: The study findings suggest that combinational therapy, utilizing NKG2D-based CAR T cells and lenalidomide, has a high potential for effectively eliminating tumor cells in vitro.

Comparison of the Dynamic Postural Control During Lifting and Lowering an External Load in Low Back Pain: A Cross-Sectional Study.

OBJECTIVE: This study aimed to compare dynamic postural control between individuals with and without chronic low back pain (LBP) through load lifting and lowering. METHODS: This cross-sectional study included 52 male patients with chronic LBP (age: 33.37 ± 9.23 years) and 20 healthy male individuals (age: 31.75 ± 7.43 years). The postural control parameters were measured using a force plate system. The participants were instructed to stand barefoot (hip-width apart) on the force plate and lift a box (10% of the weight of the participants) from the waist height to overhead and then lower it from overhead to waist height. The interaction between the groups and tasks was determined using a 2-way repeated-measures analysis of variance. RESULTS: There was no significant interaction between the groups and tasks. Regardless of the groups, postural control parameters including amplitude (P = .001) and velocity (P < .001) in anterior-posterior (AP) direction, phase plane in medial-lateral (ML) direction (P = .001), phase plane in AP-ML direction (P = .001), and the mean total velocity (P < .001) were lesser during the lowering compared with lifting. The results indicated that, regardless of the tasks, the postural control parameters including velocity (P = .004) and phase plane in AP direction (P = .004), velocity in ML direction (P < .001), phase plane (AP-ML) (P = .028), and mean total velocity (P = .001) in LBP were lesser compared with the normal group. CONCLUSION: Different tasks affected postural control differently in patients with LBP and healthy individuals. Moreover, postural control was more challenged during the load-lowering than the load-lifting task. This may have been a result of a stiffening strategy. It may be that the load-lowering task might be considered as a more influential factor for the postural control strategy. These results may provide a novel understanding of selecting the rehabilitation programs for postural control disorders in patients.

Analytical chromatography approaches during the synthesis and conjugation of methoxypolyethylene glycol-succinimidyl butanoate (mPEG-SBA) to epoetin beta.

Conjugation of epoetin beta (EPO) with methoxypolyethylene glycol-succinimidyl butanoate (mPEG-SBA) was studied. The compound mPEG-SBA was synthesized from mPEG, and the obtained intermediates and final product were analyzed by a reversed-phase chromatographic system equipped with an evaporative light scattering detector. Labeling the hydroxyl group in PEGs with benzoyl chloride and succinimide with benzylamine was applied to resolve and characterize different PEGs. The synthesized mPEG-SBA was used for the PEGylation of EPO. A size-exclusion chromatographic method monitored the reaction, simultaneously determining the PEGylated and unreacted EPO and protein aggregates. A borate buffer (0.1 M, pH 7.8) and PEG/protein molar ratio of 3:1 produced a maximum amount of monoPEGylated EPO with the minimum amount of polyPEGylated EPO variants. Although EPO is considered a stable glycoprotein hormone that remains monomeric when refrigerated, PEGylation of EPO with mPEG-SBA resulted in the significant formation of EPO dimer. The formation of EPO dimer and polyPEGylated EPO was pH-dependent, showing higher amounts of aggregates and lower amounts of polyPEGylated forms in lower pH values. Accordingly, aggregated EPO should be considered a major PEGylation-related impurity. In conclusion, the present study highlighted the importance of having suitable analytical approaches in controlling mPEG-SBA synthesis and conjugation to EPO.

Transcriptome Profiling of Cisplatin Resistance in Triple-negative Breast Cancer: New Insight into the Role of PI3k/Akt Pathway.

