Comparative Analysis of Leishmania major Nucleoside Hydrolases Toward Selecting Multi-target Strategy.

PURPOSE: Leishmania causes multiple types of leishmaniasis in different parts of the world. It has a lack of metabolic machine to produce purine bases. Therefore, the parasite produces purine bases through the breakdown of nutritional nucleotides and it makes the nucleoside hydrolases (NHs) good drug targets. They have different substrate-preferring (SP) types. Our objectives were modeling and comparative analysis of these protein structures for Leishmania major. METHOD: In this work, available sequences for all SP types of L. major NH enzymes including inosine-uridine preferring NH (IUNH), inosine-guanosine preferring NH (IGNH), and inosine-adenosine-guanosine preferring NH (IAGNH) were used to make 24 structural models via SWISS-MODEL and LOMETS. After evaluating the structural models, three enzyme structures were finalized and used to analyze substrate-binding pockets. RESULTS: The three SP types of L. major NH enzymes that can breakdown purine nucleosides were highly different in terms of sequence, structure, and profile of interacting residues within the substrate-binding pockets. In this study, new enzyme structures have been presented for three SP types and they have been compared in different aspects and it indicated that they were very different from each other. CONCLUSION: Although, previously indicated that from these three SP types in genera other than Leishmania, the role of IGNH and IAGNH was greater than IUNH in supplying purine bases, till this work, just IUNH has been structurally studied and used in drug-design investigations for Leishmania. Therefore, we are offering to use all three SP types of NHs as multi-target strategy in anti-leishmaniosis drug-design studies.

Nano-Stevia reduces the liver injury caused by streptozotocin (STZ)-induced diabetes in rats by targeting PEPCK/GCK genes, INSR pathway and apoptosis.

Objectives: Extensive application of stevia in the treatment of type 2 diabetes mellitus (DM) has been proven by a large number of previous studies. We prepared stevia loaded in nanoniosomes (nanostevia) to improve its bioavailability, functionality, and stability and explore its protective effects and underlying mechanisms in the liver of STZ-induced diabetic rats. Methods: Single-dose intraperitoneal injection of STZ (50 mg/kg body weight) was used to establish diabetic model. The mRNA levels of PEPCK and GCK genes and the protein level of INSR were evaluated by Real time-PCR and Western blot assays, respectively. TUNEL assay was used to detect apoptotic cell death in the liver tissue. Results: Diabetic rats exhibited significantly reduced levels of INSR (*** P < 0.001) as well as elevated levels of PEPCK (*** P < 0.001). Both stevia and nano-stevia were capable of increasing levels of GCK and INSR and reducing levels of PEPCK (## P < 0.01 and ### P < 0.001, respectively). In addition, significantly increased number of apoptotic cell death was seen in the liver tissue of diabetic rats (*** P < 0.001) which was markedly mitigated by treatment with both Stevia and nano-Stevia (#P < 0.05 and ## P < 0.01, respectively). Conclusion: Both stevia and nano-stevia demonstrates potent anti-apoptotic activity in the liver tissue of diabetic rats by targeting PEPCK/GCK genes and INSR pathway. These finding show that nano-stevia has more potential to reduce the liver injury caused by STZ-induced diabetes in rats and hence can be considered a valid agent and alternative therapy for attenuating complications of type 2 DM. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01278-2.

Tyrosol-loaded Nano-niosomes Attenuate Diabetic Injury by Targeting Glucose Metabolism, Inflammation, and Glucose Transfer.

