Factor of time in dendritic cell (DC) maturation: short-term activation of DCs significantly improves type 1 cytokine production and T cell responses.

Dendritic cells (DCs) have been intensively studied in correlation to tumor immunology and for the development DC-based cancer vaccines. Here, we present the significance of the temporal aspect of DC maturation for the most essential subsequent timepoint, namely at interaction with responding T cells or after CD40-Ligand restimulation. Mostly, DC maturation is still being achieved by activation processes which lasts 24 h to 48 h. We hypothesized this amount of time is excessive from a biological standpoint and could be the underlying cause for functional exhaustion. Indeed, shorter maturation periods resulted in extensive capacity of monocyte-derived DCs to produce inflammatory cytokines after re-stimulation with CD40-Ligand. This effect was most evident for the primary type 1 polarizing cytokine, IL-12p70. This capacity reached peak at 6 h and dropped sharply with longer exposure to initial maturation stimuli (up to 48 h). The 6 h maturation protocol reflected superiority in subsequent functionality tests. Namely, DCs displayed twice the allostimulatory capacity of 24 h- and 48 h-matured DCs. Similarly, type 1 T cell response measured by IFN-γ production was 3-fold higher when CD4+ T cells had been stimulated with shortly matured DC and over 8-fold greater in case of CD8+ T cells, compared to longer matured DCs. The extent of melanoma-specific CD8+ cytotoxic T cell induction was also greater in case of 6 h DC maturation. The major limitation of the study is that it lacks in vivo evidence, which we aim to examine in the future. Our findings show an unexpectedly significant impact of temporal exposure to activation signals for subsequent DC functionality, which we believe can be readily integrated into existing knowledge on in vitro/ex vivo DC manipulation for various uses. We also believe this has important implications for DC vaccine design for future clinical trials.

Different glycosylation profiles of cystatin F alter the cytotoxic potential of natural killer cells.

Cystatin F, a cysteine peptidase inhibitor, is a potent modulator of NK cytotoxicity. By inhibiting granule-mediated cytotoxicity pathway, cystatin F induces formation of non-functional NK cell stage, called split-anergy. We show that N-glycosylation determines the localization and cellular function of cystatin F. Cystatin F mostly exhibited high-mannose glycosylation in U-937 cells, both high-mannose and complex glycosylation in NK-92 and primary NKs, and predominantly complex glycosylation in super-charged NKs. Manipulating N-glycosylation with kifunensine increased high-mannose glycosylation of cystatin F and lysosome localisation, which decreased cathepsin C activity and reduced NK cytotoxicity. Mannose-6-phosphate could significantly reduce the internalization of extracellular cystatin F. By comparing NK cells with different cytotoxic potentials, we found that high-mannose cystatin F was strongly associated with lysosomes and cathepsin C in NK-92 cell line. In contrast, in highly cytotoxic super-charged NKs, cystatin F with complex glycosylation was associated with the secretory pathway and less prone to inhibit cathepsin C. Modulating glycosylation to alter cystatin F localisation could increase the cytotoxicity of NK cells, thereby enhancing their therapeutic potential for treating cancer patients.

Autologous Mesenchymal Stem Cells for Treatment of Chronic Active Antibody-Mediated Kidney Graft Rejection: Report of the Phase I/II Clinical Trial Case Series.

Mesenchymal stem cell (MSCs) therapy has already been studied in kidney transplant recipients (KTRs), and the available data showed that it is safe and well tolerated. The aim of this study was to evaluate the safety and efficacy of autologous MSCs in combination with standard therapy in KTRs with biopsy-proven chronic active antibody-mediated rejection (AMR). Patients with biopsy-proven chronic active AMR received treatment with autologous bone marrow-derived MSCs (3 × 106 cells/kg iv) after completion of standard therapy and were followed for up to 12 months. The primary endpoints were safety by assessment of adverse events. Secondary endpoints included assessment of kidney graft function, immunological and histological changes related to AMR activity and chronicity assessed by conventional microscopy and molecular transcripts. A total of 3 patients were enrolled in the study before it was terminated prematurely because of adverse events. We found that AMR did not improve in any of the patients after treatment with MSCs. In addition, serious adverse events were observed in one case when autologous MSCs therapy was administered in the late phase after kidney transplantation, which requires further elucidation.

