Interactions of the Immune System with Human Kidney Organoids.

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

Mesenchymal stromal cell-derived membrane particles: A novel cell-free therapy for inflammatory bowel diseases.

Inflammatory bowel diseases (IBD), including ulcerative colitis, are chronic and idiopathic inflammations of the gastrointestinal tract. A disruption of the epithelial barrier and an imbalance between Th1 and Th2 subsets are associated with the onset and progression of these diseases. Mesenchymal stromal cells (MSC) are a promising therapy for IBD. However, cell-tracking studies have shown that intravenously infused MSC localize to the lungs and present short-term survival. To reduce practical complexities arising from living cells, we generated membrane particles (MP) from MSC membranes, which possess some of the immunomodulatory properties of MSC. This study investigated the effect of MSC-derived MP and conditioned media (CM) as cell-free therapies in the dextran sulfate sodium (DSS)-induced colitis model. Acute colitis was induced in C57BL/6 mice by oral administration of 2% DSS in drinking water ad libitum from days 0 to 7. Mice were treated with MP, CM, or living MSC on days 2 and 5. Our findings revealed that MP, CM, and living MSC ameliorated DSS-induced colitis by reducing colonic inflammation, the loss of colonic goblet cells, and intestinal mucosa permeability, preventing apoptosis of damaged colonic cells and balancing Th1 and Th2 activity. Therefore, MSC-derived MP have high therapeutic potential for treating IBD, overcoming the deficiencies of living MSC therapy, and opening novel frontiers in inflammatory diseases medicine.

Fasciola hepatica extract suppresses fibroblast-like synoviocytes in vitro and alleviates experimental arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation, fibroblast-like synoviocytes (FLS) activation and joint destruction. Fasciola hepatica is a platyhelminth that releases excretory-secretory immunomodulatory products capable of suppressing the Th1 immune response. Despite the effectiveness of available treatments for inducing disease remission, current options are not successful in all patients and may cause side effects. Thus, we evaluated the therapeutic potential of F. hepatica extract on FLS from RA patients and arthritis models. METHODS: FLS were isolated from synovial fluid of RA patients, cultured, and exposed to F. hepatica extract (60, 80, and 100 µg/ml) for different time points to assess cell viability, adherence, migration and invasion. For in vivo experiments, mice with antigen (AIA) and collagen (CIA) induced arthritis received a 200 µg/dose of F. hepatica extract daily. Statistical analysis was performed by ANOVA and Student's t-test using GraphPad Prism 6.0. RESULTS: In vitro assays showed that extract decreased FLS cell viability at concentration of 100 µg/ml (83.8% ± 5.0 extract vs. 100.0% ± 0.0 control; p < 0.05), adherence in 20% (92.0 cells ± 5.8 extract vs. 116.3 cells ± 7.9 control; p < 0.05), migratory potential (69.5% ± 17.6 extract vs. 100.0% control; p < 0.05), and cell invasiveness potential through the matrigel (76.0% ± 8.4 extract vs. 100.0% control; p < 0.01). The extract reduced leukocyte migration by 56% (40 × 104 leukocytes/knee ± 19.00) compared to control (90.90 × 104 leukocytes/knee ± 12.90) (p < 0.01) and nociception (6.37 g ± 0.99 extract vs. 3.81 g ± 1.44 control; p < 0.001) in AIA and delayed clinical onset of CIA (11.75 ± 2.96 extract vs. 14.00 ± 2.56 control; p = 0.126). CONCLUSION: Our results point out a potential immunomodulatory effect of F. hepatica extract in RA models. Therefore, the characterization of promising new immunomodulatory molecules should be pursued, as they can promote the development of new therapies. Trial registration Collection of synovial liquid and in vitro procedures were approved by the Ethics Committee with Certificate of Presentation of Ethical Appreciation in Plataforma Brasil (CAAE: 89044918.8.0000.5327; date of registration: 26/07/2018).