INTRODUCTION: Regarding the increasing prevalence of type 2 diabetes, it has become a global concern, making it imperative to control. Chemical drugs commonly recommended for diabetes treatment cause many complications and drug resistance over time. METHODS: The polyphenol tyrosol has many health benefits, including anti-diabetic properties. Tyrosol's efficacy can be significantly increased when it is used as a niosome in the treatment of diabetes. In this study, Tyrosol and nano-Tyrosol are examined for their effects on genes implicated in type 2 diabetes in streptozotocin-treated rats. Niosome nanoparticles containing 300 mg surfactant (span60: tween60) and 10 mg cholesterol were hydrated in thin films with equal molar ratios. After 72 hours, nano-niosomal formulas were assessed for their physicochemical properties. MTT assays were conducted on HFF cells to assess the cellular toxicity of the nano niosome contacting optimal Tyrosol. Finally, the expression of PEPCK, GCK, TNF-ɑ, IL6, GLUT2 and GLUT9 was measured by real time PCR. RESULTS: Physiochemical properties of the SEM images of niosomes loaded with Tyrosol revealed that the nanoparticles had a vehicular structure. In this study, there were two stages of release: initial release (8 hours) and sustainable release (72 hours). Meanwhile, free form drugs were considerably more toxic than niosomal drugs in terms of their cellular toxicity. An in vivo comparison of oral Tyrosol gavage with nano-Tyrosol showed a significant increase in GCK (P<0.001), GLUT2 (P<0.001), and GLUT9 (P<0.001). Furthermore, nano-Tyrosol decreased the expression of TNF-ɑ (P<0.05), PEPCK (P<0.001), and IL-6 (P<0.05) that had been increased by diabetes mellitus. The results confirmed nano-Tyrosol's anti-diabetic and anti-inflammatory effects. CONCLUSION: These findings suggest that nano-Tyrosol has potential applications in diabetes treatment and associated inflammation. Further research is needed to better understand the mechanism of action.

Co-encapsulation of hydrophilic and hydrophobic drugs into niosomal nanocarrier for enhanced breast cancer therapy: In silico and in vitro studies.

Combination therapy has been considered one of the most promising approaches for improving the therapeutic effects of anticancer drugs. This is the first study that uses two different antioxidants in full-characterized niosomal formulation and thoroughly evaluates their synergistic effects on breast cancer cells. In this study, in-silico studies of hydrophilic and hydrophobic drugs (ascorbic acid: Asc and curcumin: Cur) interactions and release were investigated and validated by a set of in vitro experiments to reveal the significant improvement in breast cancer therapy using a co-delivery approach by niosomal nanocarrier. The niosomal nanoparticles containing surfactants (Span 60 and Tween 60) and cholesterol at 2:1 M ratio were prepared through the film hydration method. A systematic evaluation of nanoniosomes was carried out. The release profile demonstrated two phases (initial burst followed by sustained release) and a pH-dependent release schedule over 72 h. The optimized niosomal preparation displayed superior storage stability for up to 2 months at 4 °C, exhibiting extremely minor changes in pharmaceutical encapsulation efficiency and size. Free dual drugs (Asc + Cur) and dual-drug loaded niosomes (Niosomal (Asc + Cur)) enhanced the apoptotic activity and cytotoxicity and inhibited cell migration which confirmed the synergistic effect of co-encapsulated drugs. Also, significant up-regulation of p53 and Bax genes was observed in cells treated with Asc + Cur and Niosomal (Asc + Cur), while the anti-apoptotic Bcl-2 gene was down-regulated. These results were in correlation with the increase in the enzyme activity of SOD, CAT, and caspase, and the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) upon treatment with the mentioned drugs. Furthermore, these anti-cancer effects were higher when using Niosomal (Asc + Cur) than Asc + Cur. Histopathological examination also revealed that Niosomal (Asc + Cur) had a lower mitosis index, invasion, and pleomorphism than Asc + Cur. These findings indicated that niosomal formulation for co-delivery of Asc and Cur would offer a promising delivery system for an effective breast cancer treatment.

Efficient targeting of HIF-1α mediated by YC-1 and PX-12 encapsulated niosomes: potential application in colon cancer therapy.

