Arsenic trioxide-induced cytotoxicity in A549 cells: The role of necroptosis.

INTRODUCTION: Lung cancer is one of the deadliest cancers globally. Arsenic trioxide (ATO) is still present as a highly effective drug in treating acute promyelocytic leukemia (APL). Chemotherapy resistance is one of the major problems in cancer therapy. Necroptosis, can overcomes resistance to apoptosis, and can promote cancer treatment. This study examines the necroptosis pathway in A549 cancer cells exposed to ATO. METHODS: We used the MTT test to determine the ATO effects on the viability of A549 cells at three different time intervals. Also, the reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were performed in three-time intervals. The effect of ATO on apoptosis was evaluated by Annexin V / PI staining and, the RIPK1 and MLKL gene expression were measured by Real-Time PCR. RESULTS: The ATO has dose and time-dependent cytotoxic effects, so at 24, 48, and 72 h, the IC50 doses were 33.81 '11.44 '2.535 µM respectively. A 50 µM ATO is the most appropriate to increase the MMP loss significantly at all three times. At 24 and 48 h after exposure of cells to ATO, the ROS levels increased. The RIPK1 gene expression increased significantly compared to the control group at concentrations of 50 and 100 µM; however, MLKL gene expression decreased. CONCLUSIONS: The A549 cells, after 48 h exposure to ATO at 50 and 100 µM, induces apoptosis and necroptosis. Due to the reduced expression of MLKL, it can be concluded that ATO is probably effective in the metastatic stage of cancer cells.

Targeting the tumor microenvironment by liposomal Epacadostat in combination with liposomal gp100 vaccine.

Indoleamine-2,3-dioxygenase (IDO1) pathway has vital role in cancer immune escape and its upregulation leads to immunosuppressive environment which is associated with poor prognosis and progression in various cancers like melanoma. Previously, we showed the antitumoral efficacy of nanoliposomal form of Epacadostat (Lip-EPA), as an IDO1 inhibitor. Herein, we used Lip-EPA as a combination approach with liposomal gp100 (Lip-gp100) anti-cancer vaccine in melanoma model. Here, we showed that B16F10 tumor express IDO1 so using Lip-EPA will enhance the efficacy of vaccine therapy. The biodistribution of ICG-labelled liposomal form of EPA showed the remarkable accumulation of drug at tumor site. In an in vivo study, Lip-EPA enhanced the antitumor efficacy of Lip-gp100 in which the IDO mRNA expression was decreased (~ fourfold) in tumor samples. Also, we identified a significant increase in the number of infiltrated T lymphocytes (p < 0.0001) with enhanced in interferon gamma (IFN-γ) production (p < 0.0001). Additionally, Lip-EPA + Lip-gp100 significantly modulated intratumoral regulatory T cells which altogether resulted in the highest delay in tumor growth (TGD = 56.54%) and increased life span (ILS > 47.36%) in treated mice. Our study demonstrated that novel combination of Lip-EPA and Lip-gp100 was effective treatment with capability of being used in further clinical studies.

In-vitro cytotoxicity and in-vivo antitumor activity of two platinum complexes with 1,3-dimethyl pentyl glycine ligand against breast cancer.