Intraoperative Intradermal Application of Stromal Vascular Fraction into the Abdominal Suture Line: Histological Analysis of Abdominal Scar Tissue.

BACKGROUND: Stem cell therapy is a promising new approach to wound healing. Stromal vascular fraction is a heterogeneous collection of cells, including adipose-derived stem cells, which are traditionally isolated using a manual collagenase-based technique. To our knowledge, this is the first human study that histologically assesses the potential of intraoperative intradermal injection of stromal vascular fraction on skin regeneration. METHODS: In this controlled study, 20 patients undergoing deep inferior epigastric perforator flap breast reconstruction and bilateral flank liposuction were included. Stromal vascular fraction was injected intradermally into one side of the abdominal suture line, while the other side served as a control. Outcome measures included analysis of stromal vascular fraction by flow cytometry, histological analysis of scar tissue, and scar photography. RESULTS: Cell yield for application and cell viability were 55.9 ± 28.5 × 106 and 75.1% ± 14.5%, respectively. Age and body mass index were positively correlated with the number of cells for application and adipose-derived stem cells. Mean vascular density, elastic fiber content, collagen maturity (scar index), epidermal thickness, and number of rete ridges all showed higher values on the treated side. Furthermore, the injected number of adipose-derived stem cells and pericytes positively correlated with vascular density. CONCLUSIONS: It is safe to speculate that intradermal stromal vascular fraction injection at the beginning of the healing process increases vascular density, collagen maturity and organization, elastic fiber content, epidermal thickness, epidermal-dermal anchoring of the scarring skin and is therefore responsible for improved skin regeneration. It is a viable and safe method that can be used as an adjunctive treatment in plastic surgery procedures where suboptimal wound healing is anticipated. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Overview of Cellular Immunotherapies within Transfusion Medicine for the Treatment of Malignant Diseases.

Over the years, transfusion medicine has developed into a broad, multidisciplinary field that covers different clinical patient services such as apheresis technology and the development of stem cell transplantation. Recently, the discipline has found a niche in development and production of advanced therapy medicinal products (ATMPs) for immunotherapy and regenerative medicine purposes. In clinical trials, cell-based immunotherapies have shown encouraging results in the treatment of multiple cancers and autoimmune diseases. However, there are many parameters such as safety, a high level of specificity, and long-lasting efficacy that still need to be optimized to maximize the potential of cell-based immunotherapies. Thus, only a few have gained FDA approval, while the majority of them are studied in the context of investigator-initiated trials (IITs), where modern, academically oriented transfusion centers can play an important role. In this review, we summarize existing and contemporary cellular immunotherapies, which are already a part of modern transfusion medicine or are likely to become so in the future.

Mixed cultures of allogeneic dendritic cells are phenotypically and functionally stable - a potential for primary cell-based "off the shelf" product generation.

Vaccination against tumors using antigen-pulsed dendritic cell (DC) vaccines has greatly evolved over the last decade, with hundreds of active human clinical trials well on the way. The use of an autologous source for DC-based vaccine therapeutics remains the obvious choice in the majority of clinical studies; however, novel evidence suggests that an allogeneic source of DCs can yield success if administered in the right context. One of the challenges facing successful DC vaccination protocols is the generation of large enough numbers of DCs intended for vaccination and standardization of these procedures. In addition, variations in the quality of DC vaccines due to donor-to-donor variation represent an important therapeutic factor. To this day it has not been shown whether DCs from different donors can readily co-exist within the same co-culture for the extended periods required for vaccine manufacture. We demonstrate that generation of allogeneic DC co-cultures, generated from multiple unrelated donors, allows the preservation of their phenotypical and functional properties in vitro for up to 72 hours. Therefore, in the case of an allogeneic vaccination approach, one could ensure large numbers of DCs generated from a primary cell source intended for multiple vaccinations. By generating large amounts of ex vivo manufactured DCs from multiple donors, this would represent the possibility to ensure sufficient amounts of equipotent "off the shelf" product that could e.g. be used for an entire cohort of patients within a study.