A number of molecular biofactors have been documented in pathogenesis and poor prognosis of colorectal cancer (CRC). Among them, the Hypoxia-Inducible Factor (HIF-1a) is frequently reported to become over-expressed, and its targeting could restrict and control a variety of essential hallmarks of CRC. Niosomes are innovative drug delivery vehicles with the encapsulating capacity for co-loading both hydrophilic and hydrophobic drugs at the same time. Also, they can enhance the local accumulation while minimizing the dose and side effects of drugs. YC-1 and PX-12 are two inhibitors of HIF-1a. The purpose of this work was to synthesize dual-loaded YC-1 and PX-12 niosomes to efficiently target HIF-1α in CRC, HT-29 cells. The niosomes were prepared by the thin-film hydration method, then the niosomal formulation of YC-1 and PX-12 (NIO/PX-YC) was developed and optimized by the central composition method (CCD) using the Box-Behnken design in terms of size, polydispersity index (PDI), entrapment efficiency (EE). Also, they are characterized by DLS, FESEM, and TEM microscopy, as well as FTIR spectroscopy. Additionally, entrapment efficiency, in vitro drug release kinetics, and stability were assessed. Cytotoxicity, apoptosis, and cell cycle studies were performed after the treatment of HT-29 cells with NIO/PX-YC. The expression of HIF-1αat both mRNA and protein levels were studied after NIO/PX-YC treatment. The prepared NIO/PX-YC showed a mean particle size of 185 nm with a zeta potential of about-7.10 mv and a spherical morphology. Also, PX-12 and YC-1 represented the entrapment efficiency of about %78 and %91, respectively, with a sustainable and controllable release. The greater effect of NIO/PX-YC than the free state of PX-YC on the cell survival rate, cell apoptosis, and HIF-1α gene/protein expression were detected (p < 0.05). In conclusion, dual loading of niosomes with YC-1 and PX-12 enhanced the effect of drugs on HIF-1α inhibition, thus boosting their anticancer effects.

Evaluation of the expression level of some coding and non-coding genes in oral squamous cell carcinoma.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is the most common cancers arising from the head and neck region. There is growing evidence that lncRNAs play an important role in OSCC progression. The study aims to investigate correlations between the expression levels of LncRNAs of PARROT, MYCNUT, DANCR, and KTN1-AS1 with clinicopathological characteristics and finding suitable biomarkers for OSCC. MATERIAL AND METHOD: Total lncRNAs related to cancers and HNSC trascriptomics data were downloaded from lncRNADisease v2.0 database and xenabrowser, respectively. Then, ACO was perfomed on shared of LncRNAs between two databases. Finally, some lncRNAs were proposed as potential biomarkers. Thirty biopsies samples from patients with the OSCC and 30 healthy subjects were collected by the surgery. Questionnaires including clinical and demographic data were filled for all cases. Using Real-time PCR, the expression levels of PARROT, MYCNUT, DANCR, and KTN1-AS1 lncRNAs were quantified. RESULT: According to the results,17 novel gene symbol was identified.All the candidate lncRNAs the expression levels of PARROT, MYCNUT, DANCR, and KTN1-AS1 were remarkably upregulated in OSCC tumors in comparison with control group (RQ: 10.00 (P < 0.0001), RQ: 2.920 (P < 0.0001), RQ: 1.623 (P = 0.002), and 4.467 (P < 0.0001), respectively). Also, we found significant associations between tumor lncRNAs expression of PARRPT and DANCER and tumor metastasis (P = 0.009, and P = 0.005, respectively). Additionally, lncRNA KTN1-AS1 expression level was significantly higher in the patients with tumor size more than 3 cm, in comparison with tumor less than 3 cm (P = 0.005). According ROC analysis, all these candidate lncRNAs can be a significant predictor for OSCC (AUC of PARROT lncRNA = 69.72%, AUC of MYCNUT = 98.22%, AUC of DANCR = 74.83%, and AUC of KTN1-AS1 = 99.22%). CONCLUSION: we found that overexpression levels of PARROT, MYCNUT, DANCR, and KTN1-AS1 lncRNAs were correlated with poor clinicopathological characteristics in patients with OSCC. Also, PARROT, MYCNUT, DANCR, and KTN1-AS1 are novel biomarker for the detection of OSCC.

