Polymeric Delivery Systems as a Potential Vaccine against Visceral Leishmaniasis: Formulation Development and Immunogenicity.

Recent studies suggest that the association of antigens in microparticles increases the anti-Leishmania vaccine immunogenicity. This study aims to investigate the in situ effect of the adjuvant performance consisting of chitosan-coated poly(D,L-lactic) acid submicrometric particles (SMP) and analyze the inflammatory profile and toxicity. Two formulations were selected, SMP1, containing poly(D,L-lactide) (PLA) 1% wt/v and chitosan 1% wt/v; and SMP2, containing PLA 5% wt/v and chitosan 5% wt/v. After a single dose of the unloaded SMP1 or SMP2 in mice, the SMPs promoted cell recruitment without tissue damage. In addition, besides the myeloperoxidase (MPO) activity having demonstrated similar results among the analyzed groups, a progressive reduction in the levels of N-acetyl-ß-D-glucosaminidase (NAG) until 72 h was observed for SMPs. While IL-6 levels were similar among all the analyzed groups along the kinetics, only the SMPs groups had detectable levels of TNF-α. Additionally, the Leishmania braziliensis antigen was encapsulated in SMPs (SMP1Ag and SMP2Ag), and mice were vaccinated with three doses. The immunogenicity analysis by flow cytometry demonstrated a reduction in NK (CD3-CD49+) cells in all the SMPs groups, in addition to impairment in the T cells subsets (CD3+CD4+) and CD3+CD8+) and B cells (CD19+) of the SMP2 group. The resulting data demonstrate that the chitosan-coated SMP formulations stimulate the early events of an innate immune response, suggesting their ability to increase the immunogenicity of co-administered Leishmania antigens.

Effects of Psidium guajava L. leaves extract on blood pressure control and IL-10 production in salt-dependent hypertensive rats.

Psidium guajava (guava) leaves extract displays anti-hypertensive properties by mechanisms not yet fully understood. Here, we investigated whether sympathetic drive and immune signaling mechanisms are involved with the antihypertensive effect of the guava extract in a model of salt-dependent hypertension. Raw guava extract (rPsE) was characterized by colorimetric and UPLC-MS techniques. Two doses of rPsE (100 and 200 mg/kg) were evaluated for anti-hypertensive effect using a suspension system (PsE). Weaned male Wistar rats were put on a high-salt diet (HSD, 0.90 % Na+) for 16 weeks and received gavages of PsE for the last 4 weeks. Blood pressure (BP) was measured at the end of treatment in conscious rats. The neurogenic pressor effect was assessed by ganglionic blockade with hexamethonium. Autonomic modulation of heart rate was evaluated by spectral analysis. The effects of orally administered PsE on lumbar sympathetic nerve activity (LSNA) were assessed in anesthetized rats. Blood IL-10, IL-17A, and TNF were measured. The increased neurogenic pressor effect of HSD rats was reduced by PsE 100 mg/kg, but not by 200 mg/kg. PsE (200 mg/kg) administration in anesthetized rats produced a greater fall in BP of HSD rats compared to standard salt diet (SSD) rats. PsE hypotensive response elicited an unproportionable increase in LSNA of HSD rats compared to SSD rats. PsE (200 mg/kg) increased plasma concentrations of IL-10 but had no effect on TNF or IL-17A. Our data indicate that the antihypertensive effects of PsE may involve autonomic mechanisms and immunomodulation by overexpression of IL-10 in salt-dependent hypertensive rats.

Corrigendum: Kinetics of Phenotypic and Functional Changes in Mouse Models of Sponge Implants: Rational Selection to Optimize Protocols for Specific Biomolecules Screening Purposes.

[This corrects the article DOI: 10.3389/fbioe.2020.538203.].

Kinetics of Phenotypic and Functional Changes in Mouse Models of Sponge Implants: Rational Selection to Optimize Protocols for Specific Biomolecules Screening Purposes.