Cysteine cathepsins L and X differentially modulate interactions between myeloid-derived suppressor cells and tumor cells.

Increased proteolytic activity of cysteine cathepsins has long been known to facilitate malignant progression, and it has also been associated with tumor-promoting roles of myeloid-derived suppressor cells (MDSCs). Consequently, cysteine cathepsins have gained much attention as potential targets for cancer therapies. However, cross-talk between tumor cells and MDSCs needs to be taken into account when studying the efficacy of cathepsin inhibitors as anti-cancer agents. Here, we demonstrate the potential of the MDA-MB-231 breast cancer cell line to generate functional MDSCs from CD14+ cells of healthy human donors. During this transition to MDSCs, the overall levels of cysteine cathepsins increased, with the largest responses for cathepsins L and X. We used small-molecule inhibitors of cathepsins L and X (i.e., CLIK-148, Z9, respectively) to investigate their functional impact on tumor cells and immune cells in this co-culture system. Interactions with peripheral blood mononuclear cells reduced MDA-MB-231 cell invasion, while inhibition of cathepsin X activity by Z9 restored invasion. Inhibition of cathepsin L activity using CLIK-148 resulted in significantly increased CD8+ cytotoxicity. Of note, inhibition of cathepsins L and X in separate immune or tumor cells did not promote these functional changes. Together, our findings underlie the importance of tumor cell-immune cell interactions in the evaluation of the anti-cancer potential of cysteine cathepsin inhibitors.

Dendritic Cells Generated in the Presence of Platelet Lysate Have a Reduced Type 1 Polarization Capacity.

Previously, we have shown platelet lysate (PL) can be used as a non-xenogeneic serum supplement for generation of monocyte-derived dendritic cells (DCs). Since DC-based activation protocols are extremely sensitive to microenvironmental changes such as replacement of culture medium, we wanted to examine the behavior of DCs cultured in the presence of PL under various type-1 activation conditions and assess their type 1 polarization capacity. We compared the quality of DCs cultured in 10% PL-supplemented RPMI medium (plDCs) with clinical-grade DCs obtained using commercially available serum-free medium (sfDCs), frequently used in established DC vaccine protocols. The DC maturation protocols consisted of either monophosphoryl lipid A/IFN-γ, poly I:C/TNF-α/IFN-α or poly I:C/R848. In general, plDCs were inferior to sfDCs in most aspects of their functional type 1 polarization characteristics. After maturation, the expression of co-stimulatory, HLA class II and lymph node-homing molecules was strongly up-regulated, with some noticeable differences. The expression of CD80 and CD86 was more extensive on plDCs, which was particularly evident in case of CCR7. However, after observing their functional capacity, plDCs had significantly lower allo-stimulatory capacity both in terms of CD4+ and CD8+ T cell stimulation. The high expression of CCR7 corresponded to higher CCL-19 directed DC migration of plDCs compared to sfDCs. Finally, their capacity to induce granzyme B and IFN-γ production in CD8+ T cells was significantly reduced in comparison to sfDCs. Based on these findings, the use of PL as an alternative serum supplement for generation of monocyte-derived DC anti-tumor vaccines is questionable.Abbreviations: Ag: antigen; CCL: chemokine ligand; CCR: chemokine receptor; DC: dendritic cells; DC-SIGN: dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; FBS: fetal bovine serum; GMP: good manufacturing practice; IFN: interferon; IL: interleukin; MPLA: monophosphoryl lipid A; PGE: prostaglandin E; pI:C: polyinosinic:polycytidylic acid; pl: platelet lysate; sf: serum free; TLR: toll-like receptor; TNF: tumor necrosis factor.

