Activation of murine macrophages by membrane proteins from Tritrichomonas foetus grown on iron- and calcium-rich conditions.

Tritrichomonas foetus is a protozoan parasite that causes a venereal disease in cattle limiting reproduction by abortions and sterility. The immune response against this parasite is poorly understood. Since the iron and calcium ions are important regulators of the microenvironment of the urogenital tract in cattle, we decided to evaluate the role of these divalent cations on the antigenicity of membrane proteins of T. foetus on macrophage activation as one of the first inflammatory responses towards this pathogen. Colorimetric methods and ELISA were used to detect the nitric oxide and oxygen peroxide production and expression of cytokines in culture supernatant from macrophage incubated with membrane proteins from T. foetus cultured in iron- and calcium-rich conditions. qRT-PCR assays were used to evaluate the transcript expression of genes involved in the inflammatory response on the macrophages. The membrane proteins used for in vitro stimulation caused the up-regulation of the iNOS and NOX-2 genes as well as the generation of NO and H2 O2 in murine macrophages on a dependent way of the metal concentrations. Additionally, after stimulation, macrophages showed a considerable rise in pro-inflammatory cytokines and a downregulation of anti-inflammatory cytokines, as well as up-regulation in the transcription of the TLR4 and MyD88 genes. These data suggest that membrane proteins of T. foetus induced by iron and calcium can activate an inflammatory specific macrophage response via TLR4/MyD88 signalling pathway.

Exposure of Tritrichomonas foetus to sublethal doses of metronidazole induces a specific proinflammatory response in murine macrophages.

Tritrichomonas foetus is a flagellated parasite that primarily infects the reproductive tissues of livestock, causing bovine trichomoniasis. The cytoplasmic membrane of T. foetus contains various compounds that contribute to adherence, colonization, and pathogenicity. Metronidazole (MTZ) is the main treatment for trichomoniasis, but the emergence of drug-resistant strains is a concern due to improper use and dosing. T. foetus infection induces inflammation, and macrophages are key players in the immune response. However, our understanding of the host's immune response to T. foetus is limited, and the specific mechanisms underlying these responses are not well understood. This study aimed to investigate the impact of T. foetus surface proteins from trophozoites cultured under different sublethal MTZ conditions (MTZ-treated T. foetus MPs) on macrophage activation. By analyzing cytokine levels and gene expression in murine macrophages, we demonstrated that MTZ-treated T. foetus MPs induce a specific proinflammatory response. MTZ-treated T. foetus MPs-exposed macrophages exhibited a higher NO and H2 O2 production and overexpression of iNOS and NOX-2 genes in comparison to untreated T. foetus. Additionally, MTZ-treated T. foetus MPs triggered a significant induction of the proinflammatory cytokines IL-1ß, IL-6, TNF-α, and IFN-γ, as well as the overexpression of the TLR4, MyD88, and NF-κB genes on murine macrophages. The study aimed to unravel the immunological response and potential proinflammatory pathways involved in T. foetus infection and MTZ stress. Understanding the immune responses and mechanisms through which T. foetus surface proteins activate macrophages can contribute to the development of new therapeutic strategies for controlling bovine trichomoniasis.

Macrophages morphology and cytokine reeducation by ex situ copper thiol complexes.

OBJECTIVE: To study the reeducation effect of copper thiol complexes on macrophage morphology and cytokine expression. METHODS: The effect of copper thiol complexes was assessed on murine macrophages by the cell morphology observed through optical microscopy, while the expression of cytokines by protein abundance after stimulation. A viability experiment was performed on PMBC to confirm that copper complexes do not affect other cells. RESULTS: The M1 shape was reported after treatment with copper thiol complexes at 1-200 µM, while M2 behavior was documented between 50 and 800 µM. Surprisingly, a thin elongate morphology was observed between 400-800 µM like the M2 shape. The expression of M1 cytokines was noted ranging from 1 to 100 µM, with the highest yield at 1 µM (2243 pg/µL) for the copper-penicillamine complex. M2 production behavior was observed at 1-800 µM, with the highest abundance close to 1150 pg/µL (200-400 µM) was quantified from the copper-cysteine complex. Finally, LCCu complexes did not induce a cytotoxic response on PBMC while exhibiting a high IL-4 and IL-10 production, similar to their gold analogs. CONCLUSIONS: The capacity of copper thiol complexes to reeducate M1 to M2 morphoexpression can be promising for cell protection by using copper thiol penicillamine or immuno-regeneration of tissues when using copper thiol cysteine.

