Enhancement of the Anti-Angiogenic Effects of Delphinidin When Encapsulated within Small Extracellular Vesicles

(1) Background: The anthocyanin delphinidin exhibits anti-angiogenic properties both in in vitro and in vivo angiogenesis models. However, in vivo delphinidin is poorly absorbed, thus its modest bioavailability and stability reduce its anti-angiogenic effects. The present work takes advantage of small extracellular vesicle (sEV) properties to enhance both the stability and efficacy of delphinidin. When encapsulated in sEVs, delphinidin inhibits the different stages of angiogenesis on human aortic endothelial cells (HAoECs). (2) Methods: sEVs from immature dendritic cells were produced and loaded with delphinidin. A method based on UHPLC-HRMS was implemented to assess delphinidin metabolites within sEVs. Proliferation assay, nitric oxide (NO) production and Matrigel assay were evaluated in HAoECs. (3) Results: Delphinidine, 3-O-β-rutinoside and Peonidin-3-galactoside were found both in delphinidin and delphinidin-loaded sEVs. sEV-loaded delphinidin increased the potency of free delphinidin 2-fold for endothelial proliferation, 10-fold for endothelial NO production and 100-fold for capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on the different steps of angiogenesis. (4) Conclusions: sEVs may be considered as a promising approach to deliver delphinidin to target angiogenesis-related diseases, including cancer and pathologies associated with excess vascularization.

Endoplasmic reticulum stress-induced release and binding of calreticulin from human ovarian cancer cells

Background Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone, but can appear surface bound on cancers cells, including ovarian cancers (OC). We investigated at what stage of cell viability, CRT appeared associated with surface of human OC cells. CRT on pre-apoptotic tumour cells is thought to initiate their eradication via a process termed immunogenic cell death (ICD). Methods We treated OC cells with the chemotherapeutic—doxorubicin (DX) known to induce translocation of CRT to some tumour cell surfaces, with and without the ER stressor—thapsigargin (TG)—and/or an ER stress inhibitor—TUDCA. We monitored translocation/release of CRT in pre-apoptotic cells by flow cytometry, immunoblotting and ELISA. We investigated the difference in binding of FITC-CRT to pre-apoptotic, apoptotic and necrotic cells and the ability of extracellular CRT to generate immature dendritic cells from THP-1 monocytes. Results Dx-treatment increased endogenously released CRT and extracellular FITC_CRT binding to human pre-apoptotic OC cells. DX and TG also promoted cell death in OC cells which also increased CRT release. These cellular responses were significantly inhibited by TUDCA, suggesting that ER stress is partially responsible for the changes in CRT cellular distribution. Extracellular CRT induces maturation of THP-1 towards a imDC phenotype, an important component of ICD. Conclusion Collectively, these cellular responses suggest that ER stress is partially responsible for the changes in CRT cellular distribution. ER-stress regulates in part the release and binding of CRT to human OC cells where it may play a role in ICD.

Cyclosporine A, in Contrast to Rapamycin, Affects the Ability of Dendritic Cells to Induce Immune Tolerance Mechanisms

Following organ transplantation, it is essential that immune tolerance is induced in the graft recipient to reduce the risk of rejection and avoid complications associated with the long-term use of immunosuppressive drugs. Immature dendritic cells (DCs) are considered to promote transplant tolerance and may minimize the risk of graft rejection. The aim of the study was to evaluate the effects of immunosuppressive agents: rapamycin (Rapa) and cyclosporine A (CsA) on generation of human tolerogenic DCs (tolDCs) and also to evaluate the ability of these cells to induce mechanisms of immune tolerance. tolDCs were generated in the environment of Rapa or CsA. Next, we evaluated the effects of these agents on surface phenotypes (CD11c, MHC II, CD40, CD80, CD83, CD86, CCR7, TLR2, TLR4), cytokine production (IL-4, IL-6, IL-10, IL-12p70, TGF-β), phagocytic capacity and resistant to lipopolysaccharide activation of these DCs. Moreover, we assessed ability of such tolDCs to induce T cell activation and apoptosis, Treg differentiation and production of Th1- and Th2-characteristic cytokine profile. Data obtained in this study demonstrate that rapamycin is effective at generating maturation-resistant tolDCs, however, does not change the ability of these cells to induce mechanisms of immune tolerance. In contrast, CsA affects the ability of these cells to induce mechanisms of immune tolerance, but is not efficient at generating maturation-resistant tolDCs.

Step-by-Step Immune Activation for Suicide Gene Therapy Reinforcement

Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the formation of a chemotherapeutic agent inside cancer cells. However, despite great efforts, promising preliminary results, and a long period of time (over 25 years) since the first mention of this method, GDEPT has not yet reached the clinic. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. The advent of checkpoint immunotherapy has yielded new highly promising avenues of study in cancer therapy. For such therapy, it seems reasonable to use combinations of different immunomodulators alongside traditional methods, such as chemotherapy and radiotherapy, as well as GDEPT. In this review, we focused on non-viral gene immunotherapy systems combining the intratumoral production of toxins diffused by GDEPT and immunomodulatory molecules. Special attention was paid to the applications and mechanisms of action of the granulocyte-macrophage colony-stimulating factor (GM–CSF), a cytokine that is widely used but shows contradictory effects. Another method to enhance the formation of stable immune responses in a tumor, the use of danger signals, is also discussed. The process of dying from GDEPT cancer cells initiates danger signaling by releasing damage-associated molecular patterns (DAMPs) that exert immature dendritic cells by increasing antigen uptake, maturation, and antigen presentation to cytotoxic T-lymphocytes. We hypothesized that the combined action of this danger signal and GM–CSF issued from the same dying cancer cell within a limited space would focus on a limited pool of immature dendritic cells, thus acting synergistically and enhancing their maturation and cytotoxic T-lymphocyte attraction potential. We also discuss the problem of enhancing the cancer specificity of the combined GDEPT–GM–CSF–danger signal system by means of artificial cancer specific promoters or a modified delivery system.

Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells

Introduction: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. Material and Methods: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. Results: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. Conclusion: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity.

Th17 T cells and immature dendritic cells are the preferential initial targets after rectal challenge with an SIV-based replication-defective dual-reporter vector

Understanding the earliest events of HIV sexual transmission is critical to develop and optimize HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intra-rectally challenged with a single-round SIV-based dual reporter that expresses luciferase and iRFP670 upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent, transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 hours after the rectal challenge. Detailed phenotypic analysis revealed that on average, 62% of transduced cells are CCR6 + T cells—the vast majority of which express RORγT, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at the rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission. IMPORTANCE Men and women who participate in unprotected receptive anal intercourse are at high risk for acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

A humanized monoclonal antibody against the endothelial chemokine CCL21 for the diagnosis and treatment of inflammatory bowel disease

Chemokines are small proteins that promote leukocyte migration during development, infection, and inflammation. We and others isolated the unique chemokine CCL21, a potent chemo-attractant for naïve T-cells, naïve B-cells, and immature dendritic cells. CCL21 has a 37 amino acid carboxy terminal extension that is distinct from the rest of the chemokine family, which is thought to anchor it to venule endothelium where the amino terminus can interact with its cognate receptor, CCR7. We and others have reported that venule endothelium expressing CCL21 plays a crucial role in attracting naïve immune cells to sites of antigen presentation. In this study we generated a series of monoclonal antibodies to the amino terminus of CCL21 in an attempt to generate an antibody that blocked the interaction of CCL21 with its receptor CCR7. We found one humanized clone that blocked naïve T-cell migration towards CCL21, while memory effector T-cells were less affected. Using this monoclonal antibody, we also demonstrated that CCL21 is expressed in the mucosal venule endothelium of the large majority of inflammatory bowel diseases (IBD), including Crohn’s disease, ulcerative colitis, and also in celiac disease. This expression correlated with active IBD in 5 of 6 cases, whereas none of 6 normal bowel biopsies had CCL21 expression. This study raises the possibility that this monoclonal antibody could be used to diagnose initial or recurrent of IBD. Significantly, this antibody could also be used for therapeutic intervention in IBD by selectively interfering with recruitment of naïve immune effector cells to sites of antigen presentation, without harming overall memory immunity.

Th17 T cells and immature dendritic cells are the preferential initial targets after rectal challenge with an SIV-based replication-defective dual-reporter vector

ABSTRACTUnderstanding the earliest events of HIV sexual transmission is critical to develop and optimize HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intra-rectally challenged with a single-round SIV-based dual reporter that expresses luciferase and iRFP670 upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent, transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 hours after the rectal challenge. Detailed phenotypic analysis revealed that on average, 62% of transduced cells are CCR6+ T cells—the vast majority of which express RORγT, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at the rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission.IMPORTANCEMen and women who participate in unprotected receptive anal intercourse are at high risk for acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

Higher CD1a Levels Correlate with PD-L1 Expression and Predict Worse Overall Survival in Triple-Negative Breast Carcinoma

Objective: The aim of this study was to measure the expression of PD-L1, CD1a (a marker for immature dendritic cells), and CD83 (a marker for mature dendritic cells) and further examine the associations of PD-L1, CD83, and CD1a with overall survival (OS) in triple-negative breast carcinoma patients. Methods: PD-L1, CD1a, and CD83 expression in breast carcinoma tissues and CD83 expression in lymph node tissues were examined by immunohistochemistry and tissue microarray in 159 patients. Patients were classified into the low, medium, and high PD-L1, CD1a, and CD83 levels. Pearson χ2 test was used to analyze the correlations between PD-L1, CD1a, and CD83. The Kaplan-Meier method was used to calculate the OS. Multivariate analysis was used to identify determinants of 3- and 5-year OS. Results: 25.1, 25.8, and 49.1% of the patients had low, medium, and high PD-L1 levels, respectively. PD-L1 levels significantly correlated with CD1a (r = 0.30409, p < 0.001) and CD83 levels (r = 0.6146, p < 0.001) in breast carcinoma tissue, as well as CD83 levels (r = 0.17508, p = 0.027) in lymph node. The median OS was 83 months (range 12–106), and the 3- and 5-year OS rates were 94.97% (95% CI 91.57–98.37) and 86.79% (95% CI 81.53–92.06), respectively. Moreover, patients with high median CD1a levels had a significantly lower 5-year OS rate (75.6%) than those with low median CD1a levels (93.5%, p = 0.038). Conclusion: PD-L1, CD1a, and CD83 are variably expressed in triple-negative breast carcinoma tissues, and PD-L1 expression correlates with CD1a and CD83. Higher CD1a levels correlate with PD-L1 expression and predict worse OS in triple-negative breast carcinoma.