Chimerism and gene therapy - Lessons learned from non-conditioned murine bone marrow transplantation models.

OBJECTIVE: Hematopoietic stem and progenitor cells (HSPCs) can be used as a vector for gene therapies. In order to predict the number of HSPCs cells necessary to achieve the target level of chimerism in an autologous setting, syngeneic male bone marrow (BM) cells were transplanted into 35 non-conditioned female BALB/c mice. METHOD: The resulting chimerism was determined at 6-53 weeks using qPCR, cell subpopulation sorting, and colony-forming units (CFU) analysis. RESULTS: After the transplantation of 125.8 ± 2.5 million nucleated BM cells, the BM of recipients contained 20.0 ± 2.8% donor cells, representing a chimerism of 0.16 ± 0.02% per one million transplanted nucleated BM cells. Chimerism levels in the BM, neutrophils, and B cells were comparable, whereas in T cells it was lower, and in CFU was approximately twice greater than in BM. CONCLUSION: By extrapolating our murine data, and data from some previous studies to a human non-conditioned autologous CD34+ HSPC transplantation setting, we conclude that approximately 44 million CD34+ HSPCs would be needed to achieve 20% donor chimerism in a 70-kg human, which could serve as a starting point for the future use of HSCPs in gene and cell therapy.

Engineering recombinant Lactococcus lactis as a delivery vehicle for BPC-157 peptide with antioxidant activities.

Lactic acid bacteria (LAB) are attractive hosts for the expression of heterologous proteins and can be engineered to deliver therapeutic proteins or peptides to mucosal surfaces. The gastric stable pentadecapeptide BPC-157 is able to prevent and treat gastrointestinal inflammation by reducing the production of reactive oxygen species (ROS). In this study, we used LAB Lactococcus lactis as a vector to deliver BPC-157 by surface display and trypsin shedding or by secretion to the growth medium. Surface display of BPC-157 was achieved by fusing it with basic membrane protein A (BmpA) or with the peptidoglycan binding domain of AcmA and Usp45 secretion signal. While the expression of BmpA-fusion proteins was higher than that of AcmA/Usp45-fusion protein, the surface display ability of BPC-157 was approximately 14-fold higher with AcmA/Usp45-fusion protein. Release of BPC-157 from the bacterial surface or from isolated fusion proteins by trypsinization was demonstrated with anti-BPC-157 antibodies or by mass spectrometry. The concentration of BPC-157 delivered by surface display via AcmA/Usp45-fusion was 30 ng/ml. This increased to 117 ng/ml by Usp45 signal-mediated secretion, making the latter the most effective lactococcal delivery approach for BPC-157. Secreted BPC-157 significantly decreased ROS production in 149BR fibroblast cell model, suggesting its potential benefit in the treatment of intestinal inflammations. Additionally, a comparison of different modes of small peptide delivery by L. lactis, performed in the present study, will facilitate the future use of L. lactis as peptide delivery vehicle.

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

BACKGROUND AIMS: Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. METHODS: Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. RESULTS: DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. DISCUSSION: This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols.

Synergistic complex from plants Solanaceae exhibits cytotoxicity for the human hepatocellular carcinoma cell line HepG2.

