Development of an injectable chitosan-based hydrogel containing nano-hydroxy-apatite and alendronate for MSC-based therapy.

BACKGROUND: The combination of cells and biomaterials has become a powerful approach to regenerative medicine in recent years. Understanding the in-vitro interactions between cells and biomaterials is crucial for the success of regenerative medicine. AIM: In this study, we developed an AD-pectin/chitosan/nano-crystalline cellulose scaffold with nano-hydroxy-apatite (n-HAP) and alendronate (ALN). The second step was to evaluate its effect on the immunomodulatory properties and biological behaviors of seeded adipose-derived mesenchymal stem cells (ADSCs) for bone tissue repair. MATERIAL AND METHOD: After preparing and evaluating the characterization tests of the new combined n-HAP scaffold, we established different culture conditions to evaluate ADSC growth on this scaffold with or without ALN. The main assays were MTT assay, RT-PCR, and ELISA. RESULTS: Our data regarding characterization tests (including SEM, TGA, FTIR, gelation time, swelling ratio, rheology and degradation tests) of ALN-loaded n-HAP scaffold showed the proper stability and good mechanical status of the scaffold. ADSC proliferation and viability increased in the presence of the scaffold compared with other conditions. Moreover, our data demonstrated increased gene expression and protein levels of anti-inflammatory TGF-ß, HGF, and IDO cytokines in the presence of the ALN-loaded n-HAP scaffold, indicating the increased immunosuppressive activity of ADSCs in vitro. CONCLUSION: This study demonstrates the promising abilities of the ALN-loaded n-HAP scaffold to increase the proliferation, viability, and immunomodulatory capacity of ADSCs, elucidating new aspects of cell-material interactions that can be used for bone tissue regeneration/repair, and paving the path of future research in developing new approaches for MSC- based therapy.

STA-21, a small molecule STAT3 inhibitor, ameliorates experimental autoimmune encephalomyelitis by altering Th-17/Treg balance.

BACKGROUND: Numerous studies have demonstrated the role of T helper (Th) 17 and T regulatory (reg) cells and pro-inflammatory and anti-inflammatory cytokines related to these cells in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). STAT3 is one of the downstream signaling proteins of IL-23, IL-6, and IL-21 that are required for Th17 cells differentiation. STA-21 is a STAT3 inhibitor that functions by inhibiting STAT3 dimerization and binding to DNA impairing the expression of STAT3 target genes including, RORγt, IL-21 and IL-23R that are also required for Th17 cell differentiation. AIM: In this study, we evaluated the effect of STA-21 on EAE Model and investigated how this small molecule can change Th17/Treg balance leading to amelioration of disease. METHODS: After EAE induction and treatment with STA-21, its effects were assessed. Major assays were H&E and LFB staining, Flow cytometric analysis, Reverse transcription-PCR (RT-PCR), and ELISA. RESULTS: STA-21 ameliorated the EAE severity and decreased the EAE inflammation and demyelination. It also decreased STAT3 phosphorylation, the proportion of Th17 cells and the protein level of IL-17. In contrast, the balance of Tregs and the level of anti-inflammatory cytokine, IL-10 increased in STA-21-treated mice. Moreover, STA-21 significantly decreased the expression of Th17 related transcription factors, RORɣt and IL-23R while FOXP3 expression associated with Treg differentiation was increased. CONCLUSION: This study showed that STA-21 has therapeutic effects in EAE by reducing inflammation and shifting inflammatory immune responses to anti-inflammatory and can be used as a suitable treatment strategy for the treatment of EAE. The effectiveness of inhibiting or strengthening the functional cells of the immune system by these small molecules in terms of easy to access, simple construction and inexpensive expansion make them as a suitable tool for the treatment of inflammatory and autoimmune diseases.

Clinical perspective: Antibody-drug conjugates for the treatment of HER2-positive breast cancer.

