The anti-cancer properties of miR-340 plasmid-chitosan complexes (miR-340 CC) on murine model of breast cancer.

MiRNA-340 (miR-340) has been found to have tumour-suppressing effects in breast cancer (BC). However, for clinical use, miRNAs need to be delivered safely and effectively to protect them from degradation. In our previous study, we used chitosan complexes as a safe carrier with anti-cancer properties to deliver miR-340 plasmid into 4T1 cells. This study explored further information concerning the anti-cancer impacts of both chitosan and miR-340 plasmid in a murine model of BC. Mice bearing 4T1 cells were intra-tumorally administered miR-340 plasmid-chitosan complexes (miR-340 CC). Afterwards, the potential of miR-340 CC in promoting anti-tumour immune responses was evaluated. MiR-340 CC significantly reduced tumour size, inhibited metastasis, and prolonged the survival of mice. MiR-340 CC up-regulates P-27 gene expression related to cancer cell apoptosis, and down-regulates gene expressions involved in angiogenesis and metastasis (breast regression protein-39 (BRP-39)) and CD163 as an anti-inflammatory macrophages (MQs) marker. Furthermore, CD47 expression as a MQs immune check-point was remarkably decreased after miR-340 CC treatment. The level of IL-12 in splenocytes of miR-340 CC treated mice increased, while the level of IL-10 decreased, indicating anti-cancer immune responses. Our findings display that miR-340 CC can be considered as a promising therapy in BC.

Protective effects of different lyoprotectants on survival of clinical bacterial isolates in a hospital biobank.

Nowadays the significant role of biobanks in medical, diagnostic, industrial, and environmental research is well known. Bacterial biobanks could be used as a good resource for designing new treatments, biomedical and industrial researches, and laboratory diagnostics. To have a collection of bacteria from clinical samples and maintain their long-term viability, their preservation needs appropriate protective agents, like cryoprotectants and lyoprotectants. In this study, we collected and characterized Gram-negative and Gram-positive bacteria carrying important antibiotic resistance markers from different clinical samples of hospitalized children. Sucrose (10%), skimmed milk (10%), skimmed milk plus sodium glutamate (10% + 1%), and bovine serum albumin (BSA, 10%) were used as lyoprotectants during the freeze-drying procedure. The survival rate of the lyophilized samples was calculated by dilution plating and measuring the colony forming unit (CFU) after 3 months of storage. The culture analysis results indicated that 25 of the 27 studied bacterial genera (Dilutions 10-3 to 10-6), including Shigella, Methicillin-resistant S. aureus, Acinetobacter spp., Escherichia spp., Pseudomonas spp., Klebsiella spp., Enterococcus spp., were recovered in cultured fractions from all preservation conditions, while 2 genera were only detected in a single preservation condition (2/27, 7.4%). Based on the results, sucrose (10%) and skimmed milk (10%) presented the most protective features. The survival rates varied significantly according to types of the bacteria. Collectively, our results showed a diversity in the recovery of different bacterial genera after lyophilization. While statistically no significant difference was detected among the studied protective agents, sucrose (10%) and skimmed milk (10%) exhibited more effective lyoprotective properties for both Gram-positive and Gram-negative bacteria among the clinical isolates in our study.

Comparison of the long-term and short-term protection in mouse model of Leishmania major infection following vaccination with Live Iranian Lizard Leishmania mixed with chitin microparticles.