BACKGROUND: Aggressive nature of triple negative breast cancer (TNBC) is associated with poor prognosis compared with other breast cancer types. Current guidelines recommend the use of Cisplatin for the management of TNBC. However, the development of resistance to cisplatin is the primary cause of chemotherapy failure. OBJECTIVE: In the present study, we aimed to develop a stable cisplatin-resistant TNBC cell line to investigate the key pathways and genes involved in cisplatin-resistant TNBC. METHODS: The MDA-MB-231 cell was exposed to different concentrations of cisplatin. After 33 generations, cells showed a resistant phenotype. Then, RNA-sequencing analysis was performed in cisplatin-resistant and parent cell lines. The RNA-sequencing data was verified by quantitative PCR (qPCR). RESULTS: The IC50 of the resistant cell increased to 10-fold of a parental cell (p<0.001). Also, cisplatin-resistant cells show cross-resistance to other drugs, including 5- fluorouracil, paclitaxel, and doxorubicin. Resistant cells demonstrated reduced drug accumulation compared to the parental cells. Results showed there were 116 differentially expression genes (DEGs) (p<0.01). Gene ontology analysis revealed that the DEGs have several molecular functions, including binding and transporter activity. Functional annotation showed that the DEGs were enriched in the drug resistancerelated pathways, especially the PI3K-Akt signaling pathway. The most important genes identified in the protein-protein interaction network were heme oxygenase 1 (HMOX1) and TIMP metallopeptidase inhibitor 3 (TIMP3). CONCLUSION: We have identified several pathways and DEGs associated with the PI3KAkt pathway, which provides new insights into the mechanism of cisplatin resistance, and potential drug targets in TNBC.

RT-LAMP in SARS-CoV-2 detection: point to improve primer designing and decrease molecular diagnosis pitfalls.

BACKGROUND: Due to the high transmission rate of SARS-CoV-2, diagnostic tests have become tools for identifying patients. The key points were the virus genomes survey to design RT-LAMP primers; comparing the sensitivity and specificity of RT-LAMP and RT-qPCR; and determining the relationship among clinical symptoms, CT scan, RT-qPCR, and RT-LAMP results. METHODS: This cohort study included 444 symptomatic patients. The specificity and sensitivity of RT-LAMP were assayed. The five statistical models, simultaneously, by RapidMiner to find the best method for detecting the virus were done through the correlation between the clinical symptoms, RT-LAMP, RT-qPCR, and CT scan results. The chi-square test by SPSS 26.0 was used to calculate kappa agreement. RESULTS: The virus genome was detected in all the positive samples (198) by RT-qPCR and RT-LAMP. In addition, 246 samples were negative by RT-qPCR, while 88 were positive by RT-LAMP. Data mining analysis indicated that there were most associations between the RT-LAMP and CT scan data compared to RT-qPCR and CT scan data. CONCLUSIONS: RT-LAMP could detect SARS-CoV-2 with great simplicity, speed, and cheapness. Therefore, it is logical to screen, a large number of patients by RT-LAMP, and then RT-qPCR can be used on the limited samples.

In vivo assessment of simultaneous G1 cyclins silencing by a tumor-specific bidirectional promoter on the mammary tumor in nude mice.

Dysregulation of G1 cyclins (cyclins D1 A and E) expression contributes to the loss of standard cell cycle control during tumorigenesis. This study aims to evaluate the inhibitory effect of G1 cyclins in nude mice. The human breast cancer MDA-MB-231 cells were subcutaneously transplanted into the supra-femoral right side of female Balb/c-nude mice. The dual shRNA vector harboring G1 cyclins shRNAs (bipSUR) was intratumorally injected by the in vivo jetPEI transfection reagent for 2 weeks. We have evaluated tumor growth and tumor weight as parameters of tumor progression. Finally, necropsy, histopathological analysis, and immunodetection of G1 cyclins were assessed. Also, apoptosis induction in tumor tissues was evaluated by TUNEL assay. No toxicity and metastasis was observed in the tumor-bearing mice treated by the bipSUR. Tumor weight and volume were significantly lower in the bipSUR treated mice than untreated tumor-bearing mice and control. Histopathological observations revealed more apoptotic foci and lower mitotic cells in tumor sections in the treated mice than in control groups. A significant reduction of G1 cyclins at the protein level was indicated in the bipSUR treated mice than in other groups. Apoptosis in tumor tissues was remarkably induced in response to the bipSUR (42.53%). The bipSUR reduced the protein expression of G1 cyclins and exhibited an inhibitory effect on MDA-MB-231 xenograft mice through apoptosis induction. Further research is demanded to identify the protein partners of G1 cyclins involved in the cancer pathways. These may offer new insight into the biomedical function of G1 cyclins in breast cancer progression.

3-Dimensional Model to Study Apoptosis Induction of Activated Natural Killer Cells Conditioned Medium Using Patient-Derived Colorectal Cancer Organoids.