Antibody-drug conjugates (ADCs) are a promising class of cancer biopharmaceuticals that exploit the specificity of a monoclonal antibody (mAb) to selectively deliver highly cytotoxic small molecules to targeted cancer cells, leading to an enhanced therapeutic index through increased antitumor activity and decreased off-target toxicity. ADCs hold great promise for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer after the approval and tremendous success of trastuzumab emtansine and trastuzumab deruxtecan, representing a turning point in both HER2-positive breast cancer treatment and ADC technology. Additionally and importantly, a total of 29 ADC candidates are now being investigated in different stages of clinical development for the treatment of HER2-positive breast cancer. The purpose of this review is to provide an insight into the ADC field in cancer treatment and present a comprehensive overview of ADCs approved or under clinical investigation for the treatment of HER2-positive breast cancer.

Under hypoxic conditions, MSCs affect the expression and methylation level of survival-related genes in ALL independent of apoptosis pathways in vitro.

Mesenchymal stem cells (MSCs) are one of the most prominent cells in the bone marrow. MSCs can affect acute lymphocytic leukemia (ALL) cells under hypoxic conditions. With this aim, we used MOLT-4 cells as simulators of ALL cells cocultured with bone marrow mesenchymal stem cells (BMMSCs) under hypoxic conditions in vitro. Then, mRNA and protein expression of the MAT2A, PDK1, and HK2 genes were evaluated by real-time PCR and Western blot which was also followed by apoptosis measurement by a flow-cytometric method. Next, the methylation status of the target genes was investigated by MS-qPCR. Additionally, candidate gene expressions were examined after treatment with rapamycin using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We found that the mRNA expression of the candidate genes was augmented under the hypoxic condition in which MAT2A was upregulated in cocultured cells compared to MOLT-4, while HK2 and PDK1 were downregulated. Moreover, we found an association between gene expression and promoter methylation levels of target genes. Besides, expressions of the candidate genes were decreased, while their methylation levels were promoted following treatment with rapamycin. Our results suggest an important role for the BMMSC in regulating the methylation of genes involved in cell survival in hypoxia conditions; however, we found no evidence to prove the MSCs' effect on directing malignant lymphoblastic cells to apoptosis.

Ruxolitinib attenuates experimental autoimmune encephalomyelitis (EAE) development as animal models of multiple sclerosis (MS).

AIMS: STAT3 signaling is critical for Th17 development that plays an important role in multiple sclerosis pathogenesis. To evaluate the anti-inflammatory and regulatory T cells effects of JAK1/2 and STAT3 inhibition, we assessed the JAK 1/2 inhibitor ruxolitinib effects on Th17 cell/Tregs balance. MAIN METHODS: Ruxolitinib was administered to experimental autoimmune encephalomyelitis (EAE) mice via oral gavage, and its effects were assessed. The expression of pro-inflammatory and anti-inflammatory cytokines, including IL-17A and IL-10, were analyzed by real-time PCR. The frequency of Th17 cells and Tregs were evaluated by flow cytometry. KEY FINDING: Ruxolitinib ameliorated the EAE severity and decreased the proportion of Th17 cells and inflammatory markers levels. In contrast, the balance of Tregs and the level of anti-inflammatory cytokine were increased in ruxolitinib-treated mice. Furthermore, ruxolitinib markedly decreased the expression of Th17 related transcription factor, RORÉ£t, whereas FOXP3 expression associated with Treg differentiation was increased. SIGNIFICANCE: Our results show that ruxolitinib may be a promising therapeutic strategy for multiple sclerosis.