The sponge implant has been applied as an important in vivo model for the study of inflammatory processes as it induces the migration, proliferation, and accumulation of inflammatory cells, angiogenesis, and extracellular matrix deposition in its trabeculae. The characterization of immune events in sponge implants would be useful in identifying the immunological events that could support the selection of an appropriate experimental model (mouse strain) and time post-implant analysis in optimized protocols for novel applications of this model such as in biomolecules screening. Here, the changes in histological/morphometric, immunophenotypic and functional features of infiltrating leukocytes (LEU) were assessed in sponge implants for Swiss, BALB/c, and C57BL/6 mice. A gradual increase of fibrovascular stroma and a progressive decrease in LEU infiltration, mainly composed of polymorphonuclear cells with progressive shift toward mononuclear cells at late time-points were observed over time. Usually, Swiss mice presented a more prominent immune response with late mixed pattern (pro-inflammatory/anti-inflammatory: IL-2/IFN-γ/IL-4/IL-10/IL-17) of cytokine production. While BALB/c mice showed an early activation of the innate response with a controlled cytokine profile (low inflammatory potential), C57BL/6 mice presented a typical early pro-inflammatory (IL-6/TNF/IFN-γ) response with persistent neutrophilic involvement. A rational selection of the ideal time-point/mouse-lineage would avoid bias or tendentious results. Criteria such as low number of increased biomarkers, no recruitment of cytotoxic response, minor cytokine production, and lower biomarker connectivity (described as biomarker signature analysis and network analysis) guided the choice of the best time-point for each model (Day5/Swiss; Day7/BALB/c; Day6/C57BL/6) with wide application for screening purposes, such as identification of therapeutic biomolecules, selection of antigens/adjuvants, and follow-up of innate and adaptive immune response to vaccines candidates.

Salt-dependent hypertension and inflammation: targeting the gut-brain axis and the immune system with Brazilian green propolis.

Systemic arterial hypertension (SAH) is a major health problem around the world and its development has been associated with exceeding salt consumption by the modern society. The mechanisms by which salt consumption increase blood pressure (BP) involve several homeostatic systems but many details have not yet been fully elucidated. Evidences accumulated over the last 60 decades raised the involvement of the immune system in the hypertension development and opened a range of possibilities for new therapeutic targets. Green propolis is a promising natural product with potent anti-inflammatory properties acting on specific targets, most of them participating in the gut-brain axis of the sodium-dependent hypertension. New anti-hypertensive products reinforce the therapeutic arsenal improving the corollary of choices, especially in those cases where patients are resistant or refractory to conventional therapy. This review sought to bring the newest advances in the field articulating evidences that show a cross-talking between inflammation and the central mechanisms involved with the sodium-dependent hypertension as well as the stablished actions of green propolis and some of its biologically active compounds on the immune cells and cytokines that would be involved with its anti-hypertensive properties.

Hydroalcoholic extract of Brazilian green propolis modulates inflammatory process in mice submitted to a low protein diet.

The occurrence of inflammation and protein malnutrition is an aggravating risk factor for morbidity and mortality in the clinical setting. The green propolis, a natural product made by Apis mellifera bees from Baccharis dracunculifolia resin, has therapeutic potential to modulate chronic inflammation. However, its effect on inflammation in an impaired nutritional status is not known. The aim of this study was to characterize the effects of the administration of the hydroalcoholic extract of the green propolis in the chronic inflammatory process of mice submitted to a low-protein diet. For this, we used the subcutaneous implantation of sponge disks as an inflammatory model and the animals were distributed in the following groups: standard protein diet (12% protein content), control treatment; standard protein diet, propolis treatment; low-protein diet (3% protein content), control treatment; low-protein diet, propolis treatment. Propolis was given daily at a dose of 500 mg/kg (p.o.) during a period of 7 or 15 days. Our main findings show that animals fed with standard protein diet and treated with propolis had low levels of red blood cells, hemoglobin, and hematocrit, with the subsequent reestablishment of these levels, in addition to monocyte count elevation and higher TNF levels after one week of treatment. In the low-protein diet group, the propolis treatment provided a significant recovery in weight and maintenance of total serum protein levels at the end of two weeks of treatment. Histological analysis showed propolis reduced the inflammatory infiltrate in the sponges of both standard and low-protein diet groups. In addition, the propolis extract presented antiangiogenic effect in both groups. Therefore, our data suggests that the hydroalcoholic extract of the green propolis promotes weight recovery and avoid the reduction of protein levels, in addition to inhibit inflammation and angiogenesis in animals fed with a low-protein diet.