A detailed analysis of expedited regulatory review time of marketing authorization applications for new anticancer drugs in the US and EU.

The aim of this study was to assess the effect of expedited regulatory approval programs used by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), type of product (small molecule or biotechnology-derived product) and consulting scientific advisory committees on the regulatory review time of the marketing authorization applications (MAAs) for new anticancer drugs. A dataset composed of 76 new anticancer drugs was constructed. The date of submission of the MAAs in the United States and the European Union were comparable. The typical review time of MAAs was 136 days shorter in the United States (201 days [median]) than in the European Union (337 days [median]). The type of product did not have a high impact on the review time. The review time of the MAAs for drugs undergoing priority review in the United States or accelerated assessment in the European Union at the stage of review of MAA was generally shorter than that for drugs following the standard regulatory pathway. The regulatory pathway using at least one expedited regulatory program at the stages of drug development, review of MAA, and approval of drug in the United States (172 days [median]), and that at the stages of review of MAA and approval of drug in the European Union (183 days [median]) enabled the shortest review time of MAAs. Referral to advisory committee meeting increased the review time of MAAs for drugs undergoing one or more expedited regulatory approval programs in the United States and the European Union close to that for drugs undergoing the standard regulatory approval pathway.

Mesenchymal Stromal Cell Derived Membrane Particles Are Internalized by Macrophages and Endothelial Cells Through Receptor-Mediated Endocytosis and Phagocytosis.

Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. However, MSC are large and become trapped in the lungs after intravenous infusion, where they have a short survival time. To steer MSC immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP), which have immunomodulatory properties, and investigated their internalization and mode of interaction in macrophages subtypes and human umbilical vein endothelial cells (HUVEC) under control and inflammatory conditions. We found that macrophages and HUVEC take up MP in a dose, time, and temperature-dependent manner. Specific inhibitors for endocytotic pathways revealed that MP internalization depends on heparan sulfate proteoglycan-, dynamin-, and clathrin-mediated endocytosis but does not involve caveolin-mediated endocytosis. MP uptake also involved the actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis. Anti-inflammatory M2 macrophages take up more MP than pro-inflammatory M1 macrophages. In contrast, inflammatory conditions did not affect the MP uptake by HUVEC. Moreover, MP induced both anti- and pro-inflammatory responses in macrophages and HUVEC by affecting gene expression and cell surface proteins. Our findings on the mechanisms of uptake of MP under different conditions help the development of target-cell specific MP therapy to modulate immune responses.

In Vitro Amoebicidal Activity of Imidazolium Salts Against Trophozoites.

INTRODUCTION: Several strains of the free-living genus Acanthamoeba can cause granulomatous amoebic encephalitis (GAE), a rare chronic and slowly progressive infection of the central nervous system (CNS), and Acanthamoeba keratitis (AK), a sight-threatening eye infectious disease. AK incidence has increased with the popularization of the contact lens wear and its treatment is currently limited and frequently unsuccessful. As imidazolium salts (IS), cationic imidazole derivatives, have promising antimicrobial potential. MATERIALS AND METHODS: The present study evaluated the amoebicidal activity of four IS; 1-n-hexadecyl-3-methylimidazolium methanesulfonate (C16MImMeS), chloride (C16MImCl) and bis (triluoromethylsulfonyl) imide (C16MImNTf2 ), and 1-methyl-3-n-octadecylimidazolium chloride (C18MImCl), against the Acanthamoeba castellanii (ATCC30010) environmental strain and a clinical isolate (genotype T4). RESULTS: Three IS showed being lethal to 100% of the Acanthamoeba trophozoites at the minimum inhibitory concentrations of 125 and 62.5 µg/mL (C16MImMeS), 31.25 and 62.5 µg/mL (C16MImCl), and 125 and 125 µg/mL (C18MImCl) for ATCC30010 and isolate T4, respectively. C16MImNTf2 did not demonstrate amoebicidal activity. All active IS caused the hemolysis of erythrocytes. The cytotoxic effect of the IS was tested in RAW macrophages and human brain microvascular endothelial cells, which demonstrated cytotoxicity in all concentrations tested against both cell lines. As a consequence, these IS with amoebicidal activity presented low selectivity index values (SI) (SI < 1.0), demonstrating lack of parasite selectivity. CONCLUSION: Thus, C16MImMeS, C16MImCl, and C18MImCl seem to hold greater promise as components for contact lens cleaning and disinfection solutions, instead of direct human application.

