Small extracellular vesicle TGF-ß in cancer progression and immune evasion.

Transforming growth factor-ß (TGF-ß) is a well-known cytokine that controls various processes in normal physiology and disease context. Strong preclinical and clinical literature supports the crucial roles of the TGF-ß in several aspects of cancer biology. Recently emerging evidence reveals that the release of TGF-ß from tumor/immune/stromal cells in small extracellular vesicles (sEVs) plays an important part in tumor development and immune evasion. Hence, this review aims to address the packaging, release, and signaling pathways of TGF-ß carried in sEVs (sEV-TGF-ß) in cancer, and to explore its underpinning roles in tumor development, growth, progression, metastasis, etc. We also highlight key progresses in deciphering the roles of sEV-TGF-ß in subverting anti-tumor immune responses. The paper ends with a focus on the clinical significance of TGF-ß carried in sEVs and draws attention to its diagnostic, therapeutic, and prognostic importance.

Blockade of exosome release alters HER2 trafficking to the plasma membrane and gives a boost to Trastuzumab

Objective(s) Exosomal HER2 has been evidenced to interfere with antibody-induced anti-tumor effects. However, whether the blockade of HER2+ exosomes release would affect antibody-mediated tumor inhibition has yet to be investigated. Methods Exosomes derived from BT-474, SK-BR3 and SK-OV3 (HER2-overexpressing tumor cells) and MDA-MB-231 cells (HER2 negative) were purified and characterized by bicinchoninic acid (BCA) assay, western blotting and Transmission electron microscopy (TEM). Inhibition of exosome release was achieved by neutral sphingomyelinase-2 (nSMase-2) inhibitor, GW4869. The effects of exosome blockade on the anti-proliferative effects, apoptosis induction, and antibody-mediated cellular cytotoxicity (ADCC) activity of Trastuzumab were examined using MTT, flow cytometry, and LDH release assays. Also, the effects of exosome inhibition on the surface expression and endocytosis/internalization of HER2 were studied by flow cytometry. Results Purified exosomes derived from HER2 overexpressing cancer cells were positive for HER2 protein. Blockade of exosome release was able to significantly improve apoptosis induction, anti-proliferative and ADCC responses of Trastuzumab dose dependently. The pretreatment of Trastuzumab/purified NK cells, but not PBMCs, with HER2+ exosomes could also decrease the ADCC effects of Trastuzumab. Exosome inhibition also remarkably downregulated surface HER2 levels in a time-dependent manner, but does not affect its endocytosis/internalization. Conclusion Based on our findings, HER2+ exosomes may benefit tumor progression by dually suppressing Trastuzumab-induced tumor growth inhibition and cytotoxicity of NK cells. It seems that concomitant blocking of exosome release might be an effective approach for improving the therapeutic effects of Trastuzumab, and potentially other HER2-directed mAbs (AU)

Blockade of exosome release alters HER2 trafficking to the plasma membrane and gives a boost to Trastuzumab.

OBJECTIVE(S): Exosomal HER2 has been evidenced to interfere with antibody-induced anti-tumor effects. However, whether the blockade of HER2+ exosomes release would affect antibody-mediated tumor inhibition has yet to be investigated. METHODS: Exosomes derived from BT-474, SK-BR3 and SK-OV3 (HER2-overexpressing tumor cells) and MDA-MB-231 cells (HER2 negative) were purified and characterized by bicinchoninic acid (BCA) assay, western blotting and Transmission electron microscopy (TEM). Inhibition of exosome release was achieved by neutral sphingomyelinase-2 (nSMase-2) inhibitor, GW4869. The effects of exosome blockade on the anti-proliferative effects, apoptosis induction, and antibody-mediated cellular cytotoxicity (ADCC) activity of Trastuzumab were examined using MTT, flow cytometry, and LDH release assays. Also, the effects of exosome inhibition on the surface expression and endocytosis/internalization of HER2 were studied by flow cytometry. RESULTS: Purified exosomes derived from HER2 overexpressing cancer cells were positive for HER2 protein. Blockade of exosome release was able to significantly improve apoptosis induction, anti-proliferative and ADCC responses of Trastuzumab dose dependently. The pretreatment of Trastuzumab/purified NK cells, but not PBMCs, with HER2+ exosomes could also decrease the ADCC effects of Trastuzumab. Exosome inhibition also remarkably downregulated surface HER2 levels in a time-dependent manner, but does not affect its endocytosis/internalization. CONCLUSION: Based on our findings, HER2+ exosomes may benefit tumor progression by dually suppressing Trastuzumab-induced tumor growth inhibition and cytotoxicity of NK cells. It seems that concomitant blocking of exosome release might be an effective approach for improving the therapeutic effects of Trastuzumab, and potentially other HER2-directed mAbs. In addition, the exosome secretion pathway possibly contributes to the HER2 trafficking to plasma membrane, since the blockade of exosome secretion decreased surface HER2 levels.