Inducing long-term immunity is the primary goal of vaccination. Leishmanisation using non-pathogenic to human Leishmania spp. could be considered a reliable approach to immunising subjects against Leishmania infection. Here, we evaluated the long-term immune responses (14 weeks) after immunisation with either live- or killed-Iranian Lizard Leishmania (ILL) mixed with chitin microparticles (CMPs) against L. major infection in BALB/c mice. In total, nine groups of mice were included in the study. To evaluate short-term immunity, mice were immunised with live-ILL and CMPs and 3 weeks later were challenged with L. majorEGFP . To evaluate the long-term immunity, mice were immunised with either live- or killed-ILL both mixed with CMPs, and 14 weeks after immunisation, mice were challenged with L. majorEGFP . A group of healthy mice who received no injection was also included in the study. Eight weeks after the challenge with L. majorEGFP , all subjects were sacrificed and the parasite burden (quantitative real-time PCR and in vivo imaging), cytokines levels (IFN-γ, IL-4 and IL-10), Leishmania-specific antibody concentration, and total levels of IgG1 and IgG2a were measured. In addition, nitric oxide concentration and arginase activity were evaluated. Results showed that in mice that were immunised using live-ILL+CMP, the induced protective immune response lasted at least 14 weeks; since they were challenged with L. majorEGFP at the 14th -week post-immunisation, no open lesion was formed during the 8-week follow-up, and the footpad swelling was significantly lower than controls. They also showed a significant reduction in the parasite burden in splenocytes, compared to the control groups including the group that received killed-ILL+CMP. The observed protection was associated with a higher IFN-γ and a lower IL-10 production by splenocytes. Additionally, the results demonstrated that arginase activity was decreased in the ILL+CMP group compared to other groups. Immunisation with ILL alone reduced the parasite burden compared to non-immunised control; however, it was still significantly higher than the parasite burden in the ILL+CMP groups. In conclusion, the long-term immune response against L. major infection induced by Live-ILL+CMP was more competent than the response elicited by killed-ILL+CMP to protect mice against infection with L. majorEGFP .

TGF-ß Targeted by miR-27a Modulates Anti-Parasite Responses of Immune System.

Background: Immune cells and their secreted cytokines are known as the first barrier against pathogens. Leishmania major as an intracellular protozoan produces anti-inflammatory cytokines that lead to proliferation and survival of the parasite in the macrophages. miRNAs are small non-coding RNA molecules that regulate mRNAs expression. We aimed to investigate the relationship between the expression of TGF-ß and a bioinformatically candidate miRNA, in leishmaniasis as a model of TGF-ß overexpression. Methods: The miRNAs that target TGF-ß -3'UTR were predicted and scored by bioinformatic tools. After cloning of TGF-ß-3'UTR in psi-CHECK ™- 2 vector, targeting validation was confirmed using Luciferase assay. After miRNA mimic transfection, the expression of miR-27a, TGF-ß, as well as Nitric Oxide concentration was evaluated. Results: miR-27a received the highest score for targeting TGF-ß in bioinformatic predictions. Luciferase assay confirmed that miR-27a is targeting TGF-ß-3'UTR, since miR-27a transfection decreased the luciferase activity. After miRNA transfection, TGF-ß expression and Nitric Oxide concentration were declined in L. major infected macrophages. Conclusion: Bioinformatic prediction, luciferase assay, and miRNA transfection results showed that miR-27a targets TGF-ß. Since miRNA and cytokine-base therapies are developing in infectious diseases, finding and validating miRNAs targeting regulatory cytokines can be a novel strategy for controlling and treating leishmaniasis.

Chitosan based nanoformulation expressing miR-155 as a promising adjuvant to enhance Th1-biased immune responses.

BACKGROUND AND AIM: MiR-155 could act as a key modulator of different aspects of immune system including Th1 responses. In this study, we designed chitosan nanoparticles containing miR-155-expressing plasmid and explored their effects as an adjuvant to enhance Th1 responses for potential future application against intracellular pathogens. METHODS: Nanoparticles were formulated by complex coacervation method and characterized for physicochemical and functional characteristics. Transfection efficiency in Raw 264.7 cells, effects on miR-155 target genes and NO production were evaluated. The prepared nanoparticles were co-administered as an adjuvant with ovalbumin to immunize mice and finally production of IFN-γ and IL-4 were measured by ELISA in splenocyte recall responses. RESULTS: The prepared nanoparticles had the mean size of 244 nm and zeta potential of +17 mV, respectively. Electrophoresis analysis indicated the high capability of nanoparticles to protect the plasmid from DNaseI degradation. Furthermore, nanoparticles showed an appropriate transfection efficiency in Raw 264.7 cells and could downregulate the expression of miR-155 target genes and also upregulate NO production. In vivo immunization examinations revealed successful shift of T cell responses toward Th1. CONCLUSION: Our data suggests the high potential of chitosan nanoparticles containing miR-155-expressing plasmid as an adjuvant for significantly enhanced Th1-biased immune responses upon immunization with a given antigen.