BACKGROUND: It had been demonstrated that sugars from various plants can act as potent agents, which induce apoptosis of cancer cells. METHODS: Using HPLC, we fractionated a mixture of two plant extracts from the plant family Solanaceae, namely Capsicum chinense and the plant family Amaryllidaceae namely Allium sativum. We evaluated the effect of different fractions on apoptosis of HepG2 cell line. The most effective fraction was further studied to determine its molecular composition using mass spectrometry (MS) and NMR. We further evaluated the effect of determined molecular composition found in the selected fraction by using a mixture of commercially available substances, which were found in the fraction and tested its pro-apoptotic effect on HepG2 cells. To get some insight into potential apoptotic mechanisms we studied caspase-3 activity and mitochondrial integrity in treated cells. RESULTS: Out of 93 fractions obtained by HPLC from the plant extract we found HPLC fraction 10 (10 min elution) was the most effective. MS and NMR studies revealed high presence of cellobiose together with vitamin C, sulphur (S) and trace amounts of selenium (Se). HPLC fraction 10 triggered apoptosis of HepG2 within 3 h in the 0.01-1.0 mg/mL concentration range. Furthermore, a mixture of pure cellobiose, vitamin C, S and Se (complex cellobiose/C/S/Se) had a very similar capacity in inducing apoptosis of HepG2 cells compared to HPLC fraction 10. Complex cellobiose/C/S/Se was capable of inducing caspase-3 activity and led to loss of mitochondrial integrity. The capacity of cellobiose alone to induce apoptosis of HepG2 was approximately 1000-fold lower compared to complex cellobiose/C/S/Se. CONCLUSION: In this study we present the highly synergistic effect of a unique complex consisting of cellobiose, vitamin C, sulphur and selenium on triggering the apoptosis of human hepatocellular carcinoma (HepG2) cell line.

Novel N-amidinopiperidine-based proteasome inhibitor preserves dendritic cell functionality and rescues their Th1-polarizing capacity in Ramos-conditioned tumor environment.

The tumor microenvironment represents a burden that hampers the proper activation of immune cells, including the dendritic cells (DCs). It is, therefore, desired that the important characteristics of a given anticancer drug candidate be seen as consisting not solely of its antitumor properties, but that it also lacks potential side effects that could additionally constrain the development and function of immune cells associated with tumor immunity. We have previously identified compounds with a N-amidinopiperidine scaffold that selectively induce apoptosis in Burkitt's lymphoma cells through proteasome inhibition. Here, we demonstrate that SPI-15 affected neither the viability of DCs nor their differentiation. In addition, the compound had no significant effect on their cytokine secretion or allostimulatory capacity. Moreover, DC functionality in the context of tumor microenvironment was also unaffected, as demonstrated by experiments performed on DCs differentiated in Ramos-conditioned media in the presence or absence of SPI-15. The cytokine profile and functional assays revealed that SPI-15 rescues DC differentiation from the immunosuppressive environment produced by Ramos cells; this was seen by their reacquired ability to induce IFN-γ-secretion from naïve CD4(+)CD45RA(+) T cells and the consequently induced Th1-effector differentiation. Herein, we present novel characteristics of an N-amidinopiperidine-based protease inhibitor whose anticancer properties are not associated with the immunosuppression of DCs. We propose future studies toward the design of structurally similar compounds with the aim of developing potent anticancer drugs with minimal negative effects on crucial factors involved in tumor immunity.

Internalization and Accumulation in Dendritic Cells of a Small pH-Activatable Glycomimetic Fluorescent Probe as Revealed by Spectral Detection.

DC-SIGN, an antigen-uptake receptor in dendritic cells (DCs), has a clear role in the immune response but, conversely, can also facilitate infection by providing entry of pathogens into DCs. The key action in both processes is internalization into acidic endosomes and lysosomes. Molecular probes that bind to DC-SIGN could thus provide a useful tool to study internalization and constitute potential antagonists against pathogens. So far, only large molecules have been used to directly observe DC-SIGN-mediated internalization into DCs by fluorescence visualization. We designed and synthesized an appropriate small glycomimetic probe. Two particular properties of the probe were exploited: activation in a low-pH environment and an aggregation-induced spectral shift. Our results indicate that small glycomimetic molecules could compete with antigen/pathogen for binding not only outside but also inside the DC, thus preventing the harmful action of pathogens that are able to intrude into DCs, for example, HIV-1.

Interleukins and interferons in mesenchymal stromal stem cell-based gene therapy of cancer.