Oral administration of nano-tyrosol reversed the diabetes-induced liver damage in streptozotocin-induced diabetic rats.

Objectives: The present study was designed to evaluate the effects of Tyrosol and Nano-tyrosol on the cellular arrangement, collagen disposition, protein level of insulin receptor (INSR), and superoxide dismutase (SOD) activity in both control and streptozotocin-induced diabetic rats. Methods: Type 2 Diabetes (T2D) was induced in rats by a single intraperitoneal injection of streptozotocin (50 mg/kg). Experimental rats were administered Tyrosol and Nano-tyrosol 1 ml intra-gastrically at a dose of 20 mg/kg once a day for 30 days. Then, rats were sacrificed according to ethical principles. Livers were removed and processed for histological studies using the paraffin technique. Furthermore, non-paraffin sections were used for the INSR-1 western blot technique. Results: At the end of the experiments, the rats in diabetic control and plain niosome groups exhibited a significant increase in collagen disposition (p < 0.001), and apoptotic cells (p < 0.001), as well as decreased total protein levels of INSR (p < 0.001), and SOD activity (p < 0.001) in the hepatic cells. Oral administration of Tyrosol and Nano-tyrosol to diabetic rats reversed all the above-mentioned parameters to near normal levels (p < 0.001). Nano-tyrosol showed the highest significant effect rather than Tyrosol. Conclusion: The results of the present study suggested the beneficial effects of Tyrosol and especially Nano-tyrosol on decreasing the adverse effects of diabetes.

DNAzyme loaded nano-niosomes attenuate myocardial ischemia/reperfusion injury by targeting apoptosis, inflammation in a NF-κB dependent mechanism.

Although DNAzymes have been found to reduce injury after myocardial ischemia/reperfusion (MI/R), their efficiency have been limited due to rapid degradation in vivo. Thus, this study was conducted to extend their half-life by encapsulation into nano­niosomes and examine their cardioprotective effects in a rat model of myocardial infarction (MI). In order to synthesize nano­niosomes, surface active agent film hydration method was used. Characterization of nano­niosomes was performed using the atomic force microscopy (AFM). In order to establish MI/R model in rats, left anterior descending coronary artery (LAD) was ligated for 30 min. A single dose (150µL) of drug formulations was injected into the infarcted region. The cardiac function was evaluated using echocardiography. The expression of pro-inflammatory cytokines, apoptotic factors, and nuclear factor-κB (NF-κB) were evaluated using Western blot and immunohistochemistry, respectively. Particle size of only nano-niosomes was in the range of 60-90 nm, while a shift to 70-110 nm was seen after DNAzyme encapsulation. MI rats treated with DNAzyme­loaded nano­niosomes could markedly reduce Bax, caspase3, TNF-α, IL-1ß, and NF-κB as well as increase Bcl-2 compared to only MI/R group. Collectively, our finding show that nano­niosomes can be considered excellent drug delivery platforms to extend half-life and stability of DNAzyme, when it is used to reduce myocardial I/R injury.

Chitosan -based nanoniosome for potential wound healing applications: Synergy of controlled drug release and antibacterial activity.