COVID-19: Our Current Knowledge of Epidemiology, Pathology, Therapeutic Approaches, and Diagnostic Methods.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) firstly emerged in Wuhan, China at the end of 2019. After going through the experimental process, the virus was named the novel coronavirus (2019-nCoV) by the World Health Organization (WHO) in February 2020 which has created a global pandemic. The coronavirus disease 2019 (COVID-19) infection is challenging the people who are especially suffering from chronic health problems such as asthma, diabetes, and heart disease or immune system deteriorating disorders, including cancers, Alzheimer's, etc. Other predisposing/risk factors consist of smoking and age (elderly people are at higher risk). The 2019-nCoV attacks epithelial cells in all organs, particularly epithelial cells in the lungs, resulting in viral pneumonia. The 2019-nCoV starts its invasion with the attachment and entry into the respiratory tract epithelial cells via Angiotensin-Converting Enzyme 2 (ACE2) receptors on the epithelial cells. The critical problem with 2019-nCoV is its ability in human to human asymptomatic transmission which causes the rapid and hidden spread of the virus among the population. Also, there are several reports of highly variable and tightly case-dependent clinical manifestations caused by SARS-CoV2, which made the virus more enigmatic. The clinical symptoms are varied from common manifestations which occurred in flu and cold, such as cough, fever, body-ache, trembling, and runny nose to severe conditions, like the Acute Respiratory Distress Syndrome (ARDS) or even uncommon/unusual symptoms such as anosmia, skin color change, and stroke. In fact, besides serious injuries in the respiratory system, COVID-19 invades and damages various organs, including the kidney, liver, gastrointestinal, and nervous system. Accordingly, to cut the transmission chain of disease and control the infection spread. One of the major solutions seems to be early detection of the carriers, particularly the asymptomatic people, with sensitive and accurate diagnostic techniques. Moreover, developing novel and appropriate therapeutic approaches will contribute to the suitable management of the pandemic. Therefore, there is an urgent necessity to make comprehensive investigations and study reviews about COVID-19, offering the latest findings of novel therapies, drugs, epidemiology, and routes of virus transmission and pathogenesis. In this review, we discuss new therapeutic outcomes and cover and the most significant aspects of COVID-19, including the epidemiology, biological features, organs failure, and diagnostic techniques.

Development and biological assessment of MMAE-trastuzumab antibody-drug conjugates (ADCs).

BACKGROUND: Trastuzumab, a humanized monoclonal antibody targeting Human Epidermal growth factor Receptor 2 (HER2), is a therapeutic option used for the treatment of patients with HER2-overexpressing breast cancers. The primary purpose of the present study was to establish a trastuzumab-based antibody drug conjugate (ADC) to enhance the biopharmaceutical profile of trastuzumab. METHODS: In this study, trastuzumab was linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) through a peptide linker. Following conjugation, MMAE-trastuzumab ADCs were characterized using SDS-PAGE, UV/VIS, and cell-based ELISA. The inhibitory effects of the ADCs were measured on MDA-MB-453 (HER2-positive cells) and HEK-293 (HER2-negative cells) using in vitro cell cytotoxicity and colony formation assays. RESULTS: Our findings showed that approximately 3.4 MMAE payloads were conjugated to trastuzumab. MMAE-trastuzumab ADCs produced six bands, including H2L2, H2L, HL, H2, H, and L in non-reducing SDS-PAGE. The conjugates exhibited the same binding ability to MDA-MB-453 as unconjugated trastuzumab. The MTT assay showed a significant improvement in the trastuzumab activity following MMAE conjugation, representing a higher antitumor activity as compared with unconjugated trastuzumab. Furthermore, ADCs were capable of potentially inhibiting colony formation in HER2-positive cells, as compared with trastuzumab. CONCLUSION: MMAE-trastuzumab ADCs represent a promising therapeutic strategy to treat HER2-positive breast cancer.

Epigenetic mechanisms shape the underlining expression regulatory mechanisms of the STAT3 in multiple sclerosis disease.

OBJECTIVES: Immunological tolerance is mediated by CD4+CD25+ regulatory T (Treg) cells. Studies have shown that thymic and peripheral generations of Treg cells depend on the CD28 signaling pathway. T helper 17 (Th17) cells are involved in the pathophysiology of various inflammatory diseases. Cytokines, such as interleukin (IL)-6 and TGF-ß, regulate the reciprocal development of Th17 and Treg cells. In CD4+ T cells, signal transducer and activator of transcription 3 (STAT3) play a critical role in the induction of Th17 cell differentiation and inhibition of Treg cell development. RESULTS: In this study, we investigated the STAT3 methylation and gene expression status in patients with MS. Our study demonstrated that the level of STAT3 methylation decreased in relapsing-remitting MS patient compared to control groups, which the decreases were statistically significant. STAT3 gene expression increased in patient group relative to healthy one, and the increases were found to be statistically significant. According to our findings, it can be suggested that DNA hypermethylation of STAT3 affects the gene expression. In addition, there is a strong and significant negative correlation between the methylation status and mRNA level of STAT3.