The tumor microenvironment is importantly shaped by various cytokines, where interleukins (ILs) and interferons (IFNs) shape the balance of immune activity within tumor niche and associated lymphoid organs. Their importance in activation and tuning of both innate and adaptive immune responses prompted their use in several clinical trials, albeit with limited therapeutic efficacy and risk of toxicity due to systemic administration. Increasing preclinical evidence suggests that local delivery of ILs and IFNs could significantly increase their effectiveness, while simultaneously attenuate the known side effects and issues related to their biological activity. A prominent way to achieve this is to use cell-based delivery vehicles. For this purpose, mesenchymal stromal stem cells (MSCs) are considered an almost ideal candidate. Namely, MSCs can be obtained in large quantities and from obtainable sources (e.g. umbilical cord or adipose tissue), their ex vivo expansion is relatively straightforward compared to other cell types and they possess very low immunogenicity making them suitable for allogeneic use. Importantly, MSCs have shown an intrinsic capacity to respond to tumor-directed chemotaxis. This review provides a focused and detailed discussion on MSC-based gene therapy using ILs and IFNs, engineering techniques and insights on potential future advancements.

Structural Optimization and Biological Evaluation of Isoxazolo[5,4-d]pyrimidines as Selective Toll-Like Receptor 7 Agonists.

Toll-like receptors (TLRs) are components of innate immunity that play a crucial role in several diseases, including chronic inflammatory and infectious diseases, autoimmune diseases, and cancer. In particular, TLR7 has been identified as a key player in the innate immune response against viral infections and small-molecule TLR7 agonists have shown potential for vaccine therapy, for treatment of asthma and allergies, and as anticancer drugs. Inspired by our previous discovery of selective TLR7 agonists, our goal was to develop and introduce a new chemotype of TLR7 agonists by replacing the quinazoline ring with a new heterocycle isoxazolo[5,4-d]pyrimidine. Here, we report design, optimized synthesis, and structure-activity relationship studies of a novel class of TLR7 agonists based on the 6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine-4-amine scaffold that demonstrate high selectivity and low micromolar potencies. The best-in-class agonist 21a, with an EC50 value of 7.8 µM, also proved to be noncytotoxic and induced secretion of cytokines, including IL-1ß, IL-12p70, IL-8, and TNF-α, indicating its potential to modulate the immune response.

Three-dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes.

Podosomes, specialized actin-rich structures in macrophages (Mfs), degrade the extra-cellular matrix (ECM) and are involved in cell migration. On two-dimensional (2D) surfaces Mfs form spot-like podosomes at the ventral cell surface that develop into protrusive structures in a three-dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H, and L, both in human blood Mfs and in human monocytic cell line U-937. Monocyte-to-Mf differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X, and S significantly impairs Mf invasion through the 3D matrix. Time-lapse live-cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.

Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines.

Endosomal TLRs play an important role in innate immune response as well as in autoimmune processes. In the therapy of systemic lupus erythematosus, antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine have been used for a long time. Their suppression of endosomal TLR activation has been attributed to the inhibition of endosomal acidification, which is a prerequisite for the activation of these receptors. We discovered that chloroquine inhibits only activation of endosomal TLRs by nucleic acids, whereas it augments activation of TLR8 by a small synthetic compound, R848. We detected direct binding of antimalarials to nucleic acids by spectroscopic experiments and determined their cellular colocalization. Further analysis revealed that other nucleic acid-binding compounds, such as propidium iodide, also inhibited activation of endosomal TLRs and colocalized with nucleic acids to endosomes. We found that imidazoquinolines, which are TLR7/8 agonists, inhibit TLR9 and TLR3 even in the absence of TLR7 or TLR8, and their mechanism of inhibition is similar to the antimalarials. In contrast to bafilomycin, none of the tested antimalarials and imidazoquinolines inhibited endosomal proteolysis or increased the endosomal pH, confirming that inhibition of pH acidification is not the underlying cause of inhibition. We conclude that the direct binding of inhibitors to nucleic acids mask their TLR-binding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases.