This study aims to develop a niosomal platform which can delivery drugs such as tetracycline hydrochloride (TCH) to treat bacterial infections in wounds. To this end, chitosan (CS) was used to obtain a controlled drug release and at the same time antibacterial activity. By design of experiments the niosome encapsulated TCH (TCH-Nio) were optimized for their particle size and encapsulation efficiency, followed by analysis of the release profile of TCH and stability of TCH-Nio and TCH-Nio@CS. The antibacterial activity and cytotoxicity of the fabricated nanoparticles were investigated as well. The release rate of TCH from TCH-Nio@CS in all conditions is less than TCH-Nio. In addition, higher temperature increases the release rate of drug from these formulations. The size, polydispersity index, and encapsulation efficacy of TCH-Nio and TCH-Nio@CS were more stable in 4 °C compared to 25 °C. TCH, TCH-Nio, and TCH-Nio@CS had MIC values of 7.82, 3.91, and 1.95 µg/mL for Escherichia coli, 3.91, 1.95, and 0.98 µg/mL for Pseudomonas aeruginosa, and 1.96, 0.98, and 0.49 µg/mL for Staphylococcus aureus, respectively. Coating of chitosan on niosome encapsulated TCH (TCH-Nio@CS) led to a reduced burst release of TCH from niosome (TCH-Nio), and enabled 2-fold higher antibacterial and anti-biofilm activity against the tested bacterial pathogens E. coli, P. aeruginosa and S. aureus, compared to the uncoated TCH-Nio, and 4-folder higher than the TCH solution, suggesting the synergetic effect of niosome encapsulation and chitosan coating. Moreover, the formulated niosomes displayed no in vitro toxicity toward the human foreskin fibroblast cells (HFF). Both TCH-Nio and TCH-Nio@CS were found to down-regulate the expression of certain biofilm genes, i.e., csgA, ndvB, and icaA in the tested bacteria, which might partially explain the improved antibacterial activity compared to TCH. The obtained results demonstrated that TCH-Nio@CS is capable of controlled drug release, leading to high antibacterial efficacy. The established platform of TCH-Nio@CS enlighten a clinic potential toward the treatment of bacterial infections in skin wounds, dental implants and urinary catheter.

Intra-gastrically administration of Stevia and particularly Nano-Stevia reversed the hyperglycemia, anxiety, and memory impairment in streptozotocin-induced diabetic rats.

Type II diabetes mellitus is a group of metabolic disorders considered chronic hyperglycemia resulting from deficits in insulin secretion or insulin function. This disease usually links with various psychological problems such as anxiety and cognitive dysfunctions. Stevia (Stevia rebaudiana Bertoni) is a natural and healthy substitute sweetener for sugar and artificial sweeteners. It has become essential for human diets and food manufacturers. The aim of this research was to investigate the effects of Stevia and Nano-stevia on the regulation of anxiety and memory processes in male diabetic rats. The elevated plus-maze (EPM) test-retest procedure was used to assess anxiety and memory in male diabetic rats. The findings exhibited that induction of diabetes caused a distorted cellular arrangement in the liver tissue of male rats. On the other hand, intra-gastrically administration of Stevia (1 ml/kg) and nano-Stevia (1 ml/kg) indicated a normal appearance in the liver tissue of male diabetic rats. Moreover, induction of diabetes caused the augmentation of blood glucose, reduction in time spent in%open-arm time (%OAT) on the test day, and enhancement of%OAT on the retest day. Therefore, induction of diabetes in rats produced hyperglycemia, anxiogenic effect, and memory impairment and these responses were reversed by drug treatment. Furthermore, intra-gastrically application of Stevia (1 ml/kg) and nano-Stevia (1 ml/kg) reversed the hyperglycemia, anxiogenic effect, and memory impairment in male diabetic rats. Interestingly, Nano-Stevia exhibited the highest significant response rather than Stevia. In conclusion, the results of this research suggested the beneficial properties of Stevia and particularly Nano-Stevia on inducing anti-diabetic effects, anxiolytic behavior, as well as memory improvement in male diabetic rats.

Association of D299G Polymorphism of TLR4 Gene and CagA Virulence Factor of H. pylori among the Iranian Patients with Colorectal Cancer.