Innate and adaptive immune responses against coronavirus.

Coronaviruses (CoVs) are a member of the Coronaviridae family with positive-sense single- stranded RNA. In recent years, the CoVs have become a global problem to public health. The immune responses (innate and adaptive immunity) are essential for elimination and clearance of CoVs infections, however, uncontrolled immune responses can result in aggravating acute lung injury and significant immunopathology. Gaining profound understanding about the interaction between CoVs and the innate and adaptive immune systems could be a critical step in the field of treatment. In this review, we present an update on the host innate and adaptive immune responses against SARS-CoV, MERS-CoV and newly appeared SARS-CoV-2.

ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders.

OBJECTIVE: Zoledronic Acid (ZA) is one of the common treatment choices used in various boneassociated conditions. Also, many studies have investigated the effect of ZA on Osteoblastic-Differentiation (OSD) of Mesenchymal Stem Cells (MSCs), but its clear molecular mechanism(s) has remained to be understood. It seems that the methylation of the promoter region of key genes might be an important factor involved in the regulation of genes responsible for OSD. The present study aimed to evaluate the changes in the mRNA expression and promoter methylation of central Transcription Factors (TFs) during OSD of MSCs under treatment with ZA. MATERIALS AND METHODS: MSCs were induced to be differentiated into the osteoblastic cell lineage using routine protocols. MSCs received ZA during OSD and then the methylation and mRNA expression levels of target genes were measured by Methylation Specific-quantitative Polymerase Chain Reaction (MS-qPCR) and real-time PCR, respectively. The osteoblastic differentiation was confirmed by Alizarin Red Staining and the related markers to this stage. RESULTS: Gene expression and promoter methylation level for DLX3, FRA1, ATF4, MSX2, C/EBPζ, and C/EBPa were up or down-regulated in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21. ATF4, DLX3, and FRA1 genes were significantly up-regulated during the OSD processes, while the result for MSX2, C/EBPζ, and C/EBPa was reverse. On the other hand, ATF4 and DLX3 methylation levels gradually reduced in both ZA-treated and untreated cells during the osteodifferentiation process on days 0 to 21, while the pattern was increasing for MSX2 and C/EBPa. The methylation pattern of C/EBPζ was upward in untreated groups while it had a downward pattern in ZA-treated groups at the same scheduled time. The result for FRA1 was not significant in both groups at the same scheduled time (days 0-21). CONCLUSION: The results indicated that promoter-hypomethylation of ATF4, DLX3, and FRA1 genes might be one of the mechanism(s) controlling their gene expression. Moreover, we found that promoter-hypermethylation led to the down-regulation of MSX2, C/EBP-ζ and C/EBP-α. The results implicate that ATF4, DLX3 and FRA1 may act as inducers of OSD while MSX2, C/EBP-ζ and C/EBP-α could act as the inhibitor ones. We also determined that promoter-methylation is an important process in the regulation of OSD. However, yet there was no significant difference in the promoter-methylation level of selected TFs in ZA-treated and control cells, a methylation- independent pathway might be involved in the regulation of target genes during OSD of MSCs.

Targeting STAT3 in cancer and autoimmune diseases.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated downstream of a broad range of receptors particularly interleukin-6 (IL-6) family. STAT3 is the key regulator of cell proliferation, survival and apoptosis and is constitutively activated in most human cancers, indicating that it can be an important potential therapeutic target for cancer treatment. STAT3 also has important roles in lymphocyte biology, regulation of immune responses and autoimmunity. Considering the vital role of STAT3 in tumor progression and autoimmunity, scientists have focused to develop small molecules that suppress STAT3 function. In this review, we firstly discussed the predominant role of STAT3 in cancer and autoimmune diseases. Subsequently, we discussed the efficacy and therapeutic potential of different STAT3 inhibitors in cancer and autoimmune diseases in preclinical studies and clinical trials offering an insight into novel approaches for development of new STAT3 inhibitors.