The immunomodulatory potential of murine adipose-derived mesenchymal stem cells is enhanced following culture on chitosan film.

INTRODUCTION: Recent studies show that the paracrine immunomodulatory effects of mesenchymal stem cells (MSCs) are mediated by the secretion of interleukin-10 (IL-10), transforming growth factor-beta (TGF ß), and nitric oxide (NO). The preconditioning of MSCs improves their immunomodulatory characteristics. Chitosan is a biopolymer with low toxicity and biodegradability, used as a membrane for MSCs three-dimensional culture. The present study aimed to evaluate the levels of immunomodulatory mediators of mesenchymal cells cultured on the chitosan film. MATERIALS & METHODS: MSCs were isolated from abdominal adipose tissue of BALB/c mice. Flow cytometry and differential culture medium were used to confirm the identity of isolated mesenchymal stem cells. The MSCs were divided into three groups; The first group was treated with 10 ng/mL LPS. The second group was seeded in the flasks coated with the chitosan film (3% w/v). The last group was cultured in the flasks without any preconditioning. After 72 h, IL-10, TGF-ß, and NO concentrations were measured in the conditioned media. In addition, the arginase activity in mesenchymal stem cells was measured using a colorimetric method. RESULTS: The proliferative spindle-shaped MSCs formed several three-dimensional spheroids on the chitosan film. It was shown that the level of TGF-ß and IL-10 were increased significantly after treatment with LPS (P = 0.02) and spheroid formation (P = 0.01). In addition, the arginase activity was enormously augmented in spheroids compared to controls (7.13-fold increase; 1.71 ± 0.08 and 0.24 ± 0.01 respectively; P = 0.021). On the other hand, the LPS treatment but not the culture on chitosan film increased the NO level significantly (P = 0.02 and P = 0.14, respectively). CONCLUSION: Using chitosan film as a three-dimensional culture strategy significantly affects the production of immunosuppressive factors by MSCs in vitro through increased secretion of TGF-ß and IL-10 and arginase activity.

Intranasal administration of immunogenic poly-epitope from influenza H1N1 and H3N2 viruses adjuvanted with chitin and chitosan microparticles in BALB/c mice.

OBJECTIVES: Prevalence of influenza virus, creates the need to achieve an efficient vaccine against it. We examined whether the predicted antigenic epitopes of HA, NP, and M2 proteins of the influenza H1N1 and H3N2 viruses accompanied by chitin and chitosan biopolymers might be relevant to the induction of effective proper mucosal responses. MATERIALS AND METHODS: The construct was prepared using B and T cell predicted epitopes of HA, NP, and M2 proteins from the influenza H1N1 and H3N2 viruses by considering haplotype "d" as a dominant allele in the BALB/c mice. Intranasal immunization with purified LPS free recombinant protein together with chitin and chitosan microparticles as adjuvants was administered at an interval of 2 weeks in thirty-five BALB/c female mice which were divided into seven groups. Ten days after the last immunization, humoral and cellular immune responses were examined. RESULTS: Elevated systemic IgG2a, IgA, and mucosal IgA revealed a humoral response to the construct. An increase in the number of IFN-γ-producing cells in re-stimulation of splenocytes in the culture medium by poly-tope as well as rise in the concentrations of IL-6, IL-17, and TNF-α along with the regulatory response of IL-10, presented the capacity of the designed protein to provoke significant immune responses. The neutralization test ultimately confirmed the high efficacy of the protein in inhibiting the virus. CONCLUSION: The results support the fact that immunogenic poly-tope protein in the presence of chitin and chitosan microparticles as mucosal adjuvants is able to induce humoral and cell-mediated responses in BALB/c mice.

