Umbilical Cord Blood-Derived Monocytes as A Reliable Source of Functional Macrophages for Biomedical Research.

OBJECTIVE: Macrophages are multifunctional immune cells widely used in immunological research. While autologous macrophages have been widely used in several biomedical applications, allogeneic macrophages have also demonstrated similar or even superior therapeutic potential. The umbilical cord blood (UCB) is a well-described source of abundant allogenic monocytes and macrophages that is easy to collect and can be processed without invasive methods. Current monocyte isolation procedures frequently result in heterogenous cell products, with limited yields, activated cells, and high cost. This study outlines a simple isolation method that results in high yields and pure monocytes with the potential to differentiate into functional macrophages. MATERIALS AND METHODS: In the experimental study, we describe a simple and efficient protocol to isolate highpurity monocytes. After collection of human UCB samples, we used a gradient-based procedure composed of three consecutive gradient steps: i. Hydroxyethyl starch-based erythrocytes sedimentation, followed by ii. Mononuclear cells (MNCs) isolation by Ficoll-Hypaque gradient, and iii. Separation of monocytes from lymphocytes by a slight hyperosmolar Percoll gradient (0.573 g/ml). Then the differentiation potential of isolated monocytes to pro- and antiinflammatory macrophages were evaluated in the presence of granulocyte colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF), respectively. The macrophages were functionally characterized as well. RESULTS: A high yield of monocytes after isolation (25 to 50 million) with a high purity (>95%) could be obtained from every 100-150 ml UCB. Isolated monocytes were defined based on their phenotype and surface markers expression pattern. Moreover, they possess the ability to differentiate into pro- or anti-inflammatory macrophages with specific phenotypes, gene/surface protein markers, cytokine secretion patterns, T-cell interactions, and phagocytosis activity. CONCLUSION: Here we describe a simple and reproducible procedure for isolation of pure monocytes from UCB, which could be utilized to provide functional macrophages as a reliable and feasible source of allogenic macrophages for biomedical research.

Effect of risperidone on morphine-induced conditioned place preference and dopamine receptor D2 gene expression in male rat hippocampus.

BACKGROUND: Previous studies suggest the possible effect of risperidone on brain reward system and D1 and D2 dopamine receptors' involvement in morphine-induced conditioned place preference (CPP). AIMS: The present study was designed to investigate the effect of risperidone as an atypical antipsychotic drug on morphine-induced CPP and D2-like dopamine receptor gene expression in rat. MATERIALS AND METHODS: An unbiased CPP paradigm was used to study the effect of risperidone. Intraperitoneal (i.p.) injection of risperidone (1, 2, and 4 mg/kg) was performed 30 min before the morphine (10 mg/kg, i.p.) injection and just after the rat was placed in the CPP box. The open field test was used to assay the locomotor activity of animal. The gene expression of D2 dopamine receptor in hippocampus was measured by real-time PCR technique. The hippocampi of rats were also used for histology evaluation. RESULTS: Morphine-produced (10 mg/kg) CPP and morphine-induced CPP were reversed only by the administration of a low dose of risperidone (1 mg/kg). Low dose of risperidone (1 mg/kg) showed no effect on locomotor activity but a higher dose of risperidone (2 and 4 mg/kg) decreased locomotor activity. Real-time PCR data analysis revealed that the gene expression of D2 dopamine receptor had significant difference between morphine and a 1 mg/kg dose of risperidone. Moreover, in histological evaluation, apoptosis was observed in the morphine group, whereas there was no evidence of apoptosis in the risperidone-treated groups. CONCLUSION: Our results suggest that risperidone (1 mg/kg) reverses the morphine-induced CPP and may reduce the rewarding properties of morphine. It is also demonstrated that risperidone decreases the expression of D2 receptor in rat hippocampus. Therefore, risperidone can be considered potential adjunct therapy in morphine dependence.

The protection quest is a primary key to sharing the neutralizing antibody response to cover against all emerging VOCs based on BIV1-CovIran studies.

Over time, the antigenic evolution of emerging variants of SARS-CoV-2 has demanded the development of potential protective vaccines. Administration of additional doses of current vaccines based on the WT spike protein may boost immunity, but their effectiveness has dwindled for patients with more recent variants. Here, we studied the neutralization activity of post-WT strain-based vaccination and a structural simulation in-silico based on the interactions of the RBD-hACE2 as the key to initiating infection among the VOCs of SARS-CoV-2. Our data display shows that WT sera showed a markedly greater reduction in Delta and Omicron, suggesting that the Wuhan-based vaccines may be more susceptible to breakthrough and new VOCs. According to the MD simulation, mutations of Omicron result in a significant change in the variant charge distribution throughout the binding interface that consequently alters the critical interface electrostatic potential in comparison to other variants. This observation provides new insights into immunization policy and next-generation vaccine development.