Natural killer (NK) cells are innate lymphocytes that can kill tumor cells via different pathways, including the secretion of cytotoxic granules in immunological synapses and the binding of apoptosis-inducing ligands with cognate death receptors on tumor cells. These ligands are also soluble in NK cells conditioned medium (NK-CM). However, novel preclinical in vitro models are required for solid tumors such as colorectal cancer (CRC) to investigate apoptosis induction of activated NK-CM in a tissue-like structure. In the present study, we established a patient-derived CRC organoid culture system as a new tool for CRC research in the last decade. Tumor organoids were stained with hematoxylin and eosin (H&E) and compared with the original tumor taken from the patient. Goblet cell differentiation and mucus secretion were evaluated using periodic acid-Schiff and alcian blue histochemical staining. Moreover, tumor organoids were stained for CDX2 and Ki67 markers with immunohistochemistry (IHC) to investigate gastrointestinal origin and proliferation. Histopathological evaluations indicated tumor organoids represent patient tumor characteristics. Primary NK cells were isolated and characterized using CD56 marker expression and the lack of the CD3 marker. Flow cytometry results showed the purity of isolated CD3-and CD56 + NK cells about 93%. After further ex vivo expansion, IL-2-activated NK-CM was collected. Secretions of IFN-γ and TNF-α were measured to characterize activated NK-CM. Cytokines levels were significantly elevated in comparison to the control group. Soluble forms of apoptosis-inducing ligands, including TNF-related apoptosis-inducing ligand (TRAIL) and FasL, were detected by western blot assay. Colon cancer organoids were treated by IL-2-activated NK-CM. Apoptosis was assessed by Annexin V-FITC/PI staining and quantified by flow cytometry. In conclusion, despite the activated NK-CM containing apoptosis-inducing ligands, these ligands' soluble forms failed to induce apoptosis in patient-derived colon cancer organoids. Nevertheless, we report a reliable in vitro assessment platform in a personalized setting.

Treatment of EAE mice with Treg, G-MDSC and IL-2: a new insight into cell therapy for multiple sclerosis.

Background: This study investigates the therapeutic and protective effects of Tregs, myeloid-derived suppressor cells (MDSCs) and IL-2 on multiple sclerosis (MS) disease model. Materials & methods: C57BL/6 mice were immunized to develop an experimental autoimmune encephalomyelitis (EAE) model. We then investigated effects of pre- and post-treatment EAE mice with Tregs, MDSCs and IL-2 on inflammation and demyelination in brain tissue, and on the number of Treg, granulocytic-MDSC and Th-17 cells in spleen. Results: Pre- and post-treatment of EAE mice by Tregs, MDSCs and IL-2 resulted in no weight change, reduced Th-17 cells and suppression of pathological properties. Conclusion: Pre- and post-treatment of immunized mice by Tregs, MDSCs and IL-2 prevent EAE induction.

This study investigates the therapeutic and protective effects of suppressive immune cells and pivotal cytokines on multiple sclerosis disease model. In this study, mice were immunized to develop experimental autoimmune model. We then investigated effects of pre- and post-treatment model mice with suppressive immune cells and pivotal cytokines on immunomodulatory and pro-inflammatory cells. Pre- and post-treatment of model mice resulted in no weight change, reduced pro-inflammatory cells and suppression of undesired pathological properties.

Fibroblast Growth Factor-2 Levels are Elevated in The Serum of Patients with Multiple Sclerosis

BACKGROUND: Various factors contribute to the pathogenesis of Multiple Sclerosis (MS), one of which is Fibroblast Growth Factor 2 (FGF2). The function of FGF2 is pleiotropic. The investigation of the role of this factor in the myelination has produced conflicting results. OBJECTIVE: To investigate the serum levels of FGF2 in patients with MS. SUBJECTS AND METHODS: Eighty patients with MS and eighty healthy volunteers with no history of inflammation or demyelinating disorders were included, and serum samples were collected to evaluate serum levels of FGF2 using the ELISA technique. Both groups had the same age and gender distribution. For analysis, the Mann-Whitney U test was used. RESULTS: Patients with MS had considerably greater serum FGF2 levels than the control group (p = 0.005). There was no difference between the FGF2 level in men and women. CONCLUSION: Our data indicate that FGF2 levels may be related to the susceptibility of Iranian patients with MS. Further studies are required to analyze the involvement of FGF2 in enhancing the inflammatory process in MS.

Genetically Engineered Mesenchymal Stem Cell Therapy Against Murine Experimental Autoimmune Encephalomyelitis.