Umbelliprenin Increases the M1/M2 Ratio of Macrophage Polarization and Improves the M1 Macrophage Activity in THP-1 Cells Cocultured with AGS Cells.

BACKGROUND: Gastric adenocarcinoma is the fifth most diagnosed malignancy in the world. The immune system consists of a heterogeneous mixture of macrophages that defense the body through phagocytosis and the production of different cytokines and chemokines. Tumors cause macrophages to polarize differently in the manner of their favorite growth and angiogenesis. Umbelliprenin, a natural sesquiterpene coumarin, has been shown to have anticancer properties against some tumors, including gastric adenocarcinoma. The aim of our study was to investigate the effect of umbelliprenin on the polarization of macrophages in addition to the measurement of some of the soluble factors they produce. METHOD: The values of IC5 and IC50 for umbelliprenin in the AGS and THP-1 cells were estimated using the MTT assay. THP-1 cells were treated with 10 µM umbelliprenin, either alone or cocultured with AGS cells. Flow cytometry analysis of treated THP-1 cells was performed for CD68, CD86, and CD206 markers to evaluate M0, M1, and M2 macrophages polarization, respectively. AGS cells were assessed for apoptosis and necrosis by flow cytometry after labeling with Annexin V-FITC and propidium iodide. Interleukin- (IL-) 10 and IL-12 contents were measured in the supernatant by the ELISA method. Griess Reaction assay technique was used to determine nitric oxide (NO) concentration. RESULTS: The results of the MTT showed lower toxicity of umbelliprenin in THP-1 (IC50 = 75.79) compared to the AGS cell line (IC50 = 48.81). Umbelliprenin significantly increased the M1/M2 ratio. IL-10 content decreased significantly in the supernatant of M1 and M2 cells after umbelliprenin treatment, while IL-12 increased in the supernatant of M1 cells and decreased in the supernatant of the M2 cells. Umbelliprenin caused an increase in the NO in the supernatant of the M1 cells. CONCLUSION: Umbelliprenin alters the macrophage's secretions and its phenotypes in favor of tumor suppression.

Chitin and chitosan as tools to combat COVID-19: A triple approach.

The progressive and fatal outbreak of the newly emerged coronavirus, SARS-CoV-2, necessitates rigorous collaboration of all health care systems and researchers from all around the world to bring such a devastating pandemic under control. As there is so far no officially approved drug or ideal vaccine for this disease, investigations on this infectious disease are actively pursued. Chitin and chitosan have shown promising results against viral infections. In this review, we first delve into the problematic consequences of viral pandemics followed by an introduction on SARS-CoV-2 taxonomical classification. Then, we elaborate on the immunology of COVID-19. Common antiviral therapies and their related limitations are described and finally, the potential applicability of chitin and chitosan to fight this overwhelming viral pandemic is addressed.

Culture strategy as a modulator of target assessments: Functionality of suspension versus hanging drop-derived choriocarcinoma spheroids as in vitro model of embryo implantation.