Background: Colorectal cancer (CRC) represents 9% of all malignancies globally. TLR4 gene defenses against Helicobacter pylori infection (HPI), so its mutations are a risk factor for CRC. As there is a correlation between (HPI) and gastric cancer, we investigated whether there is an association between CagA virulence factor in HPI and D299G polymorphism of TLR4 gene with developing CRC among Iranians. Methods: This retrospective study included 85 biopsies of confirmed colorectal lesions out of 230 subjects, which were divided into two age groups. Single nucleotide polymorphism (SNP) D299G in the TLR4 gene was assessed using Tetra-primer ARMS-PCR. The expression of TLR4 and the CagA virulence factor in H.pylori was assessed using real-time PCR (RT-PCR). Results: Chi-squared test showed genotype frequencies of GG were 79% and 62%in patients 51> and 51

The effects of supplementation of Nannochloropsis oculata microalgae on biochemical, inflammatory and antioxidant responses in diabetic rats.

Diabetes is accompanied by inflammation and oxidation. Supplementation of anti-inflammatory and antioxidant compounds can prevent the progression of diabetes. This study aimed to investigate the effects of supplementation of Nannochloropsis oculata microalgae (NOM) on the inflammatory and antioxidant responses in diabetic rats. Sixty male rats were divided into six groups as diabetic and non-diabetic rats receiving 0, 10 and 20 mg/kg of body weight of NOM daily for 21 days. Body weight, the serum concentrations of insulin and glucose and the tissue concentrations of interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), interleukin-6 (IL-6), malondialdehyde (MDA), ferric reducing antioxidant power (FRAP), superoxide dismutase (SOD), glutathione peroxidase (GPx) were assessed. The results showed that induction of diabetes significantly reduced the body weight, the serum concentrations of insulin and the tissue concentrations of SOD, FRAP and GPx while increasing the concentrations of glucose, MDA, IL-1ß, IL-6, NF-κB and TNF-α. Daily oral administration of NOM (10 and 20 mg/kg) significantly maintained the body weight, the serum concentrations of insulin and the tissue concentrations of SOD, FRAP and GPx while preventing the increase in the concentrations of glucose, MDA, IL-1ß and TNF-α. In conclusion, diabetes caused inflammation and oxidation while NOM worked as a natural anti-inflammatory and antioxidant compound.

Simvastatin-loaded nano-niosomes efficiently downregulates the MAPK-NF-κB pathway during the acute phase of myocardial ischemia-reperfusion injury.

BACKGROUND: Simvastatin can potentially mitigate acute inflammatory phase of myocardial ischemia-reperfusion injury. However, these effects negatively influenced by its poor bioavailability, low water solubility and high metabolism. Here, we investigated the effects of SIM-loaded nano-niosomes on a rat model of MI/R injury to find a drug delivery method to tackle the barriers. METHODS: Nano-niosomes' characteristics were identified using dynamic light scattering and transmission electron microscopy. Fifty male Wistar rats were divided into five groups: Sham; MI/R; MI/R + nano-niosome; MI/R + SIM; MI/R + SIM-loaded nano-niosomes. Left anterior descending artery was ligated for 45 min, and 3 mg/kg SIM, nano-niosomes, or SIM-loaded nano-niosomes was intramyocardially injected ten min before the onset of reperfusion. ELISA assay was used to assess cardiac injury markers (cTnI, CK-MB) and inflammatory cytokines (TNF-α, IL-6, TGF-ß, MPC-1). Expression level of MAPK-NF-κB and histopathological changes were evaluated by western blot and hematoxylin & eosin staining, respectively. RESULTS: the size of nano-niosome was 137 nm, reached to 163 nm when simvastatin was loaded. To achieve optimized niosomes span 80, a drug/cholesterol ratio of 0.4 and seven min of sonication time was applied. Optimized entrapment efficiency of SIM-loaded nano-niosomes was 98.21%. Inflammatory cytokines and the expression level of MAPK and NF-κB were reduced in rats receiving SIM-loaded nano-niosomes compared to MI/R + SIM and MI/R + SIM-loaded nano-niosomes. CONCLUSION: Our results showed that SIM-loaded nano-niosomes could act more efficiently than SIM in alleviating the acute inflammatory response of reperfusion injury via downregulating the activation of MAPK-NF-κB.