Optimization of an Efficient Cell Culture Hepatitis B Infection System for Assessment of Hepatitis B Virus Neutralizing Monoclonal Antibodies.

BACKGROUND: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection. OBJECTIVE: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs. METHODS: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications. RESULTS: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system. CONCLUSION: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics.

Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae.

Leishmania (L.) species are protozoan parasites with a complex life cycle consisting of a number of developmental forms that alternate between the sand fly vector and their host. The non-pathogenic species L. tarentolae is not able to induce an active infection in a human host. It has been observed that, in pathogenic species, extracellular vesicles (EVs) could exacerbate the infection. However, so far, there is no report on the identification, isolation, and characterization of L. tarentolae EVs. In this study, we have isolated and characterized EVs from L. tarentolae GFP+ (tEVs) along with L. major GFP+ as a reference and positive control. The EVs secreted by these two species demonstrated similar particle size distribution (approximately 200 nm) in scanning electron microscopy and nanoparticle tracking analysis. Moreover, the said EVs showed similar protein content, and GFP and GP63 proteins were detected in both using dot blot analysis. Furthermore, we could detect Leishmania-derived GP63 protein in THP-1 cells treated with tEVs. Interestingly, we observed a significant increase in the production of IFN-γ, TNF-α, and IL-1ß, while there were no significant differences in IL-6 levels in THP-1 cells treated with tEVs following an infection with L. major compared with another group of macrophages that were treated with L. major EVs prior to the infection. Another exciting observation of this study was a significant decrease in parasite load in tEV-treated Leishmania-infected macrophages. In addition, in comparison with another group of Leishmania-infected macrophages which was not exposed to any EVs, tEV managed to increase IFN-γ and decrease IL-6 and the parasite burden. In conclusion, we report for the first time that L. tarentolae can release EVs and provide evidence that tEVs are able to control the infection in human macrophages, making them a great potential platform for drug delivery, at least for parasitic infections.

Blood transcriptional profiles distinguish different clinical stages of cutaneous leishmaniasis in humans.

Cutaneous leishmaniasis (CL) is a neglected tropical disease with severe morbidity and socioeconomic sequelae. A better understanding of underlying immune mechanisms that lead to different clinical outcomes of CL could inform the rational design of intervention measures. While transcriptomic analyses of CL lesions were recently reported by us and others, there is a dearth of information on the expression of immune-related genes in the blood of CL patients. Herein, we investigated immune-related gene expression in whole blood samples collected from individuals with different clinical stages of CL along with healthy volunteers in an endemic CL region where Leishmania (L.) tropica is prevalent. Study participants were categorized into asymptomatic (LST+) and healthy uninfected (LST-) groups based on their leishmanin skin test (LST). Whole blood PAXgene samples were collected from volunteers, who had healed CL lesions, and patients with active L. tropica cutaneous lesions. Quality RNA extracted from 57 blood samples were subjected to Dual-color reverse-transcription multiplex ligation-dependent probe amplification (dcRT-MLPA) assay for profiling 144 immune-related genes. Results show significant changes in the expression of genes involved in interferon signaling pathway in the blood of active CL patients, asymptomatics and healed individuals. Nonetheless, distinct profiles for several immune-related genes were identified in the healed, the asymptomatic, and the CL patients compared to the healthy controls. Among others, IFI16 and CCL11 were found as immune transcript signatures for the healed and the asymptomatic individuals, respectively. These results warrant further exploration to pinpoint novel blood biomarkers for different clinical stages of CL.

Leishmania tarentolae as Potential Live Vaccine Co-Expressing Distinct Salivary Gland Proteins Against Experimental Cutaneous Leishmaniasis in BALB/c Mice Model.

Leishmaniasis is a neglected vector-borne disease caused by Leishmania parasites transmitted through the infected sand flies bite. Current treatments are limited, partly due to their high cost and significant adverse effects, and no human vaccine is yet available. Sand flies saliva has been examined for their potential application as an anti-Leishmania vaccine. The salivary protein, PpSP15, was the first protective vaccine candidate against L. major. Additionally, PsSP9 was already introduced as a highly immunogenic salivary protein against L. tropica. Herein, we aimed to develop an effective multivalent live vaccine to control Cutaneous Leishmaniasis induced by two main species, L. major and L. tropica. Hence, the two above-mentioned salivary proteins using T2A linker were incorporated inside the L. tarentolae genome as a safe live vector. Then, the immunogenicity and protective effects of recombinant L. tarentolae co-expressing PpSP15 and PsSP9 were evaluated in pre-treated BALB/c mice with CpG against L. major and L. tropica. Following the cytokine assays, parasite burden and antibody assessment at different time-points at pre and post-infection, promising protective Th1 immunity was obtained in vaccinated mice with recombinant L. tarentolae co-expressing PpSP15 and PsSP9. This is the first study demonstrating the potency of a safe live vaccine based on the combination of different salivary proteins against the infectious challenge with two different species of Leishmania.