Platinum (Pt) derivatives are good candidates for discovering new anti-tumor agents. The present research aims to explore the in-vivo and in-vitro anticancer activity of two platinum complexes with 1,3-dimethyl pentyl glycine ligand (DMPG), [Pt(bpy)(13DMPG)]NO3 and [Pt(dach)(13DMPG)]NO3, against breast cancer cells. The present study was conducted to investigate the cytotoxic potential of these compounds (2-400 µM) compared to standard drugs (cisplatin, oxaliplatin, and carboplatin) on SKBR3 cells using the methyl thiazol-tetrazolium (MTT) assay. Furthermore, the gene expression changes of Bak, Bim, Bcl-2, Caspase-3, and Caspase-9 were carried out by real-time polymerase chain reaction (PCR), and flow cytometric analysis was performed to confirm the cell apoptosis in the presence of the compounds. For more validation, in-vivo anticancer activities of both compounds were investigated against breast transplanted tumors in the BALB/c mice model. The cytotoxic studies by MTT assay revealed the anti-proliferative potential of both derivatives. [Pt(dach)(13DMPG)]NO3 with an IC50 value of 15 µM, exhibited higher cytotoxicity against SKBR3 cells as compared to [Pt(bpy)(13DMPG)]NO3, oxaliplatin, and carboplatin. Based on the flow cytometry analysis, both derivatives demonstrated apoptotic effects. Also, real-time PCR analysis revealed an up-regulation of Bak, Bim, Bax, Caspases-3, and Caspase-9 genes and a significant reduction in Bcl-2 gene expression in treated cells with both compounds compared to the control group. In-vivo results validated in-vitro analysis and showed the anticancer activity of compounds against breast transplanted tumors in the BALB/c mice model. According to the results, [Pt(dach)(13DMPG)]NO3 displayed a significant anticancer activity.

Study of frequency and inheritance model of ACE1 I/D and ACE2 rs2285666 polymorphisms in COVID-19 patients with varying severity of lung involvement and its effect on serum cytokines levels.

The angiotensin-converting enzyme (ACE) has been shown to play a role as a receptor for the COVID-19 virus. This virus usually gets into cells and infects them by attaching to their glycoprotein receptors, which are found on the ACE2 receptor. The aim of this study was to evaluate the frequency and inheritance of ACE1 I/D and ACE2 rs2285666 polymorphisms in COVID-19 patients with varying severity of lung involvement and its effect on serum cytokines levels of interleukin (IL)-1 and IL-6 and laboratory parameters. One hundred eighty-five COVID-19 patients were grouped according to the severity of lung involvement. (I/D) polymorphism of the ACE1 gene and rs2285666 polymorphism of the ACE2 gene were determined by single specific primer-polymerase chain reaction and restriction fragment length reaction-polymerase chain reaction methods, respectively. Serum levels of IL-1 and IL-6 were also measured by the enzyme linked immunosorbent assay technique. No statistically significant association of ACE2 rs2285666 polymorphism genotypes and ACE1 I/D with the severity of lung involvement was noted. However, there was a statistically significant association between I/D ACE1 polymorphism genotypes and IL-6, white blood cells (WBC), and neutrophil-to-lymphocyte ratio (NLR) levels. Also, there was no statistically significant association between rs2285666 polymorphism genotypes and patients' blood oxygen saturation level, IL-6, IL-1ß, lactate dehydrogenase activity, WBC count, and NLR. In patients with COVID-19, the rs2285666 polymorphism of the ACE2 gene and the I/D polymorphism of the ACE1 gene were not significantly associated with the severity of COVID-19 disease and serum IL-6 and IL-1 cytokine levels.

CD73 downregulation by EGFR-targeted liposomal CD73 siRNA potentiates antitumor effect of liposomal doxorubicin in 4T1 tumor-bearing mice.