We used recombinant interleukin 23 receptor (RIL-23R)-engineered mesenchymal stem cells (MSCs) to study its therapeutic role in enhancing inflammation of nervous tissue in the mouse model (EAE) of multiple sclerosis (MS). Recombinant IL-23 receptor construct was designed to enter MSCs. The bioactivity of the constructs was assessed by the co-culture of MSCs/CD4 + T cells. The EAE model was induced in mice. After cell transplantation, clinical scores were evaluated, and tissue demyelination was measured by Luxol fast blue staining. The transfection of RIL-23R mRNA improved MSC properties significantly to the inflamed regions of EAE mice, and it performed an increased suppressive function on the T lymphocyte proliferation. Furthermore, in vivo therapy with RIL-23R MSCs in EAE mice showed an enhanced therapeutic action than MSCs, proven by improved myelination and a reduction in the penetration of inflammatory cells into the white matter. Our targeted transplantation procedure of modified MSC can be applied to improve the effectiveness of cellular therapy for multiple sclerosis and other autoimmune disorders.

Contribution of HLA class II genes, DRB4*01:01, DRB1*07:01, and DQB1*03:03:2 to clinical features of Vitiligo disease in Iranian population.

BACKGROUND: Vitiligo is a multifactorial depigmentation condition, which is due to skin melanocyte destruction. Increased expression of HLA class II genes in patients with pre-lesions of Vitiligo suggests a crucial role for the participation of immune response in Vitiligo development. Recent studies progressively focused on HLA-DRB1 and DQB1 genes. In this study, we have evaluated the association and role of HLA-DRB4*01:01, -DRB1*07:01, and -DQB1*03:03:2 genes in different clinical subtypes of Vitiligo in the Iranian population. METHODS: First, Genomic DNA from peripheral blood of 125 unrelated Vitiligo patients and 100 unrelated healthy controls were extracted through the salting-out method. Then, HLA class II genotyping was performed using the sequence-specific primer PCR method. Finally, the clinical relevance of the testing for these genotypes was evaluated by applying the PcPPV (prevalence-corrected positive predictive value) formula. RESULTS: Our results indicated the positive associations of DRB4*01:01 and DRB1*07:01 allelic genes with early-onset Vitiligo (p = 0.024 and 0.022, respectively). DRB4*01:01 also showed strong protection against late-onset Vitiligo (p = 0.0016, RR = 0.360). Moreover, our data revealed that the DRB1*07:01 increases the susceptibility to Sporadic Vitiligo (p = 0.030, RR = 1.702). Furthermore, our findings proposed that elevated vulnerability of Vitiligo patients due to DRB4*01:01 and DRB1*07:01 alleles maybe is correlated with the presence of amino acid Arginine at position 71 at pocket 4 on the antigen-binding site of the HLA-DRB1 receptor. CONCLUSION: Our findings on different subtypes of Vitiligo suggest that, despite a more apparent autoimmune involvement, a non-autoimmune nature for the etiology of Vitiligo should also be considered.

Biosimilarity Assessment of 2 Filgrastim Therapeutics in Healthy Volunteers.

This study aimed to compare the pharmacokinetic, pharmacodynamic, and safety profiles of a proposed biosimilar and innovator filgrastim therapeutics in healthy volunteers. In a crossover design, 23 subjects received a single subcutaneous injection of 300-µg filgrastim, followed by a 7-day washout period. Assessed pharmacokinetic parameters were the maximum observed filgrastim serum concentration (Cmax ), time to reach Cmax (tmax ), the area under the concentration-time curve (AUC), and elimination half-life. Pharmacodynamics were assessed by the maximum observed absolute neutrophil count effect (Emax ), tmax,E (time to reach Emax ), and the area under the effect of the absolute neutrophil count -time curve. The test/reference ratio (90% confidence intervals) of Cmax of 0.992 (0.860-1.143), AUC0-inf of 0.995 (0.891-1.111), Emax of 0.952 (0.841, 1.078), and area under the effect of the absolute neutrophil count -time curve from time zero to 96 hours of 0.939 (0.854-1.032), were all well within the predefined equivalence boundaries of 80% and 125%. Obtained values for tmax (∼4 hours), tmax,E (∼15 hours), and elimination half-life (∼3.5 hours) were comparable between 2 treatment groups. The local tolerability and incidence of adverse events were comparable, with no clinically meaningful difference between biosimilar and innovator products. Altogether, the results suggested a high similarity of the proposed biosimilar to the innovator filgrastim in healthy volunteers.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.