Cancer exosomes and natural killer cells dysfunction: biological roles, clinical significance and implications for immunotherapy.

Tumor-derived exosomes (TDEs) play pivotal roles in several aspects of cancer biology. It is now evident that TDEs also favor tumor growth by negatively affecting anti-tumor immunity. As important sentinels of immune surveillance system, natural killer (NK) cells can recognize malignant cells very early and counteract the tumor development and metastasis without a need for additional activation. Based on this rationale, adoptive transfer of ex vivo expanded NK cells/NK cell lines, such as NK-92 cells, has attracted great attention and is widely studied as a promising immunotherapy for cancer treatment. However, by exploiting various strategies, including secretion of exosomes, cancer cells are able to subvert NK cell responses. This paper reviews the roles of TDEs in cancer-induced NK cells impairments with mechanistic insights. The clinical significance and potential approaches to nullify the effects of TDEs on NK cells in cancer immunotherapy are also discussed.

The roles of tumor-derived exosomes in altered differentiation, maturation and function of dendritic cells.

Tumor-derived exosomes (TDEs) have been shown to impede anti-tumor immune responses via their immunosuppressive cargo. Since dendritic cells (DCs) are the key mediators of priming and maintenance of T cell-mediated responses; thus it is logical that the exosomes released by tumor cells can exert a dominant influence on DCs biology. This paper intends to provide a mechanistic insight into the TDEs-mediated DCs abnormalities in the tumor context. More importantly, we discuss extensively how tumor exosomes induce subversion of DCs differentiation, maturation and function in separate sections. We also briefly describe the importance of TDEs at therapeutic level to help guide future treatment options, in particular DC-based vaccination strategy, and review advances in the design and discovery of exosome inhibitors. Understanding the exosomal content and the pathways by which TDEs are responsible for immune evasion may help to revise treatment rationales and devise novel therapeutic approaches to overcome the hurdles in cancer treatment.

Influence of Pattern Recognition Receptor Ligands on Induction of Innate Immunity and Control of Hepatitis B Virus Infection.

Failure of current therapies to cure chronic hepatitis B has led to renewed interest in therapies that stimulate the host immune system. APOBEC3 (A3) family enzymes have been shown to induce mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) leading to inhibition of HBV transcription and replication. Pattern recognition receptor (PRR) agonists have been reported to suppress HBV, but it is unclear whether these agonists induce A3 gene expression in hepatocytes. We, therefore, evaluated whether PRR signaling activates the expression of A3 genes and other innate immunity genes and restricts HBV infection. HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were infected with HBV and treated with various PRR agonists. The level of HBV infection was subsequently assessed by measurement of HBV biomarkers, including HBV DNA, cccDNA, HBs, and HBe antigens in infected hepatocytes. Among all tested PRR ligands, only Poly(I:C)-HMW/LyoVec and Poly(I:C)-HMW significantly inhibited hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), HBV DNA, and cccDNA, whereas R848 and lipopolysaccharide (LPS) only showed significant inhibition on HBsAg and HBeAg, but not virus DNA. CpG and Pam3CSK4, on the other hand, had no significant inhibitory effect on any of the HBV infection parameters. Moreover, Poly(I:C)-HMW/LyoVec and Poly(I:C)-HMW were the only ligands that significantly increased IL-8 secretion. Interestingly, HBV infection reduced IL-8 secretion induced by Poly(I:C)-HMW and to a lesser extent Poly(I:C)-HMW/LyoVec. Poly(I:C)-HMW/LyoVec had a significant effect on increasing the expression level of A3F, A3G, A3H, TLR3, RIG-1, and MDA5 genes. Our data suggest that PRR agonists may control HBV infection through different mechanisms. The RIG-1 and MDA5 agonist, Poly(I:C)-HMW/LyoVec, seems to downregulate HBV infection through induction of A3 genes.

Emerging Molecular Functions of MicroRNA-9: Cancer Pathology and Therapeutic Implications.