Cell membrane and bioactive factors derived from mesenchymal stromal cells: Cell-free based therapy for inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract associated with multifactorial conditions such as ulcerative colitis and Crohn's disease. Although the underlying mechanisms of IBD remain unclear, growing evidence has shown that dysregulated immune system reactions in genetically susceptible individuals contribute to mucosal inflammation. However, conventional treatments have been effective in inducing remission of IBD but not in preventing the relapse of them. In this way, mesenchymal stromal cells (MSC) therapy has been recognized as a promising treatment for IBD due to their immunomodulatory properties, ability to differentiate into several tissues, and homing to inflammatory sites. Even so, literature is conflicted regarding the location and persistence of MSC in the body after transplantation. For this reason, recent studies have focused on the paracrine effect of the biofactors secreted by MSC, especially in relation to the immunomodulatory potential of soluble factors (cytokines, chemokines, and growth factors) and extracellular vehicles that are involved in cell communication and in the transfer of cellular material, such as proteins, lipids, and nucleic acids. Moreover, treatment with interferon-γ, tumor necrosis factor-α, and interleukin-1ß causes MSC to express immunomodulatory molecules that mediate the suppression via cell-contact dependent mechanisms. Taken together, we present an overview of the role of bioactive factors and cell membrane proteins derived from MSC as a cell-free therapy that can improve IBD treatment.

Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway.

BACKGROUND: Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, diarrhoea, weight loss and bloody stools, where sustained remission is not currently achievable. Dextran Sulphate Sodium (DSS)-induced colitis is an animal model that closely mimics human UC. Ultrasound (US) has been shown to prevent experimental acute kidney injury through vagus nerve (VN) stimulation and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients may present dysfunctional VN activity, our aim was to determine the effects of therapeutic ultrasound (TUS) in DSS-induced colitis. METHODS: Acute colitis was induced by 2% DSS in drinking water for 7 days and TUS was administered to the abdominal area for 7 min/day from days 4-10. Clinical symptoms were analysed, and biological samples were collected for proteomics, macroscopic and microscopic analysis, flow cytometry and immunohistochemistry. FINDINGS: TUS attenuated colitis by reducing clinical scores, colon shortening and histological damage, inducing proteomic tolerogenic response in the gut during the injury phase and early recovery of experimental colitis. TUS did not improve clinical and pathological outcomes in splenectomised mice, while α7nAChR (α7 nicotinic acetylcholine receptor - indicator of CAIP involvement) knockout animals presented with disease worsening. Increased levels of colonic F4/80+α7nAChR+ macrophages in wild type mice suggest CAIP activation. INTERPRETATION: These results indicate TUS improved DSS-induced colitis through stimulation of the splenic nerve along with possible contribution by VN with CAIP activation. FUND: Intramural Research Programs of the Clinical Centre, the National Institute of Biomedical Imaging and Bioengineering at the NIH and CAPES/Brazil.

Vitamin D supplementation ameliorates arthritis but does not alleviates renal injury in pristane-induced lupus model.