Blocking CD73 ectonucleotidase has been proposed as a potential therapeutic approach for cancer treatment. The present study aimed to investigate the antitumor effect of a novel EGFR-Targeted liposomal CD73 siRNA formulation in combination therapy with liposomal doxorubicin in the 4T1 mouse model. CD73 siRNA was encapsulated into nanoliposomes by the ethanol injection method. After preparation, characterization, morphology, and stability evaluation of formulations, the toxicity was measured by MTT assay. Uptake assay and efficiency of the liposomal formulations were investigated on the 4T1 cell line. The liposomal formulation containing CD73 siRNA was targeted with GE11 peptide for in vivo evaluations. Following biodistribution analysis, the antitumor activity of prepared formulations in combination with liposomal doxorubicin was studied in mice bearing 4T1 metastatic breast cancer cells. Finally, the induction of immune response of formulations in concomitant treatment with liposomal doxorubicin was evaluated in the tumor microenvironment of a mouse model of breast cancer. The size of prepared liposomal formulations at N/P = 16 for the liposomal CD73 siRNA and GE11-liposomal CD73 siRNA groups were 89 nm ± 4.4 and 95 nm ± 6.6, respectively. The nanoparticle's PDI was less than 0.3 and their surface charge was below 10 mV. The results demonstrated that N/P = 16 yielded the best encapsulation efficiency which was 94% ± 3.3. AFM results showed that the liposomes were spherical in shape and were less than 100 nm in size. The results of the MTT assay showed significant toxicity of the liposomes containing CD73 siRNA during the 48-h cell culture. Real-time PCR and flow cytometry results showed that liposomes containing CD73 siRNA could effectively downregulate CD73 expression. Liposomal formulations were able to significantly downregulate CD73 gene expression, in vivo. However, CD73 downregulation efficiency was significantly higher for the targeted form compared to the non-targeted formulation (P value < 0.01). The combination showed maximum tumor growth delay with remarkable survival improvement compared to the control group. Studying the immune responses in the treatment groups which received doxorubicin, showed decreased number of lymphocytes in the tumor environment. However, this decrease was lower in the combination therapy group. Finally, our results clearly showed that CD73 downregulation increases the activity of CD8+ lymphocytes (IFN-ℽ production) and also significantly decreases the Foxp3 in the CD25+ lymphocytes compared to the control group. GE11-Lipo CD73 siRNA formulation can efficiently knockdown CD73 ectonucleotidase. Also, the efficacy of liposomal doxorubicin is significantly enhanced via the downregulation of CD73 ectonucleotidase.

Enhanced antitumor immune response in melanoma tumor model by anti-PD-1 small interference RNA encapsulated in nanoliposomes.

Programmed cell death protein-1 (PD-1), as an immune checkpoint molecule, attenuates T-cell activity and induces T-cell exhaustion. Although siRNA has a great potential in cancer immunotherapy, its delivery to target cells is the main limitation of using siRNA. This study aimed to prepare a liposomal formulation as a siRNA carrier to silence PD-1 expression in T cells and investigate it's in vivo antitumor efficacy. The liposomal siRNA was prepared and characterized by size, zeta potential, and biodistribution. Following that, the uptake assay and mRNA silencing were evaluated in vitro at mRNA and protein levels. siRNA-PD-1 (siPD-1)-loaded liposome nanoparticles were injected into B16F0 tumor-bearing mice to evaluate tumor growth, tumor-infiltrating lymphocytes, and survival rate. Liposomal siPD-1 efficiently silenced PD-1 mRNA expression in T cells (P < 0.0001), and siPD-1-loaded liposomal nanoparticles enhanced the infiltration of T-helper 1 (Th 1) and cytotoxic T lymphocytes into the tumor tissue (P < 0.0001). Liposome-PD-1 siRNA monotherapy and PD-1 siRNA-Doxil (liposomal doxorubicin) combination therapy improved the survival significantly, compared to the control treatment (P < 0.001). Overall, these findings suggest that immunotherapy with siPD-1-loaded liposomes by enhancing T-cell-mediated antitumor immune responses could be considered as a promising strategy for the treatment of melanoma cancer.

Specific Clinical and Immunological Changes Following Mesenchymal Stem Cell Transplantation in COVID-19-induced Acute Respiratory Distress Syndrome Patients: A Phase-I Clinical Trial.