The role of B cells in the immunopathogenesis of multiple sclerosis.

There is ongoing debate on how B cells contribute to the pathogenesis of multiple sclerosis (MS). The success of B-cell targeting therapies in MS highlighted the role of B cells, particularly the antibody-independent functions of these cells such as antigen presentation to T cells and modulation of the function of T cells and myeloid cells by secreting pathogenic and/or protective cytokines in the central nervous system. Here, we discuss the role of different antibody-dependent and antibody-independent functions of B cells in MS disease activity and progression proposing new therapeutic strategies for the optimization of B-cell targeting treatments.

CTLA-4: From mechanism to autoimmune therapy.

CD28 and CTLA-4 are both important stimulatory receptors for the regulation of T cell activation. Because receptors share common ligands, B7.1 and B7.2, the expression and biological function of CTLA-4 is important for the negative regulation of T cell responses. Therefore, elimination of CTLA-4 can result in the breakdown of immune tolerance and the development of several diseases such as autoimmunity. Inhibitory signals of CTLA-4 suppress T cell responses and protect against autoimmune diseases in many ways. In this review, we summarize the structure, expression and signaling pathway of CTLA-4. We also highlight how CTLA-4 defends against potentially self-reactive T cells. Finally, we discuss how the CTLA-4 regulates a number of autoimmune diseases that indicate manipulation of this inhibitory molecule is a promise as a strategy for the immunotherapy of autoimmune diseases.

Janus kinase inhibitors: A therapeutic strategy for cancer and autoimmune diseases.

Many cytokines are crucial drivers of cancers and autoimmune conditions. These proteins bind to receptors and signal their responses through Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathways. Genetic variations in the JAK-STAT pathway are correlated with the increased risk of cancers, autoimmunity as well as inflammatory diseases. Targeting JAKs and STATs can be a safe and efficacious strategy for treating these diseases. Tofacitinib, as the first JAK inhibitor, is approved for rheumatoid arthritis therapy. Also, many other JAK inhibitors have been proven or are in various phases of clinical trials for various diseases. At present, small-molecule JAK inhibitors are considered as a novel category of drugs in the treatment of cancer and immune-mediated diseases.

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.

Mesenchymal stem cells as carrier of the therapeutic agent in the gene therapy of blood disorders.

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.

IL-21 and IL-21-producing T cells are involved in multiple sclerosis severity and progression.

Multiple sclerosis is a common neuroinflammatory disease of the central nervous system causing nervous system defects and severe physical disability. IL-21 is a proinflammatory cytokine produced mainly by Th-17 and Tfh cells which its exact role in MS was not yet clearly understood. In the present study we aimed to investigate the possible correlation of IL-21 gene expression, methylation, and its serum levels with MS severity and progression. The results showed that IL-21 mRNA level and serum level were significantly increased in patient group compared with control group (p = 0.02 and p < 0.0001 respectively). Moreover, we found a strong positive correlation between IL-21 mRNA levels and EDSS scores (r = 0.637, P < 0.0001), IL-21 mRNA levels and Progression Index (r = 0.540, P < 0.0001), IL-21 serum levels and EDSS scores (r = 0.617, P < 0.0001), and IL-21 serum levels and Progression Index (r = 0.527, P < 0.0001) in MS patients. Additionally, we found that the methylation level of IL-21 promoter region was decreased in patient group compared with the control group (p < 0.0001). We also found that methylation level of IL-21 gene promoter is negatively correlated with the IL-21 mRNA level (r = -0.263, p = 0.02), serum level (r = -0.249, p = 0.03), EDSS scores (r = -0.276, p = 0.01) and Progression Index (r = -0.430, p = 0.0001). Data showed that the increased percentages of IL-21-producing Tfh-like, Th-17 and Th1 cells in patients are positively correlated with MS severity and progression. The results of our study suggest a pro-inflammatory and booster role for IL-21 in the MS pathogenesis and progression.

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.