Systemic lupus erythematosus (SLE) is a multifactorial and autoimmune inflammatory disease with pleomorphic clinical manifestations involving different organs and tissues. The study of different murine models has provided a better understanding of these autoimmune phenomena. Pristane-induced lupus represents a suitable model to study factors that could influence the induction and/or progression of SLE, including genetic factors. The objective of the present study was to evaluate the development and evolution of SLE after vitamin D supplementation in PIL model. Here, we evaluated the effects of vitamin D supplementation in model of pristane-induced SLE in female BALB/c mice. The animals were randomly divided into three groups: control group (CO), pristane-induced lupus group (PIL) and pristane-induced lupus group plus vitamin D (VD). Lupus was induced in PIL and VD groups using pristane. PIL group showed arthritis and kidney injury, characterized by increased proteinuria, glomerular mesangial expansion and inflammation. Moreover, PIL model showed increased levels of IL-6, TNF-α and IFN-γ in serum. We observed that treatment with vitamin D improved arthritis through reduced of incidence and arthritis clinical score and edema, but does not influenced renal injury. Treatment with vitamin D was not able to reduce proteinuria levels, decrease mesangial hypercellularity or IgG and IgM deposition in the kidney. Vitamin D supplementation did not alter IL-6, TNF-α, IL-2 and IL-4, but reduce IFN-γ. These results support that the role of vitamin D may be different depending on acting site, what could explain different responses according clinical phenotype. Therefore, further investigations of vitamin D are needed to explore the supplement dosage, timing, and the molecular basis in SLE.

Bioactive factors secreted from mesenchymal stromal cells protect the intestines from experimental colitis in a three-dimensional culture.

BACKGROUND AIMS: Although mesenchymal stromal cells (MSCs) have shown therapeutic potential in intestinal tissue repair, controversy concerning their short survival and poor biodistribution in recipient tissues still remains. Therefore, we investigated the paracrine role of MSC in three-dimensional culture of colon with experimental colitis. METHODS: Colitis was induced in mice by oral administration of dextran sulfate sodium (DSS) for 7 days. Inflammatory responses were assessed on the basis of clinical signs, morphological, and histopathological parameters. On days 2 and 5, colonic explants were removed, and a three-dimensional culture was performed. The structural integrity of the intestinal mucosa was tested by treating the cultures with MSC or conditioned medium (CM) for 24 h, and then the colons were analyzed for histology/immunohistochemistry and interleukin (IL)-6 production. RESULTS: Histological analysis demonstrated that both MSC and CM treatment reduced colon damage in organ culture. An increase in cell proliferation (Ki-67 staining) was observed after CM treatment. Additionally, MSC treatment was able to reduce CD3+ cells. The therapeutic effect of MSC and CM was mediated by the downregulation of IL-6. DISCUSSION: The intestinal in vitro model has shown to be potentially useful for studying cellular interactions in a three-dimensional cell arrangement. Moreover, our results provide strong evidence that both MSC and CM treatments can alleviate colonic damage in organ culture. Importantly, these results suggest that MSC-secreted factors are able to protect the colon from inflammation caused by DSS-induced colitis independent of cell transplantation.

Antioxidant properties of mesenchymal stem cells against oxidative stress in a murine model of colitis.

OBJECTIVE: To investigate the effects of oxidative stress injury in dextran sulfate sodium (DSS)-induced colitis in mice treated with mesenchymal stem cells (MSC). RESULTS: Mice exposed to oral administration of 2% DSS over 7 days presented a high disease activity index and an intense colonic inflammation. Systemic infusion of MSC protected from severe colitis, reducing weight loss and diarrhea while lowering the infiltration of inflammatory cells. Moreover, toxic colitis injury increased oxidative stress. Administration of DSS decreased reduced glutathione (GSH) and superoxide dismutase (SOD) activity, and increased thiobarbituric acid-reactive substances levels in the colon. No alteration was found in catalase (CAT) and glutathione peroxidase (GPx) activity. Otherwise, MSC transplantation was able to prevent the decrease of GSH levels and SOD activity suggestive of an antioxidant property of MSC. CONCLUSION: The oxidative stress is a pathomechanism underlying the pathophysiology of colitis and MSC play an important role in preventing the impairment of antioxidants defenses in inflamed colon.