The choriocarcinoma spheroid model has been amply applied to study the underlying molecular mechanism of implantation. Reproducibility and functionality of spheroid tumor models were addressed precisely. To mimic embryo-endometrium crosstalk, no functional characteristics of spheroids have been provided based on culture strategies. In this study, choriocarcinoma spheroids were provided as suspension culture (SC) or hanging drop culture (HDC). Primary assessments were performed based on morphology, cellular density, and hormonal secretion. Spheroid-endometrial cross talk was assessed as coculture procedures. Further, alkaline phosphatase (ALP) activity and expression of genes involved in attachment, invasion, and inducing migration were quantified. We found HDC spheroids provided a homogenous-shaped aggregate with a high grade of viability, cellular integration, hormonal secretion, and the dominant role of WNTs expression in their microarchitecture. SC spheroids showed a higher level of ALP activity and the expression of integrated genes in modulating attachment, invasion, and migration abilities. Spheroid confrontation assays clearly clarified the superiority of SC spheroids to crosstalk with epithelial and stromal cells of endometrium in addition to motivating an ideal endometrial response. Conclusively, culture strategies by affecting various molecular signaling pathways should be chosen precisely according to specific target assessments. Specifically, SC assumed as an ideal model in spheroid-endometrial cross talk.

Inhibition of anti-inflammatory cytokines, IL-10 and TGF-ß, in Leishmania major infected macrophage by miRNAs: A new therapeutic modality against leishmaniasis.

Leishmania major (L. major) applies several mechanisms to escape the immune system. Interleukin-10 (IL-10) and Transforming Growth Factor (TGF-ß) downregulate nitric oxide synthase (iNOS) leading to the survival of Leishmania within macrophages. The miRNAs are known as the modulators of the immune system. The present study was conducted to assess the effect of synthetic miR-340 mimic on cytokines (IL-10 and TGF-ß1) involved in L. major infected macrophages. The miRNAs targeting of IL-10 and TGF-ß1 was predicted using bioinformatic tools. Relative expression of predicted miRNA, IL-10, and TGF-ß1 was measured by RT-qPCR before and after synthetic miRNA mimic transfection. Concentration of IL-10 and TGF-ß was measured in posttreatment condition using ELISA method. Also, infectivity was assessed by Giemsa staining. mmu-miR-340 received the highest score for targeting cytokines. The expression of miR-340 was downregulated in L. major infected macrophages. By contrast, expression of IL-10 and TGF-ß1 was upregulated in infected macrophages. After miRNA transfection, TGF-ß1 and IL-10 were both downregulated and interestingly, the combination of miR-340 and miR-27a had a stronger effect on the downregulation of target genes. This research revealed that transfection of infected macrophages with miR-340 alone or in combination with miR-27a mimic can reduce macrophage infectivity and might be introduced as a novel therapeutic agent for cutaneous leishmaniasis.

Intranasal administration of small extracellular vesicles derived from mesenchymal stem cells ameliorated the experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for the human multiple sclerosis, which is characterized by inflammation in the central nervous system (CNS), de-myelination of axonal neurons, and loss of motor coordination. The aim of the current study was to evaluate the effect of intranasal administration of mesenchymal stem cells (MSCs) and small extracellular vesicle (SEV) derived from the MSC (MSC-SEV) on disease activity and antigen-specific responses in the EAE mouse model. MSCs (5 × 105) were administered intranasally to EAE mice (n = 5) on the 15th and 24th days after immunization. In addition, the intranasal administration of MSC-SEV (10 µg) was used to treat EAE mice (n = 5) on a daily basis from the 15th to the 27th day after induction of the disease. The outcomes of therapies were evaluated using studying clinical symptoms and histological analysis of CNS lesions. Moreover, T cell proliferation, the frequency of regulatory T cells, the expression of transcription factors of T-helper subsets, and the levels of their corresponded cytokines were evaluated in splenocytes culture that was stimulated with specific-antigen. The results of treatment of EAE mice with MSC- SEV and MSC showed a significant decrease in the clinical scores, and it was found that treatment with MSC-SEV was more effective in alleviating clinical scores than MSC. In addition, the decrease in clinical symptoms was associated with an increase in immunomodulatory responses, including an increase in the frequency of Foxp3+ CD25+ regulatory T cells. Moreover, the level of TGF-ß was increased by both treatments; however, interleukin-10 was increased only by MSC treatment. Ultimately, it was achieved that the intranasal administration of MSC-SEV to EAE mice was more effective than the administration of MSC to reduce clinical scores and histological lesions of the CNS tissue.