Novel copper complexes-polyurethane composites that mimics anti-inflammatory response.

Copper is a trace element of biological significance that can form complexes with several thiol containing compounds which can be used as filler in biomedical polyurethanes. In this work, segmented polyurethanes (SPUs) were synthesized with thiol containing compounds as chain extenders including d-penicillamine (DP), l-penicillamine (LP), l-cysteine (LC) and reduced glutathione (GR). Then, the synthesized polyurethane was filled with copper chelates based on the same chain extenders. Evidence of free thiol containing chain extender in polyurethane was not observed by FTIR and Raman but EDX provided evidence of sulfur in the unfilled polyurethane and copper and sulfur in their composite. DSC and DRX showed the semi-crystalline nature of the polyurethanes which provided good mechanical properties, especially to those prepared with DP. The Tg of the PCL determined by DMA shifted toward higher temperatures by the addition of copper complexes while TGA studies showed that the thermal degradation was slightly improved when LCCu and GRCu complex were added. Macrophage viability was observed in all composition studied after longer times of extraction (72 h) and dilutions (1:2 to 1:32) but remarkably high in those prepared with LCCu and GRCu. The anti-inflammatory response was proved in LC and GR copper complex filled polyurethanes as IL-4 and IL-10 increased with time while IL-1ß and TNF-α were reduced.

Vaccine-linked chemotherapy with a low dose of benznidazole plus a bivalent recombinant protein vaccine prevents the development of cardiac fibrosis caused by Trypanosoma cruzi in chronically-infected BALB/c mice.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi and affects 6-7 million people worldwide. Approximately 30% of chronic patients develop chronic chagasic cardiomyopathy (CCC) after decades. Benznidazole (BNZ), one of the first-line chemotherapy used for CD, induces toxicity and fails to halt the progression of CCC in chronic patients. The recombinant parasite-derived antigens, including Tc24, Tc24-C4, TSA-1, and TSA-1-C4 with Toll-like receptor 4 (TLR-4) agonist-adjuvants reduce cardiac parasite burdens, heart inflammation, and fibrosis, leading us to envision their use as immunotherapy together with BNZ. Given genetic immunization (DNA vaccines) encoding Tc24 and TSA-1 induce protective immunity in mice and dogs, we propose that immunization with the corresponding recombinant proteins offers an alternative and feasible strategy to develop these antigens as a bivalent human vaccine. We hypothesized that a low dose of BNZ in combination with a therapeutic vaccine (TSA-1-C4 and Tc24-C4 antigens formulated with a synthetic TLR-4 agonist-adjuvant, E6020-SE) given during early chronic infection, could prevent cardiac disease progression and provide antigen-specific T cell immunity. METHODOLOGY/ PRINCIPAL FINDINGS: We evaluated the therapeutic vaccine candidate plus BNZ (25 mg/kg/day/7 days) given on days 72 and 79 post-infection (p.i) (early chronic phase). Fibrosis, inflammation, and parasite burden were quantified in heart tissue at day 200 p.i. (late chronic phase). Further, spleen cells were collected to evaluate antigen-specific CD4+ and CD8+ T cell immune response, using flow cytometry. We found that vaccine-linked BNZ treated mice had lower cardiac fibrosis compared to the infected untreated control group. Moreover, cells from mice that received the immunotherapy had higher stimulation index of antigen-specific CD8+Perforin+ T cells as well as antigen-specific central memory T cells compared to the infected untreated control. CONCLUSIONS: Our results suggest that the bivalent immunotherapy together with BNZ treatment given during early chronic infection protects BALB/c mice against cardiac fibrosis progression and activates a strong CD8+ T cell response by in vitro restimulation, evidencing the induction of a long-lasting T. cruzi-immunity.