Acute respiratory distress syndrome (ARDS) is a systemic inflammation resulting from immune system overactivity. ARDS is also a fatal complication of COVID-19. Mesenchymal stem cells (MSCs) have immune modulatory properties. This study evaluated the safety and efficacy of three times transplantation of umbilical cord-derived MSCs (UC-MSCs) in terms of specific immunological and clinical changes in mild-to-moderate COVID-19-induced ARDS patients. In this single-center, open-label, phase 1 clinical trial, 20 patients diagnosed with COVID-19 and mild-to-moderate ARDS were included and were divided into two groups: a control group receiving standard care and an intervention group receiving UC-MSC in addition to standard care. Three consecutive intravenous transplants of UC-MSC (1×  cells/kg body weight per each transplant) were performed in the intervention group on days 1, 3, and 5. The biological assay was investigated four times (days 0, 5, 10, and 17). UC-MSCs improved the patients' clinical and paraclinical parameters, including leukocytosis, lymphopenia, thrombocytopenia, and liver enzyme abnormalities compared to the control group. They also decreased pro-inflammatory lymphocytes (TH1 and TH17) and increased anti-inflammatory T lymphocytes. Cell therapy also reduced the mean fluorescence intensity (MFI) in overactivated CD8+ T cells.  These findings show that three UC-MSC injections could regulate a hyperactivated immune system in COVID-19-induced ARDS patients by decreasing the inflammatory T lymphocyte subset and can improve the patient's hematological condition and liver function. However, more studies are needed in this area.

Improvement of clinical outcome, laboratory findings and inflammatory cytokines levels using plasmapheresis therapy in severe COVID-19 cases.

INTRODUCTION: Cytokine storm is one of the consequences of the severe forms of COVID-19 due to excessive immune response. In this study, we investigated the therapeutic effect of plasmapheresis and its role on the inflammatory cytokines levels in patients suffering from severe COVID-19. METHODS: In plasmapheresis group, 22 severe cases of COVID-19 receiving three cycles of plasmapheresis with time interval of 24-36 h and 22 COVID-19 patients as the control group were enrolled. Clinical history and laboratory parameters as well as IL-1, IL-6, IFN-γ and IL-17 cytokines serum levels in the time points of before and after plasmapheresis were studied. RESULTS: In severe COVID-19 patients, plasmapheresis significantly improved clinical and laboratory parameters such as cough, weakness, fever, blood oxygen saturation and CRP levels. Serum levels of IL-1, IL-6, IFN-γ and IL-17 in the group of patients receiving plasmapheresis, had a significant decrease following plasmapheresis courses. Although only IL-6 level in the control group had a significant decrease between the days 1-14 of disease. Also, at both time points of before and after plasmapheresis, serum levels of IL-1, IL-6, IFN-γ and IL-17 were inversely correlated to blood oxygen saturation. CONCLUSION: Based on the obtained results, plasmapheresis therapy in severe forms of COVID-19 can effectively improve the clinical symptoms of the disease and reduce inflammatory markers. Therefore, it is suggested that plasmapheresis can be evaluated in standard treatment protocols for severe forms of COVID-19.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model.

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-ß (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.

Development of a stable and high loaded liposomal formulation of lapatinib with enhanced therapeutic effects for breast cancer in combination with Caelyx®: In vitro and in vivo evaluations.

Lapatinib, a dual tyrosine kinase inhibitor, has poor water solubility, which results in poor and incomplete absorption from the gastrointestinal tract. To overcome this obstacle, we designed a stable and high-loaded liposomal formulation encapsulating lapatinib and examined its therapeutic efficacy in vitro and in vivo on TUBO and 4T1 cell lines. We also assessed the impact of liposomal lapatinib on the extent of the tumor and spleen-infiltrating lymphocytes and the autophagy and apoptosis gene expression within the tumor site. Our results showed that liposomal lapatinib inhibits cell proliferation and significantly induces autophagy and apoptosis compared to control groups. Moreover, when it used in combination with liposomal doxorubicin, it extended the time to end from 22.4 ± 3.5 in the control group to 40 days in the TUBO cell line and from 29.2 ± 1.7 to 38.6 ± 2.2 days in 4T1 triple-negative breast cancer cell line, which reveals its promising effects on the survival of tumor-bearing mice. Our results indicated the need for further evaluations to understand liposomal lapatinib's potential effects on autophagy, apoptosis, and particularly on immune system cells.