Post-radiation xerostomia therapy with allogeneic mesenchymal stromal stem cells in patients with head and neck cancer: study protocol for phase I clinical trial.

BACKGROUND: Xerostomia is a common side effect of radiotherapy in patients with head and neck tumors that negatively affects quality of life. There is no known effective standard treatment for xerostomia. Here, we present the study protocol used to evaluate the safety and preliminary efficacy of allogeneic mesenchymal stromal stem cells (MSCs) derived from umbilical cord tissue. PATIENTS AND METHODS: Ten oropharyngeal cancer patients with post-radiation xerostomia and no evidence of disease recurrence 2 or more years after (chemo)irradiation (intervention group) and 10 healthy volunteers (control group) will be enrolled in this nonrandomized, open-label, phase I exploratory study. MSCs from umbilical cord tissue will be inserted under ultrasound guidance into both parotid glands and both submandibular glands of the patients. Toxicity of the procedure will be assessed according to CTCAE v5.0 criteria at days 0, 1, 5, 28, and 120. Efficacy will be assessed by measuring salivary flow and analyzing its composition, scintigraphic evaluation of MSC grafting, retention, and migration, and questionnaires measuring subjective xerostomia and quality of life. In addition, the radiological, functional, and morphological characteristics of the salivary tissue will be assessed before, at 4 weeks, and at 4 months after the procedure. In the control group subjects, only salivary flow rate and salivary composition will be determined. DISCUSSION: The use of allogeneic MSCs from umbilical cord tissue represents an innovative approach for the treatment of xerostomia after radiation. Due to the noninvasive collection procedure, flexibility of cryobanking, and biological advantages, xerostomia therapy using allogeneic MSCs from umbilical cord tissue may have an advantage over other similar therapies.

IFN-γ-induced increase in the mobility of MHC class II compartments in astrocytes depends on intermediate filaments.

BACKGROUND: In immune-mediated diseases of the central nervous system, astrocytes exposed to interferon-γ (IFN-γ) can express major histocompatibility complex (MHC) class II molecules and antigens on their surface. MHC class II molecules are thought to be delivered to the cell surface by membrane-bound vesicles. However, the characteristics and dynamics of this vesicular traffic are unclear, particularly in reactive astrocytes, which overexpress intermediate filament (IF) proteins that may affect trafficking. The aim of this study was to determine the mobility of MHC class II vesicles in wild-type (WT) astrocytes and in astrocytes devoid of IFs. METHODS: The identity of MHC class II compartments in WT and IF-deficient astrocytes 48 h after IFN-γ activation was determined immunocytochemically by using confocal microscopy. Time-lapse confocal imaging and Alexa Fluor546-dextran labeling of late endosomes/lysosomes in IFN-γ treated cells was used to characterize the motion of MHC class II vesicles. The mobility of vesicles was analyzed using ParticleTR software. RESULTS: Confocal imaging of primary cultures of WT and IF-deficient astrocytes revealed IFN-γ induced MHC class II expression in late endosomes/lysosomes, which were specifically labeled with Alexa Fluor546-conjugated dextran. Live imaging revealed faster movement of dextran-positive vesicles in IFN-γ-treated than in untreated astrocytes. Vesicle mobility was lower in IFN-γ-treated IF-deficient astrocytes than in WT astrocytes. Thus, the IFN-γ-induced increase in the mobility of MHC class II compartments is IF-dependent. CONCLUSIONS: Since reactivity of astrocytes is a hallmark of many CNS pathologies, it is likely that the up-regulation of IFs under such conditions allows a faster and therefore a more efficient delivery of MHC class II molecules to the cell surface. In vivo, such regulatory mechanisms may enable antigen-presenting reactive astrocytes to respond rapidly and in a controlled manner to CNS inflammation.