Exploring the Immunotherapeutic Potential of Oleocanthal against Murine Cutaneous Leishmaniasis.

Leishmaniasis is a major tropical disease with increasing global incidence. Due to limited therapeutic options with severe drawbacks, the discovery of alternative treatments based on natural bioactive compounds is important. In our previous studies we have pointed out the antileishmanial activities of olive tree-derived molecules. In this study, we aimed to investigate the in vitro and in vivo antileishmanial as well as the in vivo immunomodulatory effects of oleocanthal, a molecule that has recently gained increasing scientific attention. Pure oleocanthal was isolated from extra virgin olive oil through extraction and chromatography techniques. The in vitro antileishmanial effects of oleocanthal were examined with a resazurin-based assay, while its in vivo efficacy was evaluated in Leishmania major-infected BALB/c mice by determining footpad induration, parasite load in popliteal lymph nodes, histopathological outcome, antibody production, cytokine profile of stimulated splenocytes and immune gene expression, at three weeks after the termination of treatment. Oleocanthal demonstrated in vitro antileishmanial effect against both L. major promastigotes and intracellular amastigotes. This effect was further documented in vivo as demonstrated by the suppressed footpad thickness, the decreased parasite load and the inflammatory cell influx at the infection site. Oleocanthal treatment led to the dominance of a Th1-type immunity linked with resistance against the disease. This study establishes strong scientific evidence for olive tree-derived natural products as possible antileishmanial agents and provides an adding value to the scientific research of oleocanthal.

Spontaneous human CD8 T cell and autoimmune encephalomyelitis-induced CD4/CD8 T cell lesions in the brain and spinal cord of HLA-DRB1*15-positive multiple sclerosis humanized immune system mice.

Autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are only partially represented in current experimental models and the development of humanized immune mice is crucial for better understanding of immunopathogenesis and testing of therapeutics. We describe a humanized mouse model with several key features of MS. Severely immunodeficient B2m-NOG mice were transplanted with peripheral blood mononuclear cells (PBMCs) from HLA-DRB1-typed MS and healthy (HI) donors and showed rapid engraftment by human T and B lymphocytes. Mice receiving cells from MS patients with recent/ongoing Epstein-Barr virus reactivation showed high B cell engraftment capacity. Both HLA-DRB1*15 (DR15) MS and DR15 HI mice, not HLA-DRB1*13 MS mice, developed human T cell infiltration of CNS borders and parenchyma. DR15 MS mice uniquely developed inflammatory lesions in brain and spinal cord gray matter, with spontaneous, hCD8 T cell lesions, and mixed hCD8/hCD4 T cell lesions in EAE immunized mice, with variation in localization and severity between different patient donors. Main limitations of this model for further development are poor monocyte engraftment and lack of demyelination, lymph node organization, and IgG responses. These results show that PBMC humanized mice represent promising research tools for investigating MS immunopathology in a patient-specific approach.

A liposomal vaccine promotes strong adaptive immune responses via dendritic cell activation in draining lymph nodes.

Subunit proteins provide a safe source of antigens for vaccine development especially for intracellular infections which require the induction of strong cellular immune responses. However, those antigens are often limited by their low immunogenicity. In order to achieve effective immune responses, they should be encapsulated into a stable antigen delivery system combined with an appropriate adjuvant. As such cationic liposomes provide an efficient platform for antigen delivery. In the present study, we describe a liposomal vaccine platform for co-delivery of antigens and adjuvants able to elicit strong antigen-specific adaptive immune responses. Liposomes are composed of the cationic lipid dimethyl dioctadecylammonium bromide (DDAB), cholesterol (CHOL) and oleic acid (OA). Physicochemical characterization of the formulations showed that their size was in the range of ∼250 nm with a positive zeta potential which was affected in some cases by the enviromental pH facilitating endosomal escape of potential vaccine cargo. In vitro, liposomes were effectively taken up by bone marrow dendritic cells (BMDCs) and when encapsulated IMQ they promoted BMDCs maturation and activation. Upon in vivo intramuscular administration, liposomes' active drainage to lymph nodes was mediated by DCs, B cells and macrophages. Thus, mice immunization with liposomes having encapsulated LiChimera, a previously characterized anti-leishmanial antigen, and IMQ elicited infiltration of CD11blow DCs populations in draining LNs followed by increased antigen-specific IgG, IgG2a and IgG1 levels production as well as indcution of antigen-specific CD4+ and CD8+ T cells. Collectively, the present work provides a proof-of-concept that cationic liposomes composed of DDAB, CHOL and OA adjuvanted with IMQ provide an efficient delivery platform for protein antigens able to induce strong adaptive immune responses via DCs targeting and induction of maturation.

Clusterin overexpression in mice exacerbates diabetic phenotypes but suppresses tumor progression in a mouse melanoma model.

Clusterin (CLU) is an ATP-independent small heat shock protein-like chaperone, which functions both intra- and extra-cellularly. Consequently, it has been functionally involved in several physiological (including aging), as well as in pathological conditions and most age-related diseases, e.g., cancer, neurodegeneration, and metabolic syndrome. To address CLU function at an in vivo model we established CLU transgenic (Tg) mice bearing ubiquitous or pancreas-targeted CLU overexpression (OE). Our downstream analyses in established Tg lines showed that ubiquitous or pancreas-targeted CLU OE in mice affected antioxidant, proteostatic and metabolic pathways. Targeted OE of CLU in the pancreas, which also resulted in CLU upregulation in the liver likely via systemic effects, increased basal glucose levels in the circulation and exacerbated diabetic phenotypes. Furthermore, by establishing a syngeneic melanoma mouse tumor model we found that ubiquitous CLU OE suppressed melanoma cells growth, indicating a likely tumor suppressor function in early phases of tumorigenesis. Our observations provide in vivo evidence corroborating the notion that CLU is a potential modulator of metabolic and/or proteostatic pathways playing an important role in diabetes and tumorigenesis.