Development of a novel formulation method to prepare liposomal Epacadostat.

BACKGROUND: One of the important metabolic pathways in cancer progression is tryptophan catabolism by the indoleamin-2,3-dioxygenase (IDO) enzyme, which suppresses the immune system and induces tolerance. Inhibition of IDO1 is an important therapeutic goal for immunotherapy in many cancers such as metastatic melanoma. Epacadostat (EPA) is a very strong inhibitor of IDO1, and its clinical studies are being performed in a higher clinical phase than other inhibitors. In this study, we have developed a new liposomal EPA formulation to reduce the dose, side effects, and treatment costs. METHODS: Liposomes containing EPA were formulated using a novel remote loading method. Their morphology, particle size, surface charge, total phospholipid content, and drug loading were evaluated. Validation method studies to assay of EPA were carried out according to ICHQ2B guidelines. For in-vivo study, B16F10 melanoma bearing C57BL/6 mice were treated with the free or liposomal forms of EPA, and then monitored for tumor size and survival rate. RESULTS: A validated method for EPA determination in liposomal form using UV-visible spectrophotometry was developed which was a precise, accurate and robust method. The particle size, zeta potential, and encapsulation efficacy of liposomes was 128.1 ± 1.1 nm, -16.5 ± 1 mV, and 64.9 ± 3.5, respectively. The half maximal inhibitory concentration (IC50) of liposomal EPA was 64 ng/ml that was lower than free EPA (128 ng/ml). In-vivo results also showed that tumor growth was slower in mice receiving liposomal EPA than in the group receiving free EPA. CONCLUSION: A new method was developed to load EPA into liposomes. Moreover, the use of the nanoliposomal EPA showed more efficacy than EPA in inhibiting the tumor growth in melanoma model. Therefore, it might be used in further clinical studies as a good candidate for immunotherapy alone or in combination with other treatments.

Ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of liposomal antigen and CpG-ODN improved PD-1 blockade immunotherapy.

Lack of pre-existing tumor infiltrated T cells resulting in resistance to programmed cell death protein 1 (PD-1) blockade therapies can be solved by combining with anti-cancer vaccines and CpG-ODN in increasing T cell expansion and infiltration. Therefore, we prepared an ex vivo dendritic cell-based (DC) vaccine pulsed with a low dose of either liposomal or non-liposomal gp100 antigen (2.8 µg) plus CpG-ODN (800 ng) formulations and evaluated its anti-tumor activity in combination with anti-PD-1 therapy. Our results showed a combination of liposomal peptide plus CpG-ODN pulsed DC with anti-PD-1 antibody was more efficacious, as evidenced by a significant increase in Teff/Treg TILs with a marked fourfold elevation of IFN-γ expression level in the tumor site of treated mice which reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, this combination also led to a remarkable tumor remission and prolonged survival rate in melanoma-bearing mice compared to non-liposomal peptide plus CpG-ODN or single-treated liposomal peptide formulations. Our results provide essential insights to devise combining regimens to improve the efficacy of immune checkpoint blockers even by a low dose of peptide and CpG-ODN.

Protective immune response against P32 oncogenic peptide-pulsed PBMCs in mouse models of breast cancer.

High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.

Preventive cancer vaccination with P5 HER-2/neo-derived peptide-pulsed peripheral blood mononuclear cells in a mouse model of breast cancer.