EL4-derived Exosomes Carry Functional TNF-related Apoptosis-inducing Ligand that are Able to Induce Apoptosis and Necrosis in the Target Cells.

Exosomes released by tumor cells play critical roles in tumor progression, immune cell suppression, and cancer metastasis. The aim of the present study was to investigate whether the exosomes released by EL4 cells carry a functional TNF-related apoptosis-inducing ligand (TRAIL) molecule. Exosomes were harvested from the supernatants of EL4 cell culture, and the shape, size, and identity of EL4-derived exosomes were evaluated by utilizing scanning electron microscopy, dynamic light scattering, and dot-blot method. The expression of mRNA and TRAIL protein in EL4 cells and EL4-exosomes were investigated using real-time PCR method and dot-blot analysis. Moreover, the effects of EL4-derived exosomes on cell death in a TRAIL-sensitive cell line (4T1) were studied by using flow cytometry (annexin V/propidium iodide (PI) staining) and fluorescent microscopy analyses (acridine orange/ethidium bromide staining). The results showed that EL4 cells continuously and without the need for stimulation, produce exosomes that carry TRAIL protein. In addition, EL4-derived exosomes were capable to induce apoptosis as well as necrosis in 4T1 cells. It was ultimately revealed that EL4 cells express TRAIL protein and release exosomes containing functional TRAIL. Moreover, the released exosomes were able to induce apoptosis and necrosis in a TRAIL-sensitive cell line. Further studies are needed to reveal the potential roles of tumor-derived exosomes in the pathogenesis of cancers.

Evaluation of Leishmanization Using Iranian Lizard Leishmania Mixed With CpG-ODN as a Candidate Vaccine Against Experimental Murine Leishmaniasis.

Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice. Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania majorEGFP ; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden. Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups. Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.

MicroRNAs Expression Induces Apoptosis of Macrophages in Response to Leishmania major (MRHO/IR/75/ER): An In-Vitro and In-Vivo Study.

BACKGROUND: We aimed to investigate the effect of miR-15a mimic and inhibitor of miR-155 expression on apoptosis induction in macrophages infected with Iranian strain of Leishmania major in-vitro and in-vivo. METHODS: RAW 264.7 cells were infected with L. major promastigotes (MRHO/IR/75/ER), and then were treated with miRNAs. For in-vivo experiment, BALB/c mice were inoculated with L. major promastigotes, and then they were treated with miRNAs. For evaluation of miRNA therapeutic effect, in-vitro and in-vivo studies were performed using quantitative Real-time PCR, Flow cytometry, lesion size measurement, and Limiting Dilution Assay (LDA). This study was performed in Shahid Beheshti University of Medical Sciences in 2019. RESULTS: In-vitro results of flow cytometry showed that using miR-15a mimic, miR-155 inhibitor or both of them increased apoptosis of macrophages. In in-vivo, size of lesion increased during experiment in control groups (P<0.05) while application of both miR-155 inhibitor and miR-15a mimic inhibited the increase in the size of lesions within 6 wk of experiment (P=0.85). LDA results showed that microRNA therapy could significantly decrease parasite load in mimic or inhibitor receiving groups compared to the control group (P<0.05). CONCLUSION: miR-155 inhibitor and miR-15a mimic in L. major infected macrophages can induce apoptosis and reduce parasite burden. Therefore, miRNA-based therapy can be proposed as new treatment for cutaneous leishmaniasis.

Parasite Burden Measurement in the Leishmania major Infected Mice by Using the Direct Fluorescent Microscopy, Limiting Dilution Assay, and Real-Time PCR Analysis.

