Lower peripheral blood Toll-like receptor 3 expression is associated with an unfavorable outcome in severe COVID-19 patients.

The role of innate immunity in COVID-19 is not completely understood. Therefore, this study explored the impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection on the expression of Pattern Recognition Receptors (PRRs) in peripheral blood cells and their correlated cytokines. Seventy-nine patients with severe COVID-19 on admission, according to World Health Organization (WHO) classification, were divided into two groups: patients who needed mechanical ventilation and/or deceased (SEVERE, n = 50) and patients who used supplementary oxygen but not mechanical ventilation and survived (MILD, n = 29); a control group (CONTROL, n = 17) was also enrolled. In the peripheral blood, gene expression (mRNA) of Toll-like receptors (TLRs) 3, 4, 7, 8, and 9, retinoic-acid inducible gene I (RIGI), NOD-like receptor family pyrin domain containing 3 (NLRP3), interferon alpha (IFN-α), interferon beta (IFN-ß), interferon gamma (IFN-γ), interferon lambda (IFN-λ), pro-interleukin(IL)-1ß (pro-IL-1ß), and IL-18 was determined on admission, between 5-9 days, and between 10-15 days. Circulating cytokines in plasma were also measured. When compared to the COVID-19 MILD group, the COVID-19 SEVERE group had lower expression of TLR3 and overexpression of TLR4.

Roux-en-Y Gastric Bypass Surgery Induces Distinct but Frequently Transient Effects on Acylcarnitine, Bile Acid and Phospholipid Levels.

Roux-en-Y gastric bypass (RYGB) is an effective method to achieve sustained weight loss, but the mechanisms responsible for RYGB effects have not yet been fully characterized. In this study, we profiled the concentrations of 143 lipid metabolites in dry blood spots (DBS) of RYGB patients. DBS from obese patients (BMI range 35⁻44 kg/m²) were collected 7 days before, 15 and 90 days after the surgery. LC-MS/MS was used to quantify acylcarnitines, phosphatidylcholines, sphingomyelins and bile acids. RYGB caused a rapid increase in acylcarnitine levels that proved to be only transient, contrasting with the sustained decrease in phosphatidylcholines and increase of sphingomyelins and bile acids. A PLS-DA analysis revealed a 3-component model (R² = 0.9, Q² = 0.74) with key metabolites responsible for the overall metabolite differences. These included the BCAA-derived acylcarnitines and sphingomyelins with 16 and 18 carbons. We found important correlations between the levels of BCAA-derived acylcarnitines and specific sphingomyelins with plasma cholesterol and triacylglycerol concentrations. Along with the marked weight loss and clinical improvements, RYGB induced specific alterations in plasma acylcarnitines, bile acid and phospholipid levels. This calls for more studies on RYGB effects aiming to elucidate the metabolic adaptations that follow this procedure.

Modulation of pineal melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through NF-κB activation.

The glutamatergic modulation of melatonin synthesis is well known, along with the importance of astrocytes in mediating glutamatergic signaling in the central nervous system. Pinealocytes and astrocytes are the main cell types in the pineal gland. The objective of this work was to investigate the interactions between astrocytes and pinealocytes as a part of the glutamate inhibitory effect on melatonin synthesis. Rat pinealocytes isolated or in coculture with astrocytes were incubated with glutamate in the presence of norepinephrine, and the melatonin content, was quantified. The expression of glutamate receptors, the intracellular calcium content and the NF- κ B activation were analyzed in astrocytes and pinealocytes. TNF- α 's possible mediation of the effect of glutamate was also investigated. The results showed that glutamate's inhibitory effect on melatonin synthesis involves interactions between astrocytes and pinealocytes, possibly through the release of TNF- α . Moreover, the activation of the astrocytic NF- κ B seems to be a necessary step. In astrocytes and pinealocytes, AMPA, NMDA, and group I metabotropic glutamate receptors were observed, as well as the intracellular calcium elevation. In conclusion, there is evidence that the modulation of melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through the activation of the astrocytic NF- κ B transcription factor and possibly by subsequent TNF- α release.

Mechanisms by which fatty acids regulate leucocyte function.

Fatty acids (FAs) have been shown to alter leucocyte function and thus to modulate inflammatory and immune responses. In this review, the effects of FAs on several aspects of lymphocyte, neutrophil and macrophage function are discussed. The mechanisms by which FAs modulate the production of lipid mediators, activity of intracellular signalling pathways, activity of lipid-raft-associated proteins, binding to TLRs (Toll-like receptors), control of gene expression, activation of transcription factors, induction of cell death and production of reactive oxygen and nitrogen species are described in this review. The rationale for the use of specific FAs to treat patients with impaired immune function is explained. Substantial improvement in the therapeutic usage of FAs or FA derivatives may be possible based on an improvement in the understanding of the precise molecular mechanisms of action with respect to the different leucocyte types and outcome with respect to the inflammatory responses.

Physical training attenuates the stress-induced changes in rat T-lymphocyte function.