This study compared the prophylactic effects from vaccines based on dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) by pulsing the cells in-vitro with p5 peptide. The different test groups of mice were injected with free peptide or with peptide pulsed with DCs or PBMCs. Two weeks after the last booster dose, immunological tests were performed on splenocyte suspensions from three mice in each group and the remaining mice (5/each group) were evaluated for tumor growth and survival time. The levels of IFN-γ, granzyme B, and IL-10 were detected in T cells. Additionally, IFN-γ and perforin as well as mRNA levels of some genes associated with immune responses were assessed after challenging the splenocytes with TUBO cells. A significant increase was observed in frequency of CD4+ IFN-γ+, CD8+ IFN-γ+, and CD8+ granzyme B+ T cells, and the perforin of supernatants from mice in the DC and PBMC treatment groups. Significant expression levels of Fas ligand (FasL) and forkhead box P3 (Foxp3) were observed in the DC and PBMC groups. These responses led to smaller tumors and longer survival time in our mouse model of breast cancer. The efficacy of the PBMC-based vaccine in improving the protective immune response makes it a simpler and less expensive candidate vaccine compared with DC-based vaccines.

Immunoliposomes bearing lymphocyte activation gene 3 fusion protein and P5 peptide: A novel vaccine for breast cancer.

LAG3-Ig as an immune adjuvant has elicited potent anti-tumor immune responses in several preclinical and clinical studies, but the full potential immunostimulatory of LAG3-Ig has yet to be achieved. We hypothesized that by anchoring LAG3-Ig to the surface of liposomes, the adjuvant activity of LAG3-Ig could be improved. We also investigated the immunotherapy by co-delivery of liposome-coupled LAG3-Ig and P5 tumor antigen in mice model of TUBO breast cancer. We prepared and characterized novel PEGylated liposomes bearing surface conjugated LAG3-Ig and P5. Consistent with our hypothesis, liposomes-conjugated LAG3-Ig via multivalent binding to MHC class II molecules exerted immunostimulatory of LAG3-Ig and markedly induced maturation of dendritic cells more efficiently than free LAG3-Ig. LAG3-Ig-P5-immunoliposomes effectively elicited protective anti-tumor responses more than locally injected soluble LAG3-Ig + P5. The higher percentage of CD4+ and CD8+ T cells in the spleen and more rapid and pronounced infiltration of these effector cells into the site of the tumor were seen following immunoliposome therapy. Finally, anti-tumor immunity induced by LAG3-Ig-P5-immunoliposomes translated into the more tumor regression and prolonged survival of treated mice, compared to soluble immunotherapy. Taken together, our findings suggest that LAG3-Ig-P5-immunoliposomes can be considered as a valuable candidate for developing a liposome-based therapeutic cancer vaccine in treating HER2/ neu+ breast cancer patients.

The immunomodulatory effects of mesenchymal stromal cell-based therapy in human and animal models of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune inflammatory disease with no absolute cure. Although the exact etiopathogenesis of SLE is still enigmatic, it has been well demonstrated that a combination of genetic predisposition and environmental factors trigger a disturbance in immune responses and thereby participate in the development of this condition. Almost all available therapeutic strategies in SLE are primarily based on the administration of immunosuppressive drugs and are not curative. Mesenchymal stromal cells (MSCs) are a subset of non-hematopoietic adult stem cells that can be isolated from many adult tissues and are increasingly recognized as immune response modulating agents. MSC-mediated inhibition of immune responses is a complex mechanism that involves almost every aspect of the immune response. MSCs suppress the maturation of antigen-presenting cells (DC and MQ), proliferation of T cells (Th1, T17, and Th2), proliferation and immunoglobulin production of B cells, the cytotoxic activity of CTL and NK cells in addition to increasing regulatory cytokines (TGF-ß and IL10), and decreasing inflammatory cytokines (IL17, INF-ϒ, TNF-α, and IL12) levels. MSCs have shown encouraging results in the treatment of several autoimmune diseases, in particular SLE. This report aims to review the beneficial and therapeutic properties of MSCs; it also focuses on the results of animal model studies, preclinical studies, and clinical trials of MSC therapy in SLE from the immunoregulatory aspect.