In Vitro Activation of Macrophages by an MHC Class II-restricted Trichomonas Vaginalis TvZIP8-derived Synthetic Peptide.

BACKGROUND: Macrophages play an important role in the inflammatory response towards pathogens and their effector functions depend on the mode of activation which is mediated by recognition of pathogen-associated molecular patterns, as peptides. Trichomonas vaginalis provokes an inflammatory response in the host in which macrophages are the first line of defense. This study aimed to analyze the effect of a specific peptide derived from the transporter TvZIP8 of T. vaginalis on the activation of macrophages. METHODS: An in silico approach based on computational prediction of epitopes was applied to detect potential murine MHC class II-restricted peptides from TvZIP8 that can activate macrophages and the immunomodulatory activity was evaluated by in vitro stimulation of murine macrophages. RESULTS: Based on binding scores, one peptide denominated TvZIP8-pep was selected for further analysis. In vitro stimulation with synthetic TvZIP8-pep triggered on murine macrophages the NO and H2O2 production and an overexpression of iNOS and NOX-2 genes. Also, a significant increase of pro-inflammatory cytokines: IL-1ß, IL-6, and TNF-α, as well as, overexpression of the TLR4, MyD88, and NF-κB genes and NF-κB activation were observed on macrophages after stimulation with TvZIP8-pep in vitro. Moreover, higher levels of IFN-γ were detected in co-cultures using CD4 + T cells with TvZIP8-pep-stimulated macrophages. CONCLUSION: These results support the potential of TvZIP8 as a promising antigen to stimulates a specific macrophage response against T. vaginalis, but further analyses are required to evaluate their possible potential as a novel antigen for immunodiagnosis and/or vaccine against trichomoniasis.

Anti-inflammatory effects of Chrysophyllum cainito fruit extract in lipopolysaccharide-stimulated mouse peritoneal macrophages.

The present paper sought to investigate the in vitro and in vivo anti-inflammatory effects of the methanolic extract (ME), hexane-ethyl acetate fraction E (FE) found in Chrysophyllum cainito fruits (CCF), as well the lupeol acetate (LA) obtained from FE on lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. The macrophages were treated with ME, FE or LA at various concentrations and the viability of cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. Production of pro-inflammatory (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokines, as well as the nitric oxide (NO) and hydrogen peroxide (H2O2) levels was determined using macrophages treated with ME, FE or LA at various concentrations and stimulated with LPS as an in vitro model. Afterwards, we evaluated the anti-inflammatory effects in vivo using the TPA-induced ear edema and carrageenan-induced paw edema tests in mice and production of inflammatory mediators was estimated in serum samples. The results showed that the ME, FE and LA from fruits, FE and LA were able to trigger an inhibition in NO and H2O2 levels, as well as IL-1ß, IL-6, and TNF-α released by macrophages in a concentration-dependent manner. LA from C. cainito fruits was found to significantly attenuate carrageenan-induced paw edema and TPA-induced ear edema. Therefore, the results suggest ME, FE and LA isolated from C. cainito fruits have anti-inflammatory effects on macrophages without affecting cell viability.

Anti-inflammatory and antinociceptive effects of an ethanol extract from Senna septemtrionalis.

Senna septemtrionalis (Viv.) H.S. Irwin & Barneby (Fabaceae) is a medicinal plant used as a folk remedy for inflammation and pain. The objective of this study was to evaluate the anti-inflammatory and antinociceptive actions of an ethanol extract of Senna septemtrionalis aerial parts (SSE). The in vitro anti-inflammatory effects of SSE were assessed using LPS-stimulated macrophages and the subsequent quantification of the levels of cytokines (IL-6, IL-1ß, and TNF-α) with ELISA kits, nitric oxide (NO), and hydrogen peroxide (H2O2). The in vivo anti-inflammatory actions of SSE were evaluated with the TPA-induced ear oedema test and the carrageenan-induced paw oedema test. The antinociceptive actions of SSE (10-200 mg/kg p.o.) were assessed using three models: two chemical assays (formalin-induced orofacial pain and acetic acid-induced visceral pain) and one thermal assay (hot plate). SSE showed in vitro anti-inflammatory actions with IC50 values calculated as follows: 163.3 µg/ml (IL-6), 154.7 µg/ml (H2O2) and > 200 µg/ml (IL-1ß, TNF-α, and NO). SSE showed also in vivo anti-inflammatory actions in the TPA test (40% of inhibition of ear oedema) and the carrageenan test (ED50 = 137.8 mg/kg p.o.). SSE induced antinociceptive activity in the formalin orofacial pain test (ED50 = 80.1 mg/kg) and the acetic acid-induced writhing test (ED50 = 110 mg/kg). SSE showed no antinociceptive actions in the hot plate assay. The pre-treatment with glibenclamide abolished the antinociceptive action shown by SSE alone. Overall, SSE exerted in vitro and in vivo anti-inflammatory actions, and in vivo antinociceptive effects by the possible involvement of ATP-sensitive K + channels.