Assessment of BIV1-CovIran inactivated vaccine-elicited neutralizing antibody against the emerging SARS-CoV-2 variants of concern.

OBJECTIVES: The BIV1-CovIran vaccine is highly effective against COVID-19. The neutralizing potency of all SARS-CoV-2 vaccines seems to be decreased against variants of concern. We assessed the sensitivity of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants to neutralizing antibodies (NAbs) present in sera from individuals who had received the BIV1-CovIran candidate vaccine compared with an original Wuhan-related strain. METHODS: The ability of vaccine serum to neutralize the variants was measured using the conventional virus neutralization test. The correlation of spike (S) protein antibody and anti-receptor binding domain with neutralizing activity was investigated. RESULTS: The current study demonstrated that 29 of 32 (90.6%; 95% CI: 75.0-98.0) of the vaccinees developed NAbs against a Wuhan-related strain. It is noteworthy that 28 (87.50%) and 24 of 32 (75%) of the recipients were able to produce NAbs against Alpha, Beta, and Delta variants, respectively. Serum virus-neutralizing titres for different SARS-CoV-2 strains were weakly correlated with anti-receptor binding domain antibodies (Spearman r = 36-42, p < 0.05), but not S-binding antibodies (p > 0.05). DISCUSSION: Although there was a reduction in neutralization titres against the Alpha, Beta, and Delta variants compared with the Wuhan strain, BIV1-CovIran still exhibited potent neutralizing activity against the SARS-CoV-2 variants of concern.

Changes in immune profile affect disease progression in hepatocellular carcinoma.

Objective: Hepatocellular carcinoma (HCC) as a chronic liver condition is largely associated with immune responses. Previous studies have revealed that different subsets of lymphocytes play fundamental roles in controlling or improving the development and outcome of solid tumors like HCC. Hence, this study aimed to investigate whether immune system changes were related to disease development in HCC patients. Methods: Peripheral blood mononuclear cells were isolated from 30 HCC patients and 30 healthy volunteers using Ficoll density centrifugation. The isolated cells were stained with different primary antibodies and percentages of different immune cells were determined by flow cytometry. Results: HCC patients indicated significant reductions in the numbers of CD4+ cells, Tbet+IFNγ+cells, and GATA+IL-4+cells in peripheral blood in comparison with healthy individuals (p < 0.05). There was no significant change in IL-17+RORγt+cells between patient and healthy groups. In contrast, Foxp3+CD127lowcell frequency was significantly higher in patients than healthy subjects (p < 0.0001). The numbers of Th1, Th2, and Th17 cells were significantly lower in HCC patients than healthy control (p < 0.0001), although the reduction in Th2 cell numbers was not statistically significant. On the contrary, Treg percentage showed a significant increase in patients compared to healthy subjects (p < 0.0001). Other data revealed that Th1, Th2, and Th17 cell frequencies were significantly higher in healthy individuals than patients with different TNM stages of HCC, with the exception of Th2 in patients with stage II HCC (p < 0.01-0.05). Treg percentage was significantly increased in patients with different TNM stages (p < 0.0001). Among all CD4+ T cells, the frequency of Th2 cell was significantly associated with TNM stages of HCC (p < 0.05). Conclusion: Our data provide further evidence to show that immune changes may participate in determining HCC progression and disease outcome. However, it should be mentioned that more investigations are needed to clarify our results and explain possible impacts of other immune cells on the pathogenesis of HCC.

Hepatic stellate cell activation by TGFß induces hedgehog signaling and endoplasmic reticulum stress simultaneously.

Activation of hepatic stellates (HSCs) is known as the major cause of initiation and progression of liver fibrosis. A wide array of events occurs during HSC activation including induction of hedgehog (Hh) signaling and endoplasmic reticulum (ER) stress. Targeting HSC activation may provide promising insights into liver fibrosis treatment. In this regard, establishing in vitro models which can mimic the molecular pathways of interest is very important. We aimed to activate HSC in which Hh signaling and ER stress are stimulated simultaneously. We used 5 ng/ml TGFß to activate LX-2 cells, HSC cell line. Gene expression analysis using qRT-PCR, immunostaining and immunoblotting were performed to show HSC activation associated markers. Furthermore, the migration capacity of the TGFß treated cells is evaluated. The results demonstrated that major fibrogenic markers including collagen1a, lysyl oxidase, and tissue inhibitor of matrix metalloproteinase 1 genes are up-regulated significantly. In addition, our immunofluorescence and immunoblotting results showed that protein levels of GLI-2 and XBP1, were enhanced. Moreover, we found that TGFß treatment reduced the migration of LX-2 cells. Our results are compatible with high throughput data analysis with respect to differentially expressed genes of activated HSC compared to the quiescent ones. Moreover, our findings suggest that quercetin can reduce fibrogenic markers of activated HSCs as well as osteopontin expression, a target gene of hedgehog signaling.