The clinical effect of Nano micelles containing curcumin as a therapeutic supplement in patients with COVID-19 and the immune responses balance changes following treatment: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: To investigates the effectiveness of curcumin-containing Nanomicelles as a therapeutic supplement in the treatment of patients with COVID-19 and its effect on immune responses balance changes following treatment. TRIAL DESIGN: This study is conducted as a prospective, placebo-controlled with parallel group, single-center randomized clinical trial on COVID-19 patients. PARTICIPANTS: Patients are selected from the COVID-19 ward of Shahid Mohammadi Hospital in Bandar Abbas, Iran. INCLUSION CRITERIA: 1. Real time PCR-approved positive COVID-19 test. 2. Both gender 3. Age between 18 and 75 years 4. Signing a written consent 5. Lack of participation in other clinical trials Exclusion criteria: 1. Pregnancy or lactation 2. Allergy to turmeric or curcumin 3. Smoking 4. Patient connected to the ventilator 5. SaO2 less than 90% or PaO2 less than 8 kPa 6. Having comorbidities (such as severe renal failure, Glomerular filtration rate less than 30 ml/min, liver failure, Congestive heart failure, or Chronic obstructive pulmonary disease) 7. History of gallstones 8. History of gastritis or active gastrointestinal ulcer INTERVENTION AND COMPARATOR: In addition to the routine standard treatments for COVID-19, in the intervention group, 40mg nanomicelles containing curcumin (SinaCurcumin Capsule, Exir Nano Sina Company, Iran), four times per day (after breakfast, lunch, dinner and before bedtime) and in the placebo group as the control group, capsules with the same appearance and characteristics (Placebo capsules, Exir Nano Sina Company, Iran) are prescribed for two weeks. MAIN OUTCOMES: The effectiveness of Nano micelles containing curcumin treatment will be evaluated as daily clinical examinations of patients in both groups and, on days 0, 7 and 14, complete clinical symptoms and laboratory findings including peripheral blood and serum parameters such as inflammatory markers will be measured and recorded. Moreover, in order to evaluate the balance of immune responses changes following treatments, serum level of IFN-γ, IL-17, Il-4 and TGF-ß serum cytokines will be measured in both groups at time points of 0, 7 and 14 days post treatment. Gene expression of t-bet, GATA-3, FoxP3 and ROR- γT will also be measured at mentioned time points to assess the shift of T helper1, T helper2, T regulatory and T helper 17 immune responses following treatment. RANDOMISATION: Randomized trials will be performed on 40 COVID-19 patients which will be randomized using encoded sealed boxes with computer generated random digits with 1:1 allocation ratio. In order to randomization, placebo and SinaCurcumin Capsules will be numbered first by computer generated random digits. SinaCurcumin and placebo will then be stored and numbered in sealed packages based on generated random numbers. Finally, according to the order in which patients enter the study, packages are given to patients based on their number. BLINDING (MASKING): The present study will be blind for all patients, physicians and nurses, laboratory technicians and statisticians. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total of 40 patients will be included in the study, 20 of them will be randomly assigned to the intervention group and 20 to the placebo group. TRIAL STATUS: This is Version 1.0 of protocol dated 21 May 2020. The recruitment was started June 24, 2020 and is expected to be completed by October 31, 2020. TRIAL REGISTRATION: This present clinical trial has been registered in the Iranian Registry of Clinical Trials (IRCT) with the registration code of "IRCT20200611047735N1", https://www.irct.ir/trial/48843 . Dated: 19 June 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Encapsulated Checkpoint Blocker Before Chemotherapy: The Optimal Sequence of Anti-CTLA-4 and Doxil Combination Therapy.