Immunosuppressive effects of protein derivatives from Mucuna pruriens on a streptozotocin-induced type 1 diabetes murine model.

Type 1 diabetes is an autoimmune disease induced by abnormal insulin secretions from ß-cells in pancreas. The present study aimed to investigate the immunosuppressive effects from protein derivatives of Mucuna pruriens on a murine model of Type 1 diabetes. Hydrolyzate and five peptide fractions with different molecular weight were administered orally by 14 days, followed T1D murine model was built by intraperitoneal injection of streptozotocin over 5 days. The mice weight, blood glucose levels, anti-insulin, and anti-pancreatic islet ß-cells antibodies, pro-inflammatory cytokines as tumor necrosis factor alpha and interleukin-6 were determined in four times (0, 15, 30, and 45 day). Mice were sacrificed and pancreatic tissues samples were obtained and staining with hematoxylin and eosin to determine the degree of damage. The study demonstrated immunosuppressive activity in four of the six treatment groups: (a) T1D PPH, (b) T1D F 5-10 kDa, (c) T1D F 3-5 kDa, and (d) T1D F 1-3 kDa. PRACTICAL APPLICATIONS: Due to the high content of native protein in seeds of Mucuna pruriens, studies have reported potential in the elaboration of hydrolysates and peptides with biological activity. These protein derivatives could help in the treatment of immunological disorders that are observed in several chronic non-communicable disease and inflammatory diseases, such as T1D. Activated macrophages and lymphoplasmacytic infiltrate plays a crucial role in the initiation and maintenance of T1D; therefore, several studies has focused to reduce the effector functions of this cells for diminishing the clinical manifestations in inmmunocompromised patients. Thus, this study indicates the potential application of hydrolyzate and peptide fractions of M. pruriens in functional foods and dietary supplements could be developed for the treatment of inflammatory and chronic non-communicable diseases.

IMMUNOSUPPRESIVE EFFECTS OF THE METHANOLIC EXTRACT OF CHRYSOPHYLLUM CAINITO LEAVES ON MACROPHAGE FUNCTIONS.

BACKGROUND: The aim of this work was to evaluate the immunomodulatory effect of the methanol extract (MeOH) from Chrysophyllum cainito leaves on the MΦs functions. MATERIAL AND METHODS: Peritoneal murine MΦs isolated from Balb/c mice were treated with the MeOH extract and stimulated with LPS. The effect on the phagocytosis was evaluated by flow cytometry assay. The nitric oxide (NO) and hydrogen peroxide (H2O2) production was measured by the Griess reagent and phenol red reaction, respectively. Levels of IL-6 and TNF-α was measured using an ELISA kit. Viability of MΦs and Vero cells was determined by the MTT method. RESULTS: The MeOH extract of C. cainito leaves inhibited significantly the phagocytosis, and decreased IL-6 and TNF-α production as well as NO and H2O2 released by the MΦs, in a concentration-dependent manner. In addition, MeOH extract of C. cainito showed low cytotoxicity effect against the cells. CONCLUSION: These results suggest that MeOH extract of C. cainito leaves has an immunosuppressive effect on murine MΦs, without effects on cell viability. GC-MS chromatogram analysis of MeOH extract showed that lupeol acetate and alpha-amyrin acetate are the principal compounds.