BACKGROUND/AIMS: Modulations in the immune function by stress are a well-known phenomenon. Acute restraint stress may induce impaired T-lymphocyte responses. Moderate physical training is associated with beneficial effects on immunological functions. We investigated the effects of a moderate physical training on T-lymphocyte function in rats submitted to acute restraint stress. METHODS: Thirty male Wistar rats weighing 210-226 g were randomly divided into four groups: non-trained rats (NT, n = 7), and non-trained rats submitted to stress (NT + S, n = 8); trained rats (T, n = 7), and trained rats submitted to stress (T + S, n = 8). Trained rats were submitted to a program of moderate running over a period of 8 weeks. Rats subjected to restraint stress were kept immobilized in glass cylinders (8 cm in diameter and 24 cm long) during 60 min. Plasma corticosterone concentration, peripheral blood leukocyte number, indicators of apoptosis of T lymphocytes in blood and lymphoid organs, and mitogen-induced proliferation of T lymphocytes in lymphoid organs were evaluated. RESULTS: Acute stress exposure raised plasma corticosterone concentration (p < 0.001), but not in previously trained animals. Restraint stress induced an increase in the percentage of lymphocytes in apoptosis, and a decrease in the concanavalin-A-induced proliferation of lymphocytes from the thymus and lymph nodes, and an increase in lymphocytes of the spleen. Neither of these alterations was observed in trained animals submitted to acute restraint stress. CONCLUSIONS: Our data confirm that acute restraint stress is associated with changes in T-lymphocyte function. Moreover, moderate physical training attenuates the effects of acute stress by a mechanism that involves the hypothalamic-pituitary-adrenal axis and an increase in tolerance of leukocytes.

Mechanisms involved in Jurkat cell death induced by oleic and linoleic acids.

BACKGROUND & AIMS: Previous study from our laboratory showed the toxicity of oleic (OA) and linoleic acids (LA) on Jurkat and Raji cells and human lymphocytes in vitro. The mechanisms involved in the toxicity induced by OA and LA on Jurkat cells were determined in vitro. METHODS: Jurkat cells were treated in the presence of OA and LA (25, 50, 100 and 200muM). The parameters investigated were: triglycerides and cholesterol ester concentrations determined by enzymatic assay, activation of peroxisome proliferator activated receptor (PPAR) by electrophoretic mobility shift assay, caspase 3, 6 and 8 activities by spectrofluorometric assay, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma production by enzyme linked absorbent assay (ELISA), expression of pro- (Bax) and anti- (Bcl-2) apoptotic genes by real time polymerase chain reaction and expression of pleiotropic genes by macroarray technique RESULTS: Evidence is presented herein that the increase in triglycerides concentrations induced by OA is more pronounced than that caused by LA in Jurkat cells. Importantly, triglycerides accumulation may be a mechanism to protect lymphocytes against the toxicity induced by fatty acids. Both fatty acids raised PPAR activation, caspase 3 and 6 activities and TNF-alpha production. LA in toxic concentrations modulated the expression of genes related to cell cycle, apoptosis, proliferation, oxidative stress, and cytokine receptors. CONCLUSION: The findings reported herein support the cell death induced by OA and LA involved triglycerides accumulation, PPAR activation, caspase 3 and 6 activities and TNF-alpha production.

Comparative toxicity of fatty acids on a macrophage cell line (J774).

In the present study, the cytotoxicity of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on a macrophage cell line (J774) was investigated. The induction of toxicity was investigated by changes in cell size, granularity, membrane integrity, DNA fragmentation and phosphatidylserine externalization by using flow cytometry. Fluorescence microscopy was used to determine the type of cell death (Acridine Orange/ethidium bromide assay). The possible mechanisms involved were examined by measuring mitochondrial depolarization, lipid accumulation and PPARgamma (peroxisome-proliferator-activated receptor gamma) activation. The results demonstrate that fatty acids induce apoptosis and necrosis of J774 cells. At high concentrations, fatty acids cause macrophage death mainly by necrosis. The cytotoxicity of the fatty acids was not strictly related to the number of double bonds in the molecules: palmitic acid>docosahexaenoic acid>stearic acid=eicosapentaenoic acid=arachidonic acid>oleic acid>linoleic acid. The induction of cell death did not involve PPARgamma activation. The mechanisms of fatty acids to induce cell death involved changes in mitochondrial transmembrane potential and intracellular neutral lipid accumulation. Fatty acids poorly incorporated into triacylglycerol had the highest toxicity.

Comparative effects of eicosapentaenoic acid and docosahexaenoic acid on proliferation, cytokine production, and pleiotropic gene expression in Jurkat cells.

Comparative effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) acid on Jurkat T cells were investigated. The following parameters were evaluated: concanavalin A (Con A) induced proliferation, production of interleukin-2 (IL-2), IL-4, IL-10, and interferon-gamma (INF-gamma), and expression of pleiotropic genes by macroarray technique (83 genes in total). DHA inhibiting effect on Con A-induced proliferation was more pronounced than that of EPA. The decrease in IL-2 and INF-gamma production was observed for both fatty acids, whereas the production of IL-10 was decreased by EPA only. The expression of a significant proportion of genes was altered by the fatty acids; 30% for DHA (25 genes) and 26.5% for EPA (22 genes). DHA and EPA markedly affected the expression of genes clustered as cytokines and related receptors, signal transduction pathways, transcription factors, cell cycle, defense and repair, apoptosis, DNA synthesis, cell adhesion, cytoskeleton, and hormone receptors. Therefore, the effect of fatty acids on T-lymphocyte function involves regulation of expression of important genes. Marked differences were observed between the effects of EPA and DHA, indicating that it is an over-simplification to generalize the effects of n-3 fatty acids.

Identification of genes regulated by oleic acid in Jurkat cells by suppressive subtractive hybridization analysis.

In this study, the effect of oleic acid (50 microM) on gene expression of Jurkat cells (human T lymphocytes cell line) was examined using the suppressive subtractive hybridization approach. This technique allowed us to identify genes with higher or lower expression after cell treatment with oleic acid as compared to untreated cells. Oleic acid upregulated the expression of the translation elongation factor alpha 1 and ATP synthase 8 and downregulated gp96 (human tumor rejection antigen gp96), heat-shock protein 60 and subtilisin-like protein 4. These results suggest that oleic acid, at plasma physiological concentration, can regulate the expression of important genes to maintain the machinery that ensures cell functioning.