Metabolic hallmarks of liver regeneration.

Despite the crucial role of cell metabolism in biological processes, particularly cell division, metabolic aspects of liver regeneration are not well defined. Better understanding of the metabolic activity governing division of liver cells will provide powerful insights into mechanisms of physiological and pathological liver regeneration. Recent studies have provided evidence that metabolic response to liver failure might be a proximal signal to initiate cell proliferation in liver regeneration. In this review, we highlight how lipids, carbohydrates, and proteins dynamically change and orchestrate liver regeneration. In addition, we discuss translational studies in which metabolic intervention has been used to treat chronic liver diseases (CLDs).

Molecular detection of Trichostrongylus species through PCR followed by high resolution melt analysis of ITS-2 rDNA sequences.

Polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis is a simple, rapid and accurate method for molecular detection of various nematode species. The objective of the present study was, for the first time, to develop a PCR-HRM assay for the detection of various animal Trichostrongylus spp. A pair of primers targeting the ITS-2 rDNA region of the Trichostrongylus spp. was designed for the development of the HRM assay. DNA samples were extracted from 30 adult worms of Trichostrongylus spp., the ITS-2-rDNA region was amplified using PCR, and the resultant products were sequenced and characterized. Afterwards, the PCR-HRM analysis was conducted to detect and discriminate Trichostrongylus spp. Molecular sequence analysis revealed that 24, 4, and 1 of the samples were T. colubriformis, T. vitrinus and T. capricola, respectively. Results from PCR-HRM indicated that complete agreement was relatively found between speciation by HRM analysis and DNA sequencing for the detection of Trichostrongylus species. The PCR-HRM analysis method developed in the present study is fast and low-cost; the method can be comparable with other molecular detection techniques, representing a reliable tool for the identification of various species within the Trichostrongylus genus.

Potential drugs used in the antibody-drug conjugate (ADC) architecture for cancer therapy.

Cytotoxic small-molecule drugs have a major influence on the fate of antibody-drug conjugates (ADCs). An ideal cytotoxic agent should be highly potent, remain stable while linked to ADCs, kill the targeted tumor cell upon internalization and release from the ADCs, and maintain its activity in multidrug-resistant tumor cells. Lessons learned from successful and failed experiences in ADC development resulted in remarkable progress in the discovery and development of novel highly potent small molecules. A better understanding of such small-molecule drugs is important for development of effective ADCs. The present review discusses requirements making a payload appropriate for antitumor ADCs and focuses on the main characteristics of commonly-used cytotoxic payloads that showed acceptable results in clinical trials. In addition, the present study represents emerging trends and recent advances of payloads used in ADCs currently under clinical trials.

Adeno-associated virus as a gene therapy vector: strategies to neutralize the neutralizing antibodies.

Adeno-associated virus (AAV)-derived vectors are currently the most common type of viral vectors used in gene therapy clinical trials. The presence of neutralizing antibodies (NAbs) against wild-type AAVs in the host body is one of the limitations for the successful use of AAV vectors. AAV capsid manipulation, by which recombinant vectors lose their ability to interact with NAbs, can help overcome this obstacle. Various methods can be used for this purpose, including directed evolution as well as conjugation of certain chemical groups to AAV epitopes. The present review concisely explains the use of AAV vectors in the clinic for gene therapy of some diseases, their limitations, and solutions to these limitations.

Enrichment of cancer stem-like cells by the induction of epithelial-mesenchymal transition using lentiviral vector carrying E-cadherin shRNA in HT29 cell line.