Co-expression of Interleukin-17A molecular adjuvant and prophylactic Helicobacter pylori genetic vaccine could cause sterile immunity in Treg suppressed mice.

The increasing clinical significance of Helicobacter pylori (H. pylori) in human stomach cancer has led to global efforts to eradicate this pathogen. Recent studies have confirmed the importance of some cytokines such as Interleukin-18 (IL-18), Interleukin-8 (IL-8), Interleukin-17A (IL-17A) and Interleukin-22 (IL-22) in the pathogenesis of the so-called bacterium. This study was designed to compare the effects of Type 1T helper (Th1), Type 2T helper (Th2) cells, Regulatory T cells (Treg) and T helper 17 (Th17) modulatory effects on the efficacy of designed H. pylori vaccine by incorporating some molecular adjuvants in Treg competent and Treg suppressed groups. A bicistronic vector was used for simultaneous expression of codon-optimized Outer inflammatory protein a (OipA) gene and modified mice IL-18, IL-17A, IL-22 and Foxp3 (forkhead box P3) cytokines from four cassettes. Immunization of mice groups was performed using produced plasmids intradermally. Specific IgG1 and IgG2 and IgA antibody titers produced in mice were confirmed by enzyme-linked immunosorbent assay (ELISA) in sera and intestine obtained four weeks after the last immunization. After being stimulated with a mixture of both anti-CD28 mAb and H. pylori lysate, frequencies of single Interferon-Gamma (IFN-γ), single IL-17 and dual IFN-γ/IL-17-secreting T-cells were documented using dual-color FluoroSpot. The kinetics of Th1, Th2 and Th17 in the immunized animals was determined by relative quantification of IL-17A, IL-22, IFN-γ, IL-8, IL-2 and IL-4 specific mRNAs. Four weeks after bacterial challenge, quantitative colony count in the isolated and homogenized stomachs was utilized to assess the level of protective immunity among all groups. The results of immunologic assays showed that the highest cell-mediated immunity cytokines were produced in IL-17 receiving group in which the Treg responses were suppressed previously by the administration of the Foxp3 as an immunogen. In addition, potent clearance of Helicobacter pylori infection was seen in this group as well.

Anti-regulatory T cell vaccines in immunotherapy: focusing on FoxP3 as target.

Anti- tumor vaccination elicits imperfect immune responses against tumor cells; that is related to the presence of suppressive obstacles in the tumor microenvironment. The main members of suppressive milieu of tumor are heteroogenous groups of immune cells in which regulatory T cell is a substantial component. Tregs express different immunomodulatory molecules such as FoxP3. Transcription factor, FoxP3, is a specific intracellular marker of Treg and crucial for Treg development. Therefore it is an attractive target for cancer treatment. This article reviews some recent anti-Treg vaccine focusing on FoxP3 to ameliorate anti-tumor immune responses. Among them, fusion vaccine of FoxP3-Fc(IgG) recombinant DNA vaccine and its accordant protein vaccine represents effective results.

Increased efficacy of a dendritic cell-based therapeutic cancer vaccine with adenosine receptor antagonist and CD73 inhibitor.

Dendritic cells are important in initiating immune responses; therefore, a range of dendritic cell-based approaches have been established to induce immune response against cancer cells. However, the presence of immunosuppressive mediators such as adenosine in the tumor microenvironment reduces the efficacy of dendritic cell-based cancer immunotherapy. In this study, we investigated whether blockade of the A2A adenosine receptor with a selective antagonist and a CD73 inhibitor may increase the efficacy of a dendritic cell-based cancer vaccine. According to the findings, this therapeutic combination reduced tumor growth, prolonged survival of tumor-bearing mice, and enhanced specific antitumor immune responses. Thus, we suggest that targeting cancer-derived adenosine improves the outcomes of dendritic cell-based cancer immunotherapy.