BACKGROUND: We aimed to compare parasite burden in BALB/c mice, using three methods including the direct fluorescent microscopic using recombinant Leishmania major expressing an enhanced green fluorescent protein, limiting dilution assay, and real-time PCR technique. METHODS: The current study was carried out in 2018, to induce stable enhanced green fluorescent protein (EGFP) production. Initially, the linearized DNA expression cassette (pLEXSY-egfp-sat2) was integrated into the ssu locus of L. major. The expression of EGFP in recombinant parasite was analyzed using direct fluorescent microscopy. Afterward, BALB/c mice were infected with the L. major EGFP, and the infection was evaluated in the foot-pads and inguinal lymph-nodes using an in vivo imaging system. Subsequently, eight BALB/c mice were infected with L. major EGFP, and the results of evaluating parasite burden by a SYBR-Green based real-time PCR analysis and the limiting dilution assays were compared to the results obtained from the direct fluorescent microscopy. RESULTS: The results of the direct fluorescent microscopy showed that EGFP gene stably was expressed in parasites. Moreover, the in vivo imaging analysis of foot-pad lesions revealed that the infection caused by L. major EGFP was progressing over time. Additionally, significant correlations were observed between the results of parasite burden assay using the direct fluorescent microscopy and either limiting dilution assay (r=0.976, P<0.0001) or quantitative real-time PCR assay (r=0.857, P<0.001). CONCLUSION: Ultimately, the utilization of the direct fluorescent microscopy by employing a stable EGFP-expressing L. major is a suitable substitution for the existing methods to quantify parasite burden.

Comparative analysis between four model nanoformulations of amphotericin B-chitosan, amphotericin B-dendrimer, betulinic acid-chitosan and betulinic acid-dendrimer for treatment of Leishmania major: real-time PCR assay plus.

BACKGROUND: Amphotericin B (Amp) and Betulinic acid (BA) as antileishmanial agents have negligible water solubility and high toxicity. To solve these problems, for the first time, chitosan nanoparticles and Anionic Linear Globular Dendrimer (D) were synthesized for the treatment of Leishmania major (L. major). METHOD: Chitosan and dendrimer nanoparticles were synthesized, and Amp and BA were loaded into the nanoparticles. The particles were then characterized using various methods and their efficacy was evaluated in vitro and in vivo environments (parasite burden was confirmed using pathological studies and real-time PCR methods). RESULT: The results of docking showed that Amp and BA can be loaded into chitosan and dendrimer nanoparticles. The results of physically drug loading efficiency for AK (Amphotericin B-chitosan), BK (Betulinic acid-chitosan), AD (Amphotericin B-Dendrimer) and BD (Betulinic acid- Dendrimer) were 90, 93, 84 and 96 percent, respectively. The characterization results indicated that the drugs were loaded into nanoparticles physically. Moreover, the increased solubility rate for AD=478, BD=790, AK=80 and BK=300 folds. Furthermore, the results of the drug delivery system showed the slow controlled drug release pattern with cellular uptake of more than 90%. The treatment results showed a 100 percent decrease of toxicity for the all nanodrugs was observed in vivo and in vitro environments. Moreover, AK10 and BK20 mg/kg reduced parasite burden by 83 percent (P<0.001), while AD50 and BD40 mg/kg reduced it to a lesser extent compared to glucantime. CONCLUSION: All the synthesized nanodrugs were completely succeeded by 100% to recovery the L. major induced pathological effects in the infected footpad. Also, the results of present study were confirmed with real-time PCR and the results showed that AK and BK were succeeded in a large extent to the treatment of L. major infection (P<0.001), therefore AK and BK could be considered as proper alternatives of choices drugs.

Co-administration of chitin micro-particle and Leishmania antigen proposed a new immune adjuvant against experimental leishmaniasis.

