Summary Alveolar echinococcosis (AE) caused by the larval stage of Echinococcus multilocularis is serious parasitic diseases associated with the host´s immunosuppression. The effects of human non-immune dialyzable leukocyte extract (DLE) on immune cells in blood and spleen and parasitic cysts weight in Balb/c mice after oral (PO), subcutaneous (SC) and intraperitoneal administration (IP) were compared. The reduction in cysts weight (p < 0.01) was recorded after PO route, whereas moderate reduction was found after SC and IP routes. The elevation of lymphoid populations in blood and spleen was found after PO administration (p < 0.01) in parallel with reduced myeloid population. Infection-elicited decline in B220+B cells was partially abolished by PO route, but DLE routes did not influence the CD3+ T cells. The proportions of CD3+CD4+Th lymphocytes were moderately upregulated, whereas CD3+CD8+Tc populations were reduced after all DLE routes (p < 0.01). PO administration increased CD11b+MHCIIhigh blood monocytes, CD11b-SigleF+ cell, but not CD11b+Si-glecF+ eosinophils in the blood, stimulated after SC and IP routes. DLE induced downregulation of NO production by LPS-stimulated adherent splenocytes ex vivo. Con A-triggered T lymphocyte proliferation was associated with the elevated IFN-γ production and transcription factor Tbet mRNA expression. The alleviation of Th2 (IL-4) and Treg (TGF-β) cytokine production by lymphocytes ex vivo paralleled with downregulation of gene transcription for cytokines, GATA and FoxP3. Reduction of myeloid cells with suppressive activity was found. The SC and IP routes affected partially the cysts weights, diminished significantly gene transcription, NO levels and Th2 and Treg cytokines production. Results showed that PO route of DLE administration was the most effective in ameliorating immunosuppression via stimulation of Th1 type, reducing Th2 and Treg type of immunity and CD3+CD8+Tc lymphocytes in the blood and spleens during E. multilocularis infection in mice.

The model flatworm Mesocestoides vogae proliferating stage of infection elicits immunosuppression in the host. It was used to investigate the effects of human leukocyte extract (DLE) alone and in combination with anthelmintic albendazole (ABZ) on the reduction in peritoneal infection, peritoneal exudate cells (PECs), their adherent counterparts, and peritoneal exudates after the termination of therapy. Balb/c mice were infected with the larvae of M. vogae. PECs and adherent macrophages were studied via flow cytometry, mRNA transcript levels, and immunofluorescence. The cytokine levels were measured via ELISA and larvae were counted. ABZ significantly reduced larval counts (581.2 ± 65, p < 0.001), but the highest reduction was observed after combined treatment with ABZ and DLE (389.2 ± 119, p < 0.001) in comparison with the control. Compared to an infected group, the proportions of CD11b+CD19- myeloid cells with suppressive ability decreased after albendazole (ABZ) in combination with DLE, which was the most effective in the elevation of B cells and CD11b+F4/80mid/highMHCIIhigh macrophages/monocytes (22.2 ± 5.4%). Transcripts of the M2 macrophage markers (arginase 1, FIZZ-1, and Ym-1) were downregulated after DLE and combined therapy but not after ABZ, and the opposite trend was seen for iNOS. This contrasts with reduced ex vivo NO production by LPS-stimulated PECs from DLE and ABZ+DLE groups, where adherent macrophages/monocytes had elevated transcripts of the INF-γ receptor and STAT1 and reduced expression of STAT3, STAT6, and IL-10. Each therapy differentially modulated transcription profiles and concentrations of IFN-γ, TNF-α, IL-12p40, IL-6, IL-10, and TGF-β cytokines. DLE strongly ameliorated ABZ-induced suppression of INF-γ and IL-12 and preserved downregulation for IL-4, IL-10, and TGF-β. Epigenetic study on adherent macrophages from infected mice showed that ABZ, ABZ-sulfoxide, and DLE could interact with the mRNA of examined markers in a dose-dependent pattern. Co-administration of DLE with ABZ seemed to augment the drug’s larvicidal effect via modulation of immunity. In comparison with ABZ, combined therapy was the most effective in alleviating parasite-induced Th2/Treg/STAT3/STA6 directed immunosuppression by stimulating the Th1 cytokines, M1 macrophage polarization, and activation of the IFNγ/STAT1 signaling pathway.

Carotenoids are the most abundant lipid-soluble phytochemicals and are used as dietary supplements to protect against diseases caused by oxidative stress. Astaxanthin, a xanthophyll carotenoid, is a very potent antioxidant with numerous beneficial effects on cellular functions and signaling pathways. In this study, using spleen cells from healthy Balb/c mice, we report the bio-functional effects of an astaxanthin-rich extract (EXT) prepared from the microalga Haematococcus pluvialis and its astaxanthin monoesters-rich fraction (ME) and astaxanthin diesters-rich fraction (DE) obtained by fractionation of EXT using countercurrent chromatography (CCC). After incubation under standard culture conditions (humidity, 37 °C, 5% CO2, atmospheric oxygen), the viability of untreated splenocytes, as determined by the trypan blue exclusion assay, the MTT assay, and the neutral red assay, decreases to approximately 75% after 24 h compared with naïve splenocytes. This effect correlated with the decrease in mitochondrial membrane potential and the transition of ~59% of cells to the early stage of apoptosis, as well as with the decreased ROS production, indicating that hyperoxia in cell-culture deteriorates cell functions. They are restored or stimulated by co-cultivation with EXT, ME, and DE up to 10 µg/mL in the order EXT > DE > ME, suggesting that esterification increases bioavailability to cells in vitro. ROS and H2O2 concentrations reflect mRNA transcriptional activity of Nrf2, superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 1, as well as SOD-mediated ROS conversion, whereas they inversely correlate with iNOS-mediated NO production. The highest-tested concentration of EXT, ME, and DE (40 µg/mL) is detrimental to cells, probably because of the overwhelming scavenging activity of astaxanthin and its esters for the reactive oxygen/nitrogen species required for cellular functions and signal transduction at low physiological concentrations. In this study, we demonstrate that differential activities of ME and DE contribute to the final antioxidant and cytoprotective effects of astaxanthin extract, which is beneficial in preventing a wide range of ROS-induced adverse effects, with DE being more effective. In addition, the selection of physioxia-like conditions for pharmacological research is highlighted.