INTRODUCTION: Today, a new paradigm has emerged for cancer treatment introducing combination therapies. Doxil, a liposomal doxorubicin serving as a chemotherapeutic agent, is an effective immunogenic killer of cancer cells. Anti-CTLA-4 has been approved for the treatment of some cancers, including melanoma, but side effects have limited its therapeutic potential. METHODS: In this study, two approaches were utilized to increase treatment efficiency and decrease the side effects of anti-CTLA-4, combining it with chemotherapy and encapsulation in a PEGylated liposome. A different sequence of anti-CTLA-4 and Doxil was assessed in combination therapy using non-liposomal and liposomal anti-CTLA-4. RESULTS: Our results showed that liposomal anti-CTLA-4 reduced the size of established tumors and increased survival in comparison with non-liposomal anti-CTLA-4 in a well-established B16 mouse melanoma model. In combination therapy with Doxil, only the administration of anti-CTLA-4 before Doxil showed synergism in both non-liposomal and liposomal form and increased the CD8+/regulatory T cell ratio. DISCUSSION: In summary, our results demonstrate the potential of utilizing a nanocarrier system for the delivery of checkpoint blockers, such as anti-CTLA-4 which further showed potential in a combination therapy, especially when administered before chemotherapy.

Vaccination with dendritic cells pulsed ex vivo with gp100 peptide-decorated liposomes enhances the efficacy of anti PD-1 therapy in a mouse model of melanoma.

BACKGROUND: Targeting antigens to dendritic cells (DCs) via nanoparticles is a powerful strategy which improves the efficacy of ex vivo antigen-pulsed DC vaccines. METHODS: In this study, liposomes were first decorated with gp10025-33 self-antigen and then characterized. Then, DCs were pulsed ex vivo with liposomal gp100 and injected subcutaneously in mice bearing B16F10 established melanoma tumors in combination with anti-PD-1 therapy. RESULTS: Treatment with liposomal pulsed DC vaccine elicited the strongest anticancer immunity and enhanced intratumoral immune responses based on infiltration of gp100-specific CD4+ and CD8+ T cells to the tumor leading to significant tumor growth regression and prolonged survival rate. Treatment with liposomal pulsed DC vaccine also markedly enhanced specific cytotoxic T lymphocytes (CTL) responses with a significant higher titer of IFN-γ in the spleen. Moreover, a significant increase of PD-1 expressing CD8+ tumor infiltrating lymphocytes (TILs) was detected in tumors. CONCLUSION: Our results demonstrate an optimum dose of liposomal gp100 significantly increases the efficacy of anti-PD-1 therapy in mice and might be an effective strategy to overcome resistance to anti-PD-1 therapy.

Nanoliposomal vaccine containing long multi-epitope peptide E75-AE36 pulsed PADRE-induced effective immune response in mice TUBO model of breast cancer.

The main goal of peptide-based cancer vaccines is to induce the immune system and activation of effective T cell responses against cancerous cells. Nevertheless, the potency of peptide vaccines is insufficient in most of cases and had limited clinical success. Therefore, the optimization of peptide-based cancer vaccine is essential to achieve powerful therapeutic outcomes. One strategy to enhanced potency of peptide vaccines and induce strong immune responses is the preparation of multi-epitope peptide formulation containing both Th- and cytotoxic T lymphocyte-induced responses epitope using suitable delivery system. For this reason, we studied the effect of Dioleoylphosphatidylethanolamine-containing liposomal vaccine composed of a mixture of short peptides AE36 and E75 (HER2/neu-derived peptides) and long multi-epitope peptide E75-AE36 (linkage of short peptides) in combination with a Pan HLA-DR epitope (PADRE) peptide. These formulations were examined using a series of subcutaneously injection to HER-2+ TUBO-tumoured mice in prophylactic and therapeutic model. We observed that mice vaccinated with liposomal long peptide in combination with PADRE resulted in the superior induction of CD4+ and CD8+ T cells responses and significantly enhanced production of IFN-γ compared with liposomal short peptides and non-liposomal peptides formulations. Moreover, liposome-long peptide with PADRE led to the considerable reduction of tumour growth and lifespan induction in mouse model. In conclusion, our study indicated that liposomal formulation containing long multi-epitope peptide E75-AE36 with PADRE could be used as an effective multi-epitope prophylactic/therapeutic vaccine to generate potent antigen-specific CD8+ T-cell immune response and may be introduced as a possible candidate peptide vaccine for breast cancer.