AIMS: We investigated the protective effect of chitin micro-particle (CMP) as an adjuvant against Leishmania infection in BALB/c mice. METHODS: Mice were immunized subcutaneously with soluble Leishmania antigen (SLA) plus CMP (100 µg SLA + 100 µg CMP/100 µL) as the test group. Three weeks after the last immunization, test and control groups were infected by Leishmania major (L major). Eight weeks post-infection, evaluation of parasites load in lymph nodes was performed using limiting dilution assay. Then, the spleen cell cytokine response (TNF-α, IFN-γ, IL-4, IL-10, IL-17 and IL-27) to SLA among vaccinated and nonvaccinated groups was investigated using ELISA. Serum levels of IgG1 and IgG2a were measured as well. RESULTS: The SLA plus CMP group demonstrated the protection. The responses included reduced lesion formation and lower parasite load. Also, in comparison with control group higher levels of IFN-γ and, IL-10 in the culture of spleen cells, and lower levels of IgG1 in sera were seen in SLA plus CMP group. CONCLUSION: The data supported the possibility of using CMP as a suitable adjuvant in Leishmania vaccination.

In vitro analysis of immunomodulatory effects of mesenchymal stem cell- and tumor cell -derived exosomes on recall antigen-specific responses.

BACKGROUND: The aim of the present study was to evaluate in vitro effects of exosomes derived from mesenchymal stem cells (MSCs) or tumor cells on recall-antigen-specific immune responses. METHODS: The exosomes were isolated from the supernatant of the cultures of the adipose-derived MSCs, and 4T1 cell line. The splenocytes isolated from experimental autoimmune encephalomyelitis (EAE) mice were utilized to evaluate the effects of exosomes on recall-antigen-specific responses. The expression of master regulators for T cell sub-types and the levels of their corresponding cytokines were evaluated. RESULTS: Treatment by disease-inducing peptide (MOG35-55) combined with MSC-EXO or by MOG+TEX enhanced the expression of Foxp3 as the master regulator for Treg cells; by comparing with splenocytes which were treated by MOG. Nonetheless, the production of IL-10 and TGF-ß were increased only in splenocytes treated by MOG+TEX. Additionally, treatments of splenocytes by MOG+TEX and MOG+MSC-EXO decreased the expression of Tbx21 and Gata3, as the master regulator for T helper (TH)1 and TH2 responses. However, the IFN-γ level did not decrease. The expression of Rorc and Elf4, which are the activator and inhibitor for differentiation of TH17 respectively were increased after splenocytes was treated by MOG+TEX. However, a reduction in Rorc and Elf4 levels was observed when splenocytes were treated by MOG+MSC-EXO. Indeed, the concentration of IL-17 did not alter significantly following the treatment by MOG+exosomes. CONCLUSION: It was ultimately attained that TEX and MSC-EXO utilized various mechanisms to modulate the recall immune responses. TEX was more potent than MSC-EXO to induce regulatory responses by upregulating the production of Foxp3, IL-10, and TGF-ß.

Novel Nanosized Chitosan-Betulinic Acid Against Resistant Leishmania Major and First Clinical Observation of such parasite in Kidney.

Regarding the antiparasitic effects of Betulinic acid (B) against Leishmaniasis, it was loaded into nanochitosan (K) for the first time in order to improve its therapeutic effects and decrease its side effects for the treatment of Leishmania major-infected Balb/c mice. Improvement the therapeutic efficacy of Bas an anti-leishmania agent through increasing the effective dose was achieved by using a novel solvent and phase separation method for K synthesis. The synthesized K with the size of 102 nm and Betulinic acid-nanochitosan (BK) with the size of 124 nm and drug loading efficiency of 93%, cellular uptake of 97.5% with the slow drug release pattern was prepared. To increase the therapeutic dose, a modified 10% acetic acid solvent was used. The in vitro and in vivo results showed that the nanodrug of BK was non toxic by 100% and BK20 mg/kg could completely performed the wound healing and inhibit the parasite in a large extent (P ˂ 0.001) compared to other groups. Therefore, BK could be considered as an alternative regimen for treatment of L. major.