A better understanding of cancer stem cells (CSCs) may facilitate the prevention and treatment of cancers. Epithelial-mesenchymal transition (EMT) is a process activated during invasion and metastasis of tumors. EMT induction in normal and tumor cells makes them more resistant to chemotherapy. E-cadherin is a membrane protein and plays a role in tumor invasion, metastasis, and prognosis. Downregulation of E-cadherin is a hallmark of EMT. Here, we created a model of cancer stem-like cells enrichment via EMT induction using E-cadherin downregulation in HT29 cell line using a lentiviral vector carrying shRNA. We aimed to evaluate cancer and anti-CSC chemotherapeutics screening. The markers of EMT and CSCs were assessed and compared with control cells using flow cytometry, real-time PCR, immunocytochemistry, western blot, migration assay, invasion assay, and colony formation assay. The transduced cells showed a mesenchymal morphology. High levels of EMT-related proteins were also expressed. These results confirmed that the transduced cells underwent EMT. In addition, we observed an increased population of E-cadherin-downregulated HT29 cell line among the cells expressing colon CSC markers (CD133+ and CD44+ ) after EMT induction. E-cadherin-downregulated cells were morphologically like mesenchymal cells, and the number of CD133+ - and CD44+ -cells (CSC-like cells) increased. These cells can be used as stable models to study cancer cells and screening of antitumor therapeutics.

Antibody-drug conjugates (ADCs) for cancer therapy: Strategies, challenges, and successes.

Targeted delivery of therapeutic molecules into cancer cells is considered as a promising strategy to tackle cancer. Antibody-drug conjugates (ADCs), in which a monoclonal antibody (mAb) is conjugated to biologically active drugs through chemical linkers, have emerged as a promising class of anticancer treatment agents, being one of the fastest growing fields in cancer therapy. The failure of early ADCs led researchers to explore strategies to develop more effective and improved ADCs with lower levels of unconjugated mAbs and more-stable linkers between the drug and the antibody, which show improved pharmacokinetic properties, therapeutic indexes, and safety profiles. Such improvements resulted in the US Food and Drug Administration approvals of brentuximab vedotin, trastuzumab emtansine, and, more recently, inotuzumab ozogamicin. In addition, recent clinical outcomes have sparked additional interest, which leads to the dramatically increased number of ADCs in clinical development. The present review explores ADCs, their main characteristics, and new research developments, as well as discusses strategies for the selection of the most appropriate target antigens, mAbs, cytotoxic drugs, linkers, and conjugation chemistries.

Trastuzumab-monomethyl auristatin E conjugate exhibits potent cytotoxic activity in vitro against HER2-positive human breast cancer.

Targeted therapy using specific monoclonal antibodies (mAbs) conjugated to chemotherapeutic agents or toxins has become one of the top priorities in cancer therapy. Antibody-drug conjugates (ADCs) are emerging as a promising strategy for cancer-targeted therapy. In this study, trastuzumab, a humanized monoclonal anti-HER2 antibody, was reduced by dithiothreitol and conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a valine-citrulline peptide linker (trastuzumab-MC-Val-Cit-PABC-MMAE [trastuzumab-vcMMAE]). After conjugation, ADCs were characterized by using UV-vis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and flow cytometry. The antitumor activity of the ADC was evaluated in breast cancer cells in vitro. In addition, ADCs were further characterized using purification by the protein A chromatography, followed by assessment using apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. Hydrophobic interaction chromatography was used to determine drug-to-antibody ratio species of ADCs produced. Our finding showed that approximately 5.12 drug molecules were conjugated to each mAb. H2L2, H2L, HL, H2, H, and L forms of ADCs were detected in nonreducing SDS-PAGE. The binding of trastuzumab-vcMMAE to HER2-positive cells was comparable with that of the parental mAb. The MTT assay showed that our ADCs induced significant cell death in HER2-positive cells, but not in HER2-negative cells. The ADCs produced was a mixture of species, unconjugated trastuzumab (14.147%), as well as trastuzumab conjugated with two (44.868%), four (16.886%), six (13.238%), and eight (10.861%) molecules of MMAE. These results indicated that MMAE-conjugated trastuzumab significantly increases the cytotoxic activity of trastuzumab, demonstrating high affinity, specificity, and antitumor activity in vitro. Trastuzumab-vcMMAE is an effective and selective agent for the treatment of HER2-positive breast tumors.

A developed antibody-drug conjugate rituximab-vcMMAE shows a potent cytotoxic activity against CD20-positive cell line.