MiRNAs that are characterized by small non-coding RNAs orchestrate the expression of important genes involved in cancer cell development processes, including apoptosis, proliferation, angiogenesis, metastasis, and drug resistance at the post-transcriptional level. Dysregulation of miR-9 in various cancers has been reported. Recently, miR-9 has been considered as a key miRNA in various malignancies. However, its importance in the pathogenesis of different neoplasms is not yet well defined. Accordingly, this study was conducted in order to clarify the potential roles of miR-9 in the development of various cancers, prognosis, and treatment approaches. We have shown that a large number of miR-9 targets play fundamental roles in carcinogenesis, and that is dysregulated in various cancer cells. Our findings werefound aberrant miR-9 expression in a majority of cancers. This review article generally emphasizesthe critical roles of miR-9 in cancer cell progression. Additionally, we intended to investigate the effects of down-regulation or up-regulation of miR-9 in different types of cancers. It is hoped that a good understanding of the regulatory roles of miR-9 in various cancers ishelpful for using miR-9 in clinical settings, including prognosis, diagnosis, and miRNA-based target therapy.

Promotion of Burn Wound Healing by Local Application of Adipose-Derived Mesenchymal Stem Cells: An Experimental Study.

Background:The burn wound is one of the health problems in the world that affects physical and mental health. Today, adipose-derived mesenchymal stem cells (ADSCs) have received medical attention for their accessibility and the ability to reproduce and repair. The present study was designed to investigate the effect of ADSCs on burn wound healing. Methods : The present experimental study was performed on 36 male Wistar rats divided into 1 control group and 3 experimental groups. The second-degree burns with a radius of 10 mm were induced after anesthesia. ADSCs and Dulbecco's Modified Eagle Medium (DMEM) were injected into the dermis around the burn area in the ADSCs and DMEM groups, respectively. Silver sulfadiazine (SSD) ointment was applied topically once daily as the SSD group. The control group did not receive any treatment. The rats were evaluated for 21 days. Wound healing rate, histopathological parameters, and the number of fibroblasts were evaluated by the immunofluorescence technique and vascular endothelial growth factor and transforming growth factor ß (TGF-ß) gene expression by reverse transcription-polymerase chain reaction. The results were entered into SPSS software (SPSS Inc) and analyzed with 1-way analysis of variance and repeated measures analysis. Results: The number of fibroblasts, the number of vessels, TGF-ß, and VEGF gene expression in the burn area were significantly higher in the ADSCs group than in the SSD, DMEM, and control groups. The results also showed that the amount of inflammation was significantly lower in the ADSCs group compared with the control group (p<0.001). Moreover, the percentage of wound recovery was significantly higher in the ADSCs group compared with other groups (p<0.001). Conclusion: ADSCs accelerate and improve burn wound healing by affecting fibroblasts, keratinocytes, and inflammatory cells as well as increasing the expression of the TGF-ß and VEGF genes, and thus increase in angiogenesis.

Immunoreactivity pattern of monoclonal antibodies against Hepatitis B vaccine with global Hepatitis B virus genotypes.

Hepatitis B surface antigen (HBsAg) specific monoclonal antibodies (mAbs) are potentially valuable therapeutic and diagnostic tool. We have previously established and characterized a panel of mAbs derived from immunized BALB/c mice with a yeast-derived recombinant HB vaccine subgentoype A2 and HBsAg subtype adw2. This study was conducted to evaluate the reactivity pattern of this anti-HBs mAbs panel with various genotypes and subgenotypes of HBV using the first WHO HBV genotype reference panel containing 15 serum samples representing the subgenotypes A1, A2, B1, B2, C2, D1-D3, E, F2, and H. Ten out of 21 anti-HBs mAbs were able to strongly recognize all gentopye/subtypes of HBsAg provided in the WHO reference panel. However, 10 out of 21 anti-HBs mAbs showed a moderate to profound loss of reactivity with HBV genotypes/HBsAg subtypes D2/ayw3, E/ayw4, F2/adw4, and H/adw4. Two mAbs from the second group displayed a profoundly reduced reactivity with only 1 out of 3 C2/adr genotype/subtype samples. The amino acid alignment of these 3 samples showed that this particular sample contains amino acid substitution at residue 127, which is located inside "a" determinant. This amino acid substitution, which profoundly affected the reactivity of anti-HBs antibodies, has been previously reported only in D/ayw3, E/ayw4, F/adw4, and H. Interestingly, the amino acid alignment of the samples in this WHO panel showed that P127T substitution can also be found in C2/adr. Comparing amino acids sequences inside the antigenic loop (AGL) showed that D2/ayw3 contains a T118A/P127T double substitution, E/ayw4 contains P127L/T140S, F2/adw4 contains P127L/T140S/ F158L, and H/adw4 contains P127L substitution. Therefore, amino acid variability at positions 118, 127, 140, and 158 was found to cause significant loss of reactivity with anti-HBs mAbs. Since HBsAg variability in different genotypes of HBV can profoundly affect the reactivity of anti-HBs mAbs, analytical sensitivity for HBsAg assays should be considered based on the circulating and common HBV variants in the relevant countries.