Methotrexate Locally Released from Poly(ε-Caprolactone) Implants: Inhibition of the Inflammatory Angiogenesis Response in a Murine Sponge Model and the Absence of Systemic Toxicity.

In this study, the methotrexate (MTX) was incorporated into the poly(ε-caprolactone) (PCL) to design implants (MTX PCL implants) aiming the local treatment of inflammatory angiogenesis diseases without causing systemic side effects. Sponges were inserted into the subcutaneous tissue of mice as a framework for fibrovascular tissue growth. After 4 days, MTX PCL implants were also introduced, and anti-inflammatory, antiangiogenic, and antifibrogenic activities of the MTX were determined. MTX reduced the vascularization (hemoglobin content), the neutrophil, and monocyte/macrophage infiltration (MPO and NAG activities, respectively), and the collagen deposition in sponges. MTX reduced tumor necrosis factor-α and IL-6 levels, demonstrating its local antiangiogenic and anti-inflammatory effects. Furthermore, hepatotoxicity, nephrotoxicity, and myelotoxicity, which could be induced by the drug, were evaluated. However, MTX did not promote toxicity to these organs, as the levels of AST and ALT (hepatic markers) and creatinine and urea (renal markers) were not increased, and the complete blood count was not decreased. In conclusion, MTX PCL implants demonstrated to be effective in regulating the components of the inflammatory angiogenesis locally established, and presented an acceptable safety profile.

Leishmania metacyclogenesis is promoted in the absence of purines.

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

Brazilian green propolis inhibits inflammatory angiogenesis in a murine sponge model.

Angiogenesis and inflammation are persistent features of several pathological conditions. Propolis, a sticky material that honeybees collect from living plants, has been reported to have multiple biological effects including anti-inflammatory and anti-neoplasic activities. Here, we investigated the effects of water extract of green propolis (WEP) on angiogenesis, inflammatory cell accumulation and endogenous production of cytokines in sponge implants of mice over a 14-day period. Blood vessel formation as assessed by hemoglobin content and by morphometric analysis of the implants was reduced by WEP (500 mg kg(-1) orally) compared to the untreated group. The levels of vascular endothelial growth factor (VEGF) increased progressively in the treated group but decreased after Day 10 in the control group. Accumulation of neutrophils and macrophages was determined by measuring myeloperoxidase (MPO) and N-acetyl-ß-(D)-glucosaminidase (NAG) activities, respectively. Neutrophil accumulation was unaffected by propolis, but NAG activity was reduced by the treatment at Day 14. The levels TGF-ß1 intra-implant increased progressively in both groups but were higher (40%) at Day 14 in the control implants. The pro-inflammatory levels of TNF-α peaked at Day 7 in the control implants, and at Day 14 in the propolis-treated group. Our results indicate that the anti-inflammatory/anti-angiogenic effects of propolis are associated with cytokine modulation.

Aqueous extract of brazilian green propolis: primary components, evaluation of inflammation and wound healing by using subcutaneous implanted sponges.

Propolis is a chemically complex resinous bee product which has gained worldwide popularity as a means to improve health condition and prevent diseases. The main constituents of an aqueous extract of a sample of green propolis from Southeast Brazil were shown by high performance liquid chromatography/mass spectroscopy/mass spectroscopy to be mono- and di-O-caffeoylquinic acids; phenylpropanoids known as important constituents of alcohol extracts of green propolis, such as artepillin C and drupanin were also detected in low amounts in the aqueous extract. The anti-inflammatory activity of this extract was evaluated by determination of wound healing parameters. Female Swiss mice were implanted subcutaneously with polyesther-polyurethane sponge discs to induce wound healing responses, and administered orally with green propolis (500 mg kg(-1)). At 4, 7 and 14 days post-implantation, the fibrovascular stroma and deposition of extracellular matrix were evaluated by histopathologic and morphometric analyses. In the propolis-treated group at Days 4 and 7 the inflammatory process in the sponge was reduced in comparison with control. A progressive increase in cell influx and collagen deposition was observed in control and propolis-treated groups during the whole period. However, these effects were attenuated in the propolis-treated group at Days 4 and 7, indicating that key factors of the wound healing process are modulated by propolis constituents.