Rituximab is a chimeric monoclonal antibody directed against B-lymphocyte specific antigen CD20, which is used for the treatment of B-cell malignancies. However, the effectiveness of rituximab is limited partly due to treatment resistance. The aim of this study was to develop rituximab-based antibody drug conjugate (ADC) to enhance rituximab activity. In this study, monomethyl auristatin E (MMAE) was covalently conjugated to dithiothreitol -reduced rituximab via a valine-citrulline peptide linker (rituximab-vcMMAE). The conjugates were then characterized by using nonreducing sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and cell-based enzyme-linked immunosorbent assay (ELISA). The cytotoxic activity of the ADC was evaluated against Raji (human B-cell lymphoma; CD20-positive) and MOLT-4 (T lymphoblast; acute lymphoblastic leukemia; CD20-negative) cell lines. In addition, the colony formation assay was used to identify the propagation ability of ADC-treated cells in vitro. Results from nonreducing SDS-PAGE revealed various species of rituximab-MC-Val-Cit-PABC-MMAE (rituximab-vcMMAE), as compared with unconjugated rituximab. The binding capacity of rituximab-vcMMAE to the CD20-positive cell was similar to that of the parental rituximab. Most importantly, our results revealed that rituximab-vcMMAE was highly potent against the CD20-positive cell line, but not against the CD20-negative cell. At the same time, rituximab-vcMMAE was able to inhibit colony formation in CD20-positive cells. These data indicate that rituximab-vcMMAE may be a highly effective and selective therapy for the treatment of B-cell lymphoma.

Secretome of Aggregated Embryonic Stem Cell-Derived Mesenchymal Stem Cell Modulates the Release of Inflammatory Factors in Lipopolysaccharide-Induced Peripheral Blood Mononuclear Cells

Background: Bone marrow mesenchymal stem cells (BM-MSCs) have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs (ESC-MSCs) as an alternative for BM-MSCs. Some of the therapeutic effects of the ESC-MSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome. Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs (hESC-MSCs) spheroids on secretion of IL-1ß, IL-10, and tumor necrosis factor α (TNF-α) from lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells (PBMCs). Methods: In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were non-adherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation. Results: Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-α (301.7 ± 5.906, p < 0.0001) and IL-1ß (485.2 ± 48.38, p < 0.001) from LPS-induced PBMCs as the indicators of inflammation, in comparison with adherent culture-prepared secretome (TNF-α: 166.6 ± 8.04, IL-1ß: 125.2 ± 2.73). Conclusion: Our study indicated that cell aggregation can be an appropriate strategy to increase immunomodulatory characteristics of hESC-MSCs.

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome.

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1ß were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1ß from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.

Myc Decoy Oligodeoxynucleotide Inhibits Growth and Modulates Differentiation of Mouse Embryonic Stem Cells as a Model of Cancer Stem Cells.

BACKGROUND: Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells. OBJECTIVE: In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs. METHODS: A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting. RESULTS: EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium. CONCLUSION: The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations.

Evaluation of Factors Influencing Antibody Reduction for Development of Antibody Drug Conjugates

Background: Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates (ADCs). Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates. Methods: In the present study, we investigated the effect of various dithiothreitol (DTT) concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively. Results: DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively. Conclusion: Optimized site-specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs.

Pluripotency Crossroads: Junction of Transcription Factors, Epigenetic Mechanisms, MicroRNAs, and Long Non-coding RNAs.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) and have the potency to differentiate into three germ layers (ectoderm, endoderm, and mesoderm). This potency of ESCs, called pluripotency, is critical for maintaining stemness. Transcriptional regulatory circuitry preserving stemness consists of transcription factors (TFs), epigenetic mechanisms, microRNAs (miRNAs or miRs), and long non-coding RNAs (lncRNAs). In this circuitry, components assist each other to activate essential genes for maintaining pluripotency and suppressing lineage-specific genes. TFs act directly by binding to their binding sites in the genome or indirectly by activating another gene (such as a miR), epigenetic mechanisms play their role by providing an activatory or inhibitory context for transcription, miRNAs regulate gene expression at the post-transcriptional level, and lncRNAs act as a scaffold function for epigenetic elements, regulating gene expression in ESCs. All these factors create a crossroad and collaborate to sustain stemness in the ESCs. Herein, we explain the role of each member in this circuitry and demonstrate the significance of the crossroad for keeping stemness.

Hypoxia Pre-Conditioned Embryonic Mesenchymal Stem Cell Secretome Reduces IL-10 Production by Peripheral Blood Mononuclear Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells (ESCs) and bone marrow cells after hypoxia and normoxia preconditioning. METHODS: ESCs differentiated into MSCs and characterized by flow cytometry and differentiation into adipocytes and osteoblasts. The experimental groups consisted of individual groups of ESC-MSCs and BM-MSCs (bone marrow-derived mesenchymal stromal cells), which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell (PBMC) assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs. RESULTS: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium. CONCLUSIONS: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BM-MSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor α, which needs further investigation.