Improved Anti-Treg Vaccination Targeting Foxp3 Efficiently Decreases Regulatory T Cells in Mice.

INTRODUCTION: The critical role of regulatory T (Treg) cells in dampening immune responses against tumor cells is apparent. Therefore, several methods have been introduced for eliminating Treg. Among them, inducing immune responses against Treg cells expressing Foxp3 transcription factor is a hopeful approach to decrease the frequency of Tregs. In current study, we used the chimeric FoxP3-Fc(IgG) fusion construct/protein to effectively stimulate the immune responses against Treg cells. MATERIALS AND METHODS: Previously constructed FoxP3-Fc(IgG) DNA vaccine and its protein counterpart were injected into C57BL/6 mice in a prime/boost regimen. After 2 weeks, the mice were killed to measure the frequency of Tregs in their spleens, as well as analyze their specific cytokine production, T-cell proliferation, and CD8 T-cell cytotoxicity against FoxP3 protein. RESULTS: FACS analysis of FoxP3 CD4 cells in splenocytes revealed the efficiency of FoxP3 DNA-prime protein-boost strategy to decrease the Treg cells and further showed considerable superiority of Fc(IgG) fusion strategy. This significant reduction in Treg frequency was also concomitant with higher FoxP3-specific CTL and Th1 responses in FoxP3-Fc vaccinated animals. CONCLUSIONS: Prime/boost vaccination against FoxP3 in addition to enhanced antigen presentation by means of Fc fusion strategy could be successfully considered for Treg depletion studies. Validity of this approach should be experimentally tested in preclinical tumor models.

Construction and Production of Foxp3-Fc (IgG) DNA Vaccine/Fusion Protein.

BACKGROUND: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell (DC) based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc(IgG) can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I (cross priming). METHODS: DNA construct containing fragment C (Fc) portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli (E. coli) strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining. RESULTS: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification. CONCLUSION: Due to successful expression of Foxp3-Fc (IgG), it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means.

Mesenchymal stem cells do not suppress lymphoblastic leukemic cell line proliferation.

BACKGROUND: Several studies have demonstrated the immunosuppresive effects of mesenchymal stem cells (MSCs) in allogeneic or mitogenic interactions. Cell-cell contact inhibition and secretion of suppressive soluble factors have been suggested in this regard. OBJECTIVE: To investigate if adipose derived MSCs could inhibit Jurkat lymphoblastic leukemia T cell proliferation during coculture. METHODS: Adherent cells with the ability of cellular growth were isolated from normal adipose tissues. Initial characterization of growing cells by flow cytometry suggested their mesenchymal stem cell characteristics. Cells were maintained in culture and used during third to fifth culture passages. Jurkat or allogeneic peripheral blood mononuclear cells (PBMCs) were labeled with carboxy fluorescein diacetate succinimidyl ester and cocultured with increasing doses of MSCs or MSC culture supernatant. Proliferation of PBMCs or Jurkat cells under these conditions was assessed by flow cytometry after 2 and 3 days of coculture, respectively. RESULTS: Results showed the expression of CD105, CD166 and CD44, and the absence of CD45, CD34 and CD14 on the surface of MSC like cells. Moreover, initial differentiation studies showed the potential of cell differentiation into hepatocytes. Comparison of Jurkat cell proliferation in the presence and absence of MSCs showed no significant difference, with 70% of cells displaying signs of at least one cell division. Similarly, the highest concentration of MSC culture supernatant (50% vol/vol) had no significant effect on Jurkat cell proliferation (p>0.6). The same MSC lots significantly suppressed the allogeneic PHA activated PBMCs under similar culture conditions. CONCLUSION: Using Jurkat cells as a model of leukemia T cells, our results indicated an uncertainty about the suppressive effect of MSCs and their inhibitory metabolites on tumor or leukemia cell proliferation. Additional systematic studies with MSCs of different sources are needed to fully characterize the immunological properties of